Anti-complement C1S antibody formulations

Abstract

Provided herein are compositions comprising a humanized antibody that specifically binds to the complement component C1s (anti-C1s antibody) that are capable of stable long-term storage. In addition to humanized antibodies, these compositions may contain arginine or a salt thereof.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 352,475, filed on June 15, 2022, under 35 U.S.C. §119(e), the entire contents of which are incorporated herein by reference.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (B155370017WO00-SEQ-JRV.xml; size: 21,726 bytes; and creation date: June 9, 2023) are incorporated herein by reference in its entirety. Background Art

[0005] The complement system is a well-known immune response effector mechanism that not only provides protection against pathogens and other harmful substances, but also provides the ability to recover from injury. The classical complement pathway is triggered by the activation of the first component of complement, which is called the C1 complex, including C1q, C1r and C1s proteins. When C1 binds to the immune complex, the C1s component (a serine protease sensitive to diisopropylfluorophosphate (DFP)) cleaves complement components C4 and C2 to initiate activation of the classical complement pathway. The classical complement pathway appears to play a role in many diseases and disorders, and stable formulations of antibodies targeting this pathway are needed. Summary of the invention

[0006] The present disclosure provides compositions comprising humanized antibodies that specifically bind to complement C1s protein (i.e., humanized anti-complement C1s antibodies, also referred to herein as "humanized anti-C1s antibodies," "humanized C1s antibodies," or "subject antibodies"). The present disclosure also provides methods of treating complement-mediated diseases or disorders, which methods comprise administering the compositions of the present disclosure.

[0007] It is found that the subject antibody has a higher tendency to self-associate, and also has a tendency to isomerize (LC D32 isomerization) of the aspartic acid residue at position number 32 of the first complementary determining region (CDR1) of the light chain in the light chain variable domain sequence. A group of comprehensive studies were conducted to understand the effects of formulation composition and process conditions on the biophysical and chemical stability of the subject antibody. The research described herein proves that the presence of arginine or its salt reduces the rate of D32 isomerization. When using the composition of the present disclosure, under refrigeration, environment and accelerated storage conditions, resistance to isomerization was observed in sample formulations of the subject antibody using different batches. The composition of the present disclosure maintains antibody stability during long-term storage, and provides protection against physicochemical stress, while also suppressing LC D32 isomerization, antibody aggregation and deamidation. The composition of the present disclosure further allows the subject antibody to be formulated as a liquid.

[0008] In one aspect, the present disclosure provides a composition comprising: (a) a humanized antibody that specifically binds to complement component C1s, wherein the antibody comprises a light chain (LC) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and (b) about 50 mM to about 200 mM arginine or a salt thereof.

[0009] In certain embodiments, the composition comprises about 100mM to about 200mM or about 100mM to about 150mM arginine or its salt. In certain embodiments, the arginine salt is arginine hydrochloride, arginine citrate, arginine oxalate, arginine phosphate, arginine succinate or arginine tartrate. In certain embodiments, the arginine salt is arginine hydrochloride. In certain embodiments, the composition comprises about 150mM arginine hydrochloride. In certain embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0010] In some embodiments, the composition comprises a buffer. In some embodiments, the composition comprises a buffer having a concentration of about 1 mM to about 50 mM, about 5 mM to about 25 mM, or about 10 mM to about 20 mM. In some embodiments, the buffer is histidine, acetate, citrate, oxalate, phosphate, succinate, or tartrate. In some embodiments, the buffer is histidine. In some embodiments, the composition comprises about 10 mM histidine.

[0011] In some embodiments, the composition further comprises a stabilizer. In some embodiments, the composition further comprises a stabilizer at a concentration of about 1% to about 8% (w / v) or about 1% to about 5% (w / v). In some embodiments, the stabilizer is sucrose, sorbitol or trehalose. In some embodiments, the stabilizer is sucrose. In some embodiments, the composition comprises about 3% (w / v) sucrose.

[0012] In some embodiments, the composition further comprises a chelating agent. In some embodiments, the composition comprises a chelating agent at a concentration of about 1 μM to about 50 μM, about 5 μM to about 25 μM, or about 10 μM to about 25 μM. In some embodiments, the chelating agent is diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA). In some embodiments, the composition comprises about 10 μM of diethylenetriaminepentaacetic acid (DTPA). In some embodiments, the chelating agent is methionine. In some embodiments, the composition comprises about 5 mM to about 10 mM of methionine.

[0013] In some embodiments, the composition further comprises a surfactant. In some embodiments, the composition comprises a surfactant at a concentration of about 0.01% to about 0.1% (w / v) or about 0.03% to about 0.06% (w / v). In some embodiments, the surfactant is polysorbate 80 (PS80) or poloxamer 188 (P188). In some embodiments, the surfactant is PS80, optionally wherein the composition comprises about 0.06% (w / v) polysorbate 80.

[0014] In some embodiments, the pH of the composition is about 6 to about 7.5, about 6 to about 7, about 6.5 to about 7.5, or about 6.5 to about 7.1. In some embodiments, the pH of the composition is about 6.8.

[0015] In some embodiments, the composition comprises about 50 mg / mL to about 250 mg / mL, or about 100 mg / mL to about 200 mg / mL of the anti-C1s antibody. In some embodiments, the composition comprises about 150 mg / mL of the anti-C1s antibody.

[0016] In some embodiments, the composition comprises: (a) about 50 mg / mL to about 250 mg / mL of antibody; (b) about 50 mM to about 200 mM of arginine HCl; (c) about 1 mM to about 50 mM of histidine; (d) about 1% to about 8% (w / v) sucrose; (e) about 1 μM to about 50 μM of diethylenetriaminepentaacetic acid (DTPA); and (f) about 0.01% to about 0.1% (w / v) of polysorbate 80 (PS80), and the pH of the composition is about 6 to about 7.5. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0017] In some embodiments, the composition comprises: (a) about 150 mg / mL of antibody; (b) about 150 mM arginine HCl; (c) about 10 mM histidine; (d) about 3% (w / v) sucrose; (e) about 10 μM DTPA; and (f) about 0.06% (w / v) PS80, and the pH of the composition is about 6.5 to about 7.1, or about 6.8. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0018] In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO:7 and a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody is a Fab fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, the antibody comprises a heavy chain constant region of isotype IgG4. In some embodiments, relative to the IgG4 constant region sequence of SEQ ID NO:11, the IgG4 constant region comprises proline, glutamic acid, leucine and serine substitutions at amino acid residues 108, 115, 308 and 314, respectively. In some embodiments, the antibody comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:13. In some embodiments, the antibody comprises a light chain comprising an amino acid sequence of SEQ ID NO:9 and a heavy chain comprising an amino acid sequence of SEQ ID NO:10.

[0019] In some embodiments, the antibody has a lower isomerization rate at D32 in the light chain located in CDR1 compared to an antibody in a corresponding formulation without arginine or a salt thereof, optionally wherein D32 isomerization is determined by total peptide mapping. In some embodiments, the antibody (i) has an isomerization rate that is at least about 2%-3% lower per week at 40°C, at least about 1%-3% lower per month at 25°C, or at least about 0.4%-0.6% lower per month at 5°C compared to an antibody in a corresponding formulation without arginine or a salt thereof; or (ii) an isomerization rate that is about 10% lower after storage at 25°C for 12 weeks compared to an antibody in a corresponding formulation without arginine or a salt thereof.

[0020] In some embodiments, the isomerization rate of the antibody is less than 7.5% after storage for 12 weeks at 5°C, less than 20% after storage for 12 weeks at 25°C, or less than 35% after storage for 12 weeks at 40°C.

[0021] In some embodiments, the composition is in liquid, lyophilized, or reconstituted lyophilized form. In some embodiments, the composition is in liquid form.

[0022] In one aspect, the present disclosure provides a container containing the composition of the present disclosure. In some embodiments, the container is a vial or a syringe. In some embodiments, the container is a syringe. In some embodiments, the syringe is a prefilled syringe.

[0023] In one aspect, the disclosure provides a kit or article of manufacture comprising a container of the disclosure.

[0024] In one aspect, the present disclosure provides a pharmaceutical unit dosage form suitable for parenteral administration to a human, the pharmaceutical unit dosage form comprising a composition of the present disclosure in a container.

[0025] In one aspect, the disclosure provides a method comprising administering a composition of the disclosure to a human.

[0026] In one aspect, the disclosure provides a method of reducing the level of a complement component cleavage product in a human, the method comprising administering to the human a composition of the disclosure.

[0027] In one aspect, the disclosure provides a method of inhibiting C1s-mediated cleavage of complement components in a human, the method comprising administering to the human a composition of the disclosure.

[0028] In some embodiments, the human has cold agglutinin disease (CAD), immune thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), or antibody-mediated rejection (AMR).

[0029] In one aspect, the present disclosure provides a method of treating a complement-mediated disease in a person in need thereof, the method comprising administering to the person a composition of the present disclosure. In some embodiments, the complement-mediated disease is cold agglutinin disease (CAD), immune thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), or antibody-mediated rejection (AMR).

[0030] In some embodiments, administration is intravenous or subcutaneous.

[0031] In one aspect, the present disclosure provides a drug delivery device comprising a primary container containing a composition of the present disclosure, wherein the drug delivery device is a sleeve-activated autoinjector with a manual needle insertion.

[0032] International Publication No. WO 2014 / 066744, entitled Anti-Complement C1s Antibodies and Uses Thereof, filed on October 25, 2013, WO 2016 / 164358, entitled Humanized Anti-C1s Antibodies and Methods of Use Thereof, filed on April 5, 2016, and WO 2018 / 071676, entitled Anti-C1s Antibodies and Methods of Use Thereof, filed on October 12, 2017, are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown are Biacore binding versus LC D32 isomerization rate or forced isomerization studies.

[0034] Figure 2A-2D The effect of pH and stabilizer on the isomerization of LC D32 is shown. Figure 2A The effect of pH and stabilizer at 5°C is shown. Figure 2B The effect of pH and stabilizer at 25°C is shown. Figure 2C The effect of pH and stabilizer at 40°C is shown. Figure 2D The effects of pH and stabilizers on the isomerization rate at different temperatures are summarized.

[0035] Figure 3A-3E Analysis of an orbital oscillation study is shown. Figure 3A : HMW substances, Figure 3B : Turbidity, Figure 3C-Figure 3E : Subvisible particles (≤2μm, ≤10μm, ≤25μm particles / mL).

[0036] (h = hour; d = day).

[0037] Figure 4A-4E Analysis of wrist action oscillation studies is shown. Figure 4A : HMW substances, Figure 4B : Turbidity, Figure 4C-4E: Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (h = hour).

[0038] Figure 5A-5D Analysis of wrist oscillation + silicone spiking studies is shown. Figure 5A : Turbidity, Figure 5B-5D : Subvisible particles (≤2μm, ≤10μm, ≤25μm particles / mL).

[0039] (h = hour).

[0040] Figure 6A-6F Analysis of an IV bag dilution study is shown. Fig. 6A : HMW substances, Figure 6B : Turbidity, Figure 6C :concentration, Figure 6D-6F : Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (h = hour).

[0041] Figure 7A-7F Analysis of the photostability studies is shown. Fig. 7A : HMW substances, Figure 7B : Turbidity, Figure 7C : PS80 concentration, Figure 7D-7F : Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (h = hour; d = day).

[0042] Figure 8A-8E Analysis of the Batch 1 chelator study is shown. Fig. 8A PS80 concentrations of 10 μM EDTA compared to 10 μM DTPA are shown. Figure 8B The remaining PS 80% for 10 / 50 μM EDTA compared to 10 / 50 μM DTPA is shown. Figure 8C : Ester spectrum, Fig.8D : HMW substances, Fig. 8E : Biacore analysis. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0043] Fig. 9 Shown is a visual analysis of Batch 1 formulation stored at 40°C for 3 months.

[0044] Figures 10A-10E Analysis of the Batch 2 chelator study is shown. Fig. 10A PS80 concentrations of 10 μM EDTA compared to 10 μM DTPA are shown. Fig. 10B The remaining PS 80% for 10 / 50 μM EDTA compared to 10 / 50 μM DTPA is shown. Fig. 10C: HMW substances, Fig. 10D : LMW substances, Fig.10E : Biacore analysis. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0045] Figure 11A-11E Analysis of the Batch 3 chelator study is shown. Fig.11A PS80 concentrations of 10 μM EDTA compared to 10 μM DTPA are shown. Fig. 11B The remaining PS 80% for 10 / 50 μM EDTA compared to 10 / 50 μM DTPA is shown. Fig. 11C : HMW substances, Fig.11D : LMW substances, Fig.11E : Biacore analysis. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0046] Figure 12A-12E Analysis of Batch 4 chelator studies is shown. Fig. 12A PS80 concentrations of 10 μM EDTA compared to 10 μM DTPA are shown. Fig. 12B PS80% comparison - 10 / 50 μM EDTA vs. 10 / 50 μM DTPA is shown. Fig. 12C : HMW substances, Fig.12D : LMW substances, Fig.12E : Biacore analysis. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0047] Figure 13A-13E Shown is the analysis of the prefilled syringe (PFS) stability study conducted on Batch 1 samples by buffer type (H = histidine buffer; K = potassium phosphate buffer) and pH (value noted after buffer type). Fig.13A : HMW substances, Fig. 13B :concentration, Fig. 13C : pH, Fig.13D : Turbidity, Fig.13E : PS80 concentration. (W = week; M = month; C = degree Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0048] Figure 14A-Figure 14C Shown are sub-visible particle analyses of PFS stability studies conducted on Batch 1 samples for buffer type (H = histidine buffer; K = potassium phosphate buffer) and pH (values ​​noted after buffer type). Fig.14A : Sub-visible particles ≤ 2μm particles / mL, Fig. 14B: Sub-visible particles ≤ 10 μm particles / mL, Fig. 14C : Subvisible particles ≤ 25 μm particles / mL. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means 3 months storage at 25°C) (OMPI = OMPIPFS; BD = BD PFS).

[0049] Figure 15A-15E Shown is an analysis of the vial stability study conducted on Batch 1 samples by buffer type (H = histidine buffer; K = potassium phosphate buffer) and pH (value noted after buffer type). Fig.15A : HMW substances, Fig. 15B :concentration, Fig. 15C : pH, Fig.15D : Turbidity, Fig.15E : PS80 concentration. (W = week; M = month; C = degree Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0050] Figure 16A-16C Shown are sub-visible particle analyses of vial stability studies conducted on Batch 1 samples by buffer type (H = histidine buffer; K = potassium phosphate buffer) and pH (value noted after buffer type). Fig.16A : Sub-visible particles ≤ 2μm particles / mL, Fig. 16B : Sub-visible particles ≤ 10 μm particles / mL, Fig. 16C : Subvisible particles ≤ 25 μm particles / mL. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0051] Figures 17A-17G Analysis of the frozen storage stability study of Batch 1 samples is shown. Fig.17A : HMW substances, Fig. 17B :concentration, Fig. 17C : pH, Fig.17D : Turbidity, Figures 17E-17G : Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (M = month).

[0052] Figure 18A-18E Peptide maps and Biacore binding analysis of batch 1 samples are shown. Fig.18A :LC D32 isomerization, Fig.18B :Heavy chain (HC) M251 oxidation, Fig.18C : HC PENNYK deamidation, Fig.18D : HC VSNK deamidation, Fig.18E : Biacore analysis. (W = weeks; M = months; C = degrees Celsius; for example, "6M-25C" means storage at 25°C for 6 months).

[0053] Figure 19A-19G Analysis of the PFS stability study of Batch 2 samples is shown. Fig.19A : HMW substances, Fig.19B : LMW substances, Fig.19C :concentration, Fig.19D : pH, Fig.19E : Turbidity, Fig.19F : PS80 concentration, Figure 19G : Charge variant analysis. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0054] Figure 20A-20C Sub-visible particle analysis of Batch 2 samples from the PFS stability study is shown. Fig. 20A : Sub-visible particles ≤ 2μm particles / mL, Fig. 20B : Sub-visible particles ≤ 10 μm particles / mL, Fig. 20C : Subvisible particles ≤ 25 μm particles / mL. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0055] Figure 21A-21G Analysis of the vial stability study of Batch 2 samples is shown. Fig.21A : HMW substances, Fig.21B : LMW substances, Fig. 21C :concentration, Fig.21D : pH, Fig.21E : Turbidity, Fig.21F : PS80 concentration, Figure 21G : Charge variant analysis. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0056] Figure 22A-22C Subvisible particle analysis of vial stability study of Batch 2 samples is shown. Fig.22A : Sub-visible particles ≤ 2μm particles / mL, Fig. 22B : Sub-visible particles ≤ 10 μm particles / mL, Fig. 22C : Subvisible particles ≤ 25 μm particles / mL. (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0057] Figure 23A-23F Analysis of the frozen storage stability study of Batch 2 samples is shown. Fig.23A : HMW substances, Fig. 23B : LMW substances, Fig.23C :concentration, Fig.23D : pH, Fig.23E: Turbidity, Fig.23F : Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (M = month).

[0058] Figure 24A-Figure 24E Peptide mapping and Biacore binding analysis of batch 2 samples are shown. Fig.24A :LC D32 isomerization, Fig. 24B : HC M251 oxidation, Fig.24C : HC PENNYK deamidation, Fig.24D : HC VSNK deamidation, Fig.24E : Biacore analysis. (V = vial; P = PFS) (W = week; M = month; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0059] Figure 25A-25G Analysis of the PFS stability study of Batch 3 samples is shown. Fig.25A : HMW substances, Fig.25B : LMW substances, Fig.25C :concentration, Fig.25D : pH, Fig.25E : Turbidity, Fig.25F : PS80 concentration, Figure 25G : Subvisible particles (≤2 μm particles / mL, ≤10 μm particles / mL, ≤25 μm particles / mL). (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0060] Figure 26A-26G Analysis of the vial stability study of Batch 3 samples is shown. Fig.26A : HMW substances, Fig.26B : LMW substances, Fig.26C :concentration, Fig.26D : pH, Fig.26E : Turbidity, Fig.26F : PS80 concentration, Figure 26G : Subvisible particles (≤2 μm particles / mL, ≤10 μm particles / mL, ≤25 μm particles / mL). (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0061] Figure 27A-Figure 27E Peptide maps and Biacore binding analysis of batch 3 samples are shown. Fig.27A :LC D32 isomerization, Fig.27B : HC M251 oxidation, Fig.27C : HC PENNYK deamidation, Fig.27D : HC VSNK deamidation, Fig.27E: Biacore analysis. (V = vial; P = PFS). (W = week; M = month; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0062] Figure 28A-28G Analysis of the vial stability study of Batch 4 samples is shown. Fig.28A : HMW substances, Fig.28B : LMW substances, Fig.28C :concentration, Fig.28D : pH, Fig.28E : Turbidity, Fig.28F : PS80 concentration, Figure 28G : Subvisible particles (≤2 μm particles / mL, ≤10 μm particles / mL, ≤25 μm particles / mL). (W = weeks; M = months; C = degrees Celsius; for example, "3M-25C" means storage at 25°C for 3 months).

[0063] Figure 29A-29E Peptide mapping and Biacore binding analysis of batch 4 samples are shown. Fig.29A :LC D32 isomerization, Fig.29B : HC M251 oxidation, Fig.29C : HC PENNYK deamidation, Fig.29D : HC VSNK deamidation, Fig.29E : Biacore analysis. (V = vial). (W = week; M = month; C = degrees Celsius; for example, "1M-5C" means storage at 5°C for 1 month).

[0064] Figure 30A-30G Analysis of the vial stability study of Batch 5 samples is shown. Fig. 30A : HMW substances, Fig. 30B : LMW substances, Fig. 30C :concentration, Fig.30D : pH, E: turbidity, Fig.30F : PS80 concentration, Figure 30G : Subvisible particles (≤2 μm particles / mL, ≤10 μm particles / mL, ≤25 μm particles / mL). (W = weeks; M = months; C = degrees Celsius; for example, "1M-5C" means storage at 5°C for 1 month).

[0065] Figure 31A-Figure 31E Analysis of samples in vials with wrist shaking is shown. Fig.31A : HMW substances, Fig.31B : LMW substances, Fig.31C : Turbidity, Fig.31D : Charge variant analysis (acidic %, monomeric %, basic %), Fig.31E: Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (h = hour).

[0066] Figure 32A-Figure 32E Analysis of samples subjected to wrist oscillation in PFS is shown. Fig.32A : HMW substances, Fig.32B : LMW substances, Fig.32C : Turbidity, Fig.32D : Charge variant analysis (acidic %, monomeric %, basic %), Fig.32E : Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (h = hour).

[0067] Figure 33A-Figure 33F Analysis of samples subjected to freeze-thaw cycles is shown. Fig.33A : HMW substances, Fig.33B : LMW substances, Fig.33C : Turbidity, Fig.33D :concentration, Fig.33E : pH, Fig.33F : Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (FT=freeze-thaw cycles).

[0068] Figure 34A-Figure 34I Photostability analysis of the samples is shown. Fig.34A : HMW substances, Fig.34B : LMW substances, Fig.34C : Turbidity, Fig.34D :concentration, Fig.34E : pH, F: PS 80 concentration, Figure 34G : Charge variant analysis, Fig.34H : Total peptide map analysis, Fig.34I : Subvisible particles (≤2 μm, ≤10 μm, ≤25 μm particles / mL). (d=day). DETAILED DESCRIPTION

[0069] Humanized anti-C1s antibody

[0070] The compositions disclosed herein include humanized anti-C1s antibodies. The humanized anti-C1s antibodies disclosed herein bind to and inhibit the activated form of C1s within the classical pathway (CP). The complement system is a component of the innate immune system that mediates humoral immunity. The mechanism of the antibody is specific for CP, leaving the lectin pathway and the alternative pathway functionally intact.

[0071] In some embodiments, the humanized anti-C1s antibodies of the present disclosure inhibit C1s-mediated cleavage of complement component C4, for example, by inhibiting the enzymatic activity of the serine protease domain of C1s. In some embodiments, the humanized anti-C1s antibodies of the present disclosure inhibit C1s-mediated cleavage of complement component C2. In some embodiments, the humanized anti-C1s antibodies of the present disclosure inhibit C1s-mediated cleavage of C4 and C2.

[0072] In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to a complement C1s protein having the amino acid sequence depicted in SEQ ID NO: 15. The amino acid sequence SEQ ID NO: 15 provided below represents the complement C1s protein of Homo sapiens:

[0073] (SEQ ID NO: 15).

[0074] In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to human complement C1s protein, wherein the dissociation constant (KD) is no more than 2.5 nM. In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to human complement C1s protein, wherein the KD is no more than 2 nM. In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to human complement C1s protein, wherein the KD is no more than 1 nM. In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to human complement C1s protein, wherein the KD is no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM or no more than 0.1 nM. In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to human complement C1s protein, wherein the KD is no more than 0.3 nM. In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to human complement C1s protein, wherein the KD is no more than 0.2 nM. In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to human complement C1s protein with a KD of no more than 0.1 nM. Methods for measuring the binding of antibodies to human complement C1s protein can be determined by those skilled in the art.

[0075] In some embodiments, the humanized anti-C1s antibodies of the present disclosure bind to human complement C1s protein with a KD of no more than 90 pM, no more than 80 pM, no more than 70 pM, no more than 60 pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, no more than 20 pM, no more than 10 pM, no more than 9 pM, no more than 8 pM, no more than 7 pM, no more than 6 pM, no more than 5 pM, no more than 4 pM, no more than 3 pM, no more than 2 pM, or no more than 1 pM.

[0076] In some embodiments, the humanized anti-C1s antibodies of the present disclosure inhibit the classical complement pathway with a half-maximal inhibitory concentration (IC 50 ) is 10 -8 M or lower, 5×10 -9 M or lower, or 10 -9 M or lower.

[0077] "Antibodies" encompass antibodies or immunoglobulins of any isotype, including but not limited to humanized antibodies and chimeric antibodies. Antibodies can be single-chain antibodies (scAbs) or single-domain antibodies (dAbs) (e.g., single-domain heavy chain antibodies or single-domain light chain antibodies; see Holt et al. (2003) Trends Biotechnol. [Biotech Trends] 21:484). The term "antibody" also encompasses fragments of antibodies that retain specific binding to an antigen (antibody fragments). "Antibodies" further include single-chain variable fragments (scFv) and diabodies, scFvs are antibody heavy chains (V H ) and light chain (V L ) variable region fusion protein, diabody is composed of V H and V L Non-covalent dimers of scFv fragments of the same family (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)). Other fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein are also encompassed by the term "antibody".

[0078] "Antibody fragments" include a portion of an intact antibody, such as the antigen binding region or variable region of an intact antibody. Examples of antibody fragments include antigen binding fragments (Fab), Fab', F(ab')2, variable domain Fv fragments (Fv), Fd fragments, and antigen binding fragments of chimeric antigen receptors.

[0079] Papain digestion of antibodies produces two identical antigen-binding fragments (called "Fab" fragments), each with a single antigen-binding site, and a residual "Fc" fragment (the name reflects its ability to crystallize readily). Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0080] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This region includes a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define V H -V L The antigen binding site on the surface of the dimer. These six CDRs together give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although with a lower affinity than the entire binding site.

[0081] The "Fab" fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment by the addition of several residues including at least one cysteine ​​from the antibody hinge region at the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH herein represents Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0082] The "scFv" antibody fragment contains the V H and V L , wherein these regions are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a V H Area and V L The polypeptide linker between the two regions enables the scFv to form the required structure for antigen binding. For a review of scFv, see Pluckthun's The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0083] "Diabodies" are small antibody fragments with two antigen-binding sites. These fragments contain the same V domain as the V domain in the same polypeptide chain. L Connect V H (V H -V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the two domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, Hollinger et al. Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 90: 6444-6448 (1993).

[0084] Antibodies can be monovalent or divalent.Antibodies can be Ig monomers, "Y-shaped" molecules composed of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0085] The antibody can be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, and / or a fluorescent protein. The antibody can be further conjugated to other moieties, such as members of a specific binding pair, such as the biotin member of a biotin-avidin specific binding pair. The antibody can also be bound to a solid support, including but not limited to polystyrene plates and / or beads.

[0086] An "isolated" antibody is an antibody that has been identified and separated and / or recovered from a component of its natural environment (i.e., not naturally occurring). Contaminating components in its natural environment are materials that interfere with the use of the antibody (e.g., diagnostic or therapeutic use), and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody (1) is purified to, for example, greater than 90%, greater than 95%, greater than 98%, or greater than 99% by weight of the antibody, as determined by the Lowry method, (2) is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) is purified to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody encompasses an antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. In some embodiments, the isolated antibody is prepared by at least one purification step.

[0087] "Monoclonal antibody" is an antibody produced by a group of identical cells, all of which are produced from a single cell by repetitive cell replication. That is, the cell clone produces only a single antibody species. Although monoclonal antibodies can be produced using hybridoma production technology, other production methods known to those skilled in the art (e.g., antibodies derived from antibody phage display libraries) can also be used.

[0088] "Complementarity determining region (CDR)" is a non-contiguous antigen binding site found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Lefranc et al. (2003) Developmental and Comparative Immunology 27:55; Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared to each other. Nonetheless, the use of any of the Kabat, Lefranc, Chothia or MacCallum definitions (also referred to as numbering systems) to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to fall within the scope of the term as defined and used herein.

[0089] The terms "LC CDR1", "LC CDR2" and "LC CDR3" refer to the first, second and third CDRs in the light chain variable region, respectively. As used herein, the terms "HC CDR1", "HC CDR2" and "HC CDR3" refer to the first, second and third CDRs in the heavy chain variable region, respectively. As used herein, the terms "CDR1", "CDR2" and "CDR3" refer to the first, second and third CDRs in the variable region of either chain, respectively.

[0090] When used in relation to an antibody variable region, "framework" includes all amino acid residues outside the CDR region within the antibody variable region. The variable region framework is typically a discontinuous amino acid sequence that includes only those amino acids outside the CDR. A "framework region" includes individual framework domains separated by CDRs.

[0091] "Humanized antibody" is an antibody comprising antibody parts from different sources, wherein at least one part comprises an amino acid sequence of human origin. For example, a humanized antibody may comprise a part of an antibody derived from a non-human source (such as a mouse) having the necessary specificity and a part of an antibody sequence derived from a human source (e.g., a chimeric immunoglobulin), which is chemically linked together (e.g., synthesized) by conventional techniques or prepared as a continuous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein part of a chimeric antibody can be expressed to produce a continuous polypeptide chain). Another example of a humanized antibody is an antibody comprising at least one chain of a framework region containing a CDR derived from an antibody from a non-human source and a light chain and / or heavy chain derived from a human source (e.g., a CDR-grafted antibody with or without a framework change). Chimeric or CDR-grafted single-chain antibodies are also covered by the term humanized immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, MS et al., WO 86 / 01533; Neuberger, MS et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, EA et al., European Patent Application No. 0,519,596 A1. For single-chain antibodies, see also Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, RE et al., Science, 242:423-426 (1988).

[0092] In some embodiments, humanized antibodies are produced using synthetic and / or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chains. For example, nucleic acid (e.g., DNA) sequences encoding humanized variable regions can be constructed using PCR mutagenesis methods to change the DNA sequence encoding the human or humanized chain, such as a DNA template from a previously humanized variable region (see, e.g., Kamman, M. et al., Nucl. Acids Res. [Nucleic Acids Research], 17: 5404 (1989); Sato, K. et al., Cancer Research [Cancer Research], 53: 851-856 (1993); Daugherty, BL et al., Nucleic Acids Res. [Nucleic Acids Research], 19 (9): 2471-2476 (1991); and Lewis, AP and JS Crowe, Gene [Gene], 101: 297-302 (1991)). Using these or other suitable methods, variants can also be easily generated. For example, cloned variable regions can be mutagenized and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see, e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93 / 06213, published April 1, 1993).

[0093] In some embodiments, the humanized anti-C1s antibodies described herein are full-length IgG, Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scFv, scAb or Fv. In some embodiments, the humanized anti-C1s antibodies described herein are full-length IgG. In some embodiments, the heavy chain of any humanized anti-C1s antibody as described herein comprises a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3 or a combination thereof). The heavy chain constant region can be of any suitable origin (e.g., human, mouse, rat or rabbit). In some embodiments, the heavy chain constant region is from human IgG (gamma heavy chain), such as IgG1, IgG2 or IgG4.

[0094] In some embodiments, mutations can be introduced into the heavy chain constant region of any of the humanized anti-C1s antibodies described herein. In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the heavy chain constant region (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region, numbered according to the Kabat numbering system (e.g., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Antibody Fc region mutations that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Examples of mutations that can be made in an antibody Fc receptor to alter the affinity of an antibody for an Fc receptor are described, for example, in Smith P et al., (2012) PNAS [Proceedings of the National Academy of Sciences of the United States of America] 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which references are incorporated herein by reference.

[0095] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the heavy chain constant region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described in, e.g., U.S. Pat. No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.

[0096] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into IgG constant domains or their FcRn binding fragments to change (e.g., shorten or increase) the in vivo half-life of the antibody. In some embodiments, one or more mutations are introduced into Fc or hinge-Fc domain fragments. For examples of mutations that will change (e.g., shorten or increase) the in vivo half-life of an antibody, see, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745.

[0097] In some embodiments, the constant region antibodies described herein are IgG1 constant regions and comprise a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamate (E) substitution at position 256, numbered according to the EU index in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to exhibit a four-fold increase in half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem [Journal of Biological Chemistry] 281: 23514-24). In some embodiments, the antibody comprises an IgG constant domain containing one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436, numbered according to the EU index in Kabat. Additional mutations that can be introduced into the heavy chain constant region that will increase antibody half-life are known in the art, for example, the M428L / N434S (EU numbering, M459L / N466S Kabat numbering) mutation as described in Zalevsky et al., Nat Biotechnol. 2010 Feb;28(2):157-159.

[0098] In some embodiments, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region to change the effector function of the antibody. The effector ligand of affinity change can be, for example, an Fc receptor or the C1 component of complement. The method is further described in detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion or inactivation of the constant region domain (by point mutation or other means) can reduce the Fc receptor binding of circulating antibodies, thereby increasing tumor localization. For descriptions of mutations that cause constant domain deletion or inactivation to increase tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886. In some embodiments, at least one amino acid substitution can be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, thereby reducing Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem [Journal of Biological Chemistry] 276: 6591-604).

[0099] In some embodiments, at least one amino acid in the constant region may be replaced with a different amino acid residue so that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). This method is further described in detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, at least one amino acid residue in the N-terminal region of the CH2 domain of the antibody described herein is altered, thereby altering the ability of the antibody to fix complement. This method is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and / or increase the affinity of the antibody for Fcγ receptors. This method is further described in International Publication No. WO 00 / 42072.

[0100] In some embodiments, to avoid potential complications due to Fab arm exchange known to occur with native IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation (Angal S. et al., "A single aminoacid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. In some embodiments, to reduce residual antibody-dependent cellular toxicity, an L235E (EU numbering, corresponding to L248E in Kabat numbering) mutation is introduced into the heavy chain constant region, e.g., as described in Benhnia et al., J. Virology, December 2009, pp. 12355-12367.

[0101] In some embodiments, the humanized anti-C1s antibody comprises a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of KASQSVDYDGDSYMN (SEQ ID NO: 1). In some embodiments, the humanized anti-C1s antibody comprises a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of DASNLES (SEQ ID NO: 2). In some embodiments, the humanized anti-C1s antibody comprises a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of QQSNEDPWT (SEQ ID NO: 3). In some embodiments, the humanized anti-C1s antibody comprises a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 3.

[0102] In some embodiments, the humanized anti-C1s antibody comprises a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of DDYIH (SEQ ID NO:4). In some embodiments, the humanized anti-C1s antibody comprises a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of RIDPADGHTKYAPKFQV (SEQ ID NO:5). In some embodiments, the humanized anti-C1s antibody comprises a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of YGYGREVFDY (SEQ ID NO:6). In some embodiments, the humanized anti-C1s antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO:4, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a HC CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0103] In some embodiments, the humanized anti-C1s antibody comprises a LC CDR1 comprising the amino acid sequence of SEQ ID NO:1, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:2, a LC CDR3 comprising the amino acid sequence of SEQ ID NO:3, a HC CDR1 comprising the amino acid sequence of SEQ ID NO:4, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a HC CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0104] In some embodiments, the humanized anti-C1s antibody comprises a light chain variable region (VL) comprising the following amino acid sequence:

[0105] DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKPGQPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPWTFGGGTKVEIK (SEQ ID NO:7). In some embodiments, the humanized anti-C1s antibody comprises LC CDR1, LC CDR2, and LC CDR3 in the VL comprising the amino acid sequence of SEQ ID NO:7.

[0106] In some embodiments, the humanized anti-C1s antibody comprises a heavy chain variable region (VH) comprising the following amino acid sequence:

[0107] QVQLVQSGAEVKKPGASVKLSCTASGFNIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLRSEDTAVYYCARYGYGREVFDYWGQGTTVTVSS (SEQ ID NO: 8). HC CDR1, HC CDR2 and HC CDR3 in VH comprising the amino acid sequence of SEQ ID NO: 8.

[0108] In some embodiments, the humanized anti-C1s antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:7 and a VH comprising the amino acid sequence of SEQ ID NO:8.

[0109] In some embodiments, the humanized anti-C1s antibody comprises LC CDR1, LC CDR2, and LC CDR3 in VL comprising the amino acid sequence of SEQ ID NO:7 and HC CDR1, HC CDR2, and HC CDR3 in VH comprising the amino acid sequence of SEQ ID NO:8.

[0110] In some embodiments, the humanized anti-C1s antibody comprises a light chain (LC) comprising the following amino acid sequence:

[0111] DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKPGQPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:9).

[0112] In some embodiments, the humanized anti-C1s antibody comprises a heavy chain (HC) comprising the following amino acid sequence:

[0113] QVQLVQSGAEVKKPGASVKLSCTASGFNIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLRSEDTAVYYCARYGYGREVFDYWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK(SEQ ID NO:10).

[0114] In some embodiments, the humanized anti-C1s antibody comprises a LC comprising the amino acid sequence of SEQ ID NO:9 and a HC comprising the amino acid sequence of SEQ ID NO:10.

[0115] In some embodiments, the humanized anti-C1s antibody comprises a LC CDR1 comprising an amino acid sequence containing no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) relative to the LC CDR1 amino acid sequence of SEQ ID NO: 1. In some embodiments, the humanized anti-C1s antibody comprises a LC CDR2 comprising an amino acid sequence containing no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) relative to the LC CDR2 amino acid sequence of SEQ ID NO: 2. In some embodiments, the humanized anti-C1s antibody comprises a LC CDR3 comprising an amino acid sequence containing no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) relative to the LC CDR3 amino acid sequence of SEQ ID NO: 3.

[0116] In some embodiments, the humanized anti-C1s antibody comprises a HC CDR1 comprising an amino acid sequence containing no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) relative to the HC CDR1 amino acid sequence of SEQ ID NO: 4. In some embodiments, the humanized anti-C1s antibody comprises a HC CDR2 comprising an amino acid sequence containing no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) relative to the HC CDR2 amino acid sequence of SEQ ID NO: 5. In some embodiments, the humanized anti-C1s antibody comprises a HC CDR3 comprising an amino acid sequence containing no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) relative to the HC CDR3 amino acid sequence of SEQ ID NO: 6.

[0117] In some embodiments, the humanized anti-C1s antibody comprises a VL comprising an amino acid sequence that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) relative to the VL amino acid sequence of SEQ ID NO:7.

[0118] In some embodiments, the humanized anti-C1s antibody comprises a VH comprising an amino acid sequence that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) relative to the VH amino acid sequence of SEQ ID NO:8.

[0119] In some embodiments, the humanized anti-C1s antibody comprises a VL comprising a LC CDR1 comprising the amino acid sequence of SEQ ID NO:1, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:2, a LC CDR3 comprising the amino acid sequence of SEQ ID NO:3, and a framework region comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) relative to the VL sequence of SEQ ID NO:7.

[0120] In some embodiments, the humanized anti-C1s antibody comprises a VH comprising a HC CDR1 comprising the amino acid sequence of SEQ ID NO:4, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:5, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:6, and a framework region comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) relative to the VH sequence of SEQ ID NO:8.

[0121] In some embodiments, the humanized anti-C1s antibody comprises (a) a VL comprising a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and comprising a framework region comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) relative to the VL sequence of SEQ ID NO: 7, and (b) a VH comprising a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 6, and comprising a framework region comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) relative to the VL sequence of SEQ ID NO: The VH sequence of ID NO: 8 contains a framework region with no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation).

[0122] In some embodiments, the humanized anti-C1s antibody comprises a VL comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the VL amino acid sequence of SEQ ID NO:7.

[0123] In some embodiments, the humanized anti-C1s antibody comprises a VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the VH amino acid sequence of SEQ ID NO:8.

[0124] In some embodiments, the humanized anti-C1s antibody comprises a VL comprising an LC CDR1 comprising the amino acid sequence of SEQ ID NO:1, an LC CDR2 comprising the amino acid sequence of SEQ ID NO:2, an LC CDR3 comprising the amino acid sequence of SEQ ID NO:3, and comprising a framework region that is at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the framework region of the VL sequence of SEQ ID NO:7.

[0125] In some embodiments, the humanized anti-C1s antibody comprises a VH comprising a HC CDR1 comprising the amino acid sequence of SEQ ID NO:4, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:5, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:6, and a framework region that is at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the framework region of the VH sequence of SEQ ID NO:8.

[0126] In some embodiments, the humanized anti-C1s antibody comprises (a) a VL comprising a LC CDR1 comprising the amino acid sequence of SEQ ID NO:1, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:2, a LC CDR3 comprising the amino acid sequence of SEQ ID NO:3, and comprising framework regions that are at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the framework regions of the VL sequence of SEQ ID NO:7, and (b) a VH comprising a HC CDR1 comprising the amino acid sequence of SEQ ID NO:4, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:5, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:6, and comprising framework regions that are at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the framework regions of the VH sequence of SEQ ID NO:8.

[0127] In some embodiments, the heavy chain constant region of any humanized anti-C1s antibody described herein is an IgG4 constant region or a variant thereof. Examples of IgG4 constant regions and variants are provided in Table 1.

[0128] Table 1. Examples of heavy chain constant regions

[0129]

[0130]

[0131] In some embodiments, the light chain of any humanized anti-C1s antibody described herein may further comprise a light chain constant region (C L ). In some examples, C L is a kappa light chain. In other embodiments, C L is a lambda light chain. In some embodiments, C L is a kappa light chain, the sequence of which is provided below:

[0132] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC(SEQ ID NO:14)

[0133] Other antibody heavy and light chain constant regions are well known in the art, such as those provided in the IMGT database (imgt.org) or vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0134] Composition

[0135] The present disclosure provides compositions, including pharmaceutical compositions or formulations, comprising humanized anti-C1s antibodies. The humanized anti-C1s antibodies of the present disclosure can be formulated into pharmaceutical compositions by combining with appropriate pharmaceutically acceptable carriers, pharmaceutically acceptable diluents or other pharmaceutically acceptable excipients.

[0136] Exemplary antibody concentrations in the compositions of the present disclosure may range from about 50 mg / mL to about 250 mg / mL, about 75 mg / mL to about 225 mg / mL, about 100 mg / mL to about 200 mg / mL, about 100 mg / mL to about 150 mg / mL, about 150 mg / mL to about 200 mg / mL, about 125 mg / mL to about 175 mg / mL, or about 140 mg / mL to about 160 mg / mL. In some embodiments, the antibody concentration in the compositions of the present disclosure is about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 175 mg / mL, about 200 mg / mL, or about 250 mg / mL. In some embodiments, the compositions of the present disclosure comprise about 150 mg / mL of a humanized anti-C1s antibody.

[0137] In certain embodiments, the compositions of the present disclosure include arginine or its salt.Arginine can serve as a stabilizer.As described herein, the presence of arginine reduces the isomerization rate (D32 isomerization rate) of aspartic acid residues at position number 32 in the light chain variable domain sequence of the subject antibody.The scope of the exemplary concentration of arginine or its salt in the compositions can be about 50mM to about 200mM, about 75mM to about 175mM, about 100mM to about 150mM, about 125mM to about 175mM or about 125mM to about 150mM. In some embodiments, the compositions of the present disclosure comprise about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, or about 200 mM arginine or a salt thereof. Exemplary arginine salts include arginine citrate, arginine hydrochloride, arginine oxalate, arginine phosphate, arginine succinate or arginine tartrate. Other arginine salts may also be used. In certain embodiments, the composition of the present disclosure comprises arginine hydrochloride. In certain embodiments, the composition of the present disclosure comprises about 50mM to about 200mM, about 75mM to about 175mM, about 100mM to about 150mM, about 125mM to about 175mM or about 125mM to about 150mM arginine hydrochloride. In some embodiments, the compositions of the present disclosure include about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 130mM, about 135mM, about 140mM, about 145mM, about 150mM, about 155mM, about 160mM, about 165mM, about 170mM, about 175mM, about 180mM, about 185mM, about 190mM or about 200mM arginine hydrochloride. In some embodiments, the compositions of the present disclosure include about 150mM arginine hydrochloride. In some embodiments, arginine hydrochloride is L-arginine hydrochloride.

[0138] In certain embodiments, the compositions of the present disclosure include a buffer. The exemplary concentration of the buffer in the composition can range from about 1mM to about 50mM, about 10mM to about 40mM, about 10mM to about 30mM, about 10mM to about 25mM, about 10mM to about 25mM, about 5mM to about 25mM, about 5mM to about 20mM, or about 5mM to about 10mM. In certain embodiments, the compositions of the present disclosure include a buffer of about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM. Exemplary buffers include acetate, citrate, histidine, oxalate, phosphate, succinate, and tartrate. Other buffers may also be used. In some embodiments, the buffer is histidine. In some embodiments, the composition of the present disclosure comprises about 1mM to about 50mM, about 10mM to about 40mM, about 10mM to about 30mM, about 10mM to about 25mM, about 10mM to about 25mM, about 5mM to about 25mM, about 5mM to about 20mM, or about 5mM to about 10mM of histidine. In certain embodiments, the composition of the present disclosure comprises about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM of histidine. In certain embodiments, the composition of the present disclosure comprises about 10mM of histidine. The histidine buffer may comprise histidine and a histidine salt. The concentration of histidine refers to the concentration of the histidine present and any optional histidine salt. The contribution of a single component to the concentration depends on the target pH. In certain embodiments, the histidine buffer comprises histidine and histidine hydrochloride. In certain embodiments, the concentration of histidine refers to the concentration of both histidine and histidine hydrochloride.

[0139] In certain embodiments, in addition to arginine, the composition of the present disclosure also includes a stabilizer. The range of exemplary concentrations of stabilizers in the composition can be about 1% to about 8% (w / v), about 1% to about 5% (w / v), about 1% to about 3% (w / v) or about 3% to about 5% (w / v). In certain embodiments, the composition of the present disclosure includes about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v) or about 8% (w / v) of stabilizer. In certain embodiments, stabilizer is sugar, sugar alcohol or amino sugar. Exemplary sugar, sugar alcohol and amino sugar include fructose, galactose, glucose, lactose, maltose, mannose, raffinose, sorbitol, sorbose, sucrose, galactosamine, glucosamine, N-methylglucosamine and neuraminic acid. Other stabilizers can also be used. In some embodiments, the composition of the present disclosure includes sucrose, trehalose or sorbitol. In some embodiments, the composition of the present disclosure includes sucrose. In some embodiments, the composition of the present disclosure includes about 1% to about 8% (w / v), about 1% to about 5% (w / v), about 1% to about 3% (w / v) or about 3% to about 5% (w / v) sucrose. In some embodiments, the composition of the present disclosure includes about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v) or about 8% (w / v) sucrose. In some embodiments, the composition of the present disclosure includes about 3% (w / v) sucrose. Sucrose can also serve as a cryoprotectant. In some embodiments, the composition can include different cryoprotectants selected from ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, trehalose and propylene glycol. Other cryoprotectants can also be used. In some embodiments, the concentration of the cryoprotectant in the composition can range from about 1% to about 10% (w / v), about 1% to about 5% (w / v), about 1% to about 3% (w / v), or about 3% to about 5% (w / v). In some embodiments, the composition of the present disclosure comprises about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v) or about 10% (w / v) of cryoprotectant.

[0140] In some embodiments, the compositions of the present disclosure include chelators. The range of exemplary concentrations of chelators in the compositions can be about 1 μM to about 50 μM, about 5 μM to about 40 μM, about 5 μM to about 30 μM, about 5 μM to about 25 μM, about 5 μM to about 20 μM, about 5 μM to about 15 μM, about 10 μM to about 20 μM, about 10 μM to about 25 μM, or about 10 μM to 30 μM. In some embodiments, the compositions of the present disclosure include chelators at a concentration of about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM or about 20 μM. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), dihydroxyethylglycine, citric acid, tartaric acid and methionine. Other chelating agents may also be used. In certain embodiments, the chelating agent is methionine. In certain embodiments, the composition of the present disclosure comprises about 5mM to about 10mM, about 5mM to about 7.5mM, about 7.5mM to about 10mM, about 6mM to about 8mM or about 8mM to about 10mM methionine. In certain embodiments, the composition of the present disclosure comprises about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM or about 10mM methionine. The technician will recognize that the concentration of the chelating agent can vary according to the chelating agent used. In certain embodiments, the chelating agent is DTPA. In some embodiments, the compositions of the present disclosure include about 1 μM to about 50 μM, about 5 μM to about 40 μM, about 5 μM to about 30 μM, about 5 μM to about 25 μM, about 5 μM to about 20 μM, about 5 μM to about 15 μM, about 10 μM to about 20 μM, about 10 μM to about 25 μM, or about 10 μM to 30 μM DTPA. In some embodiments, the compositions of the present disclosure include about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, or about 20 μM DTPA. In some embodiments, the compositions of the present disclosure include about 10 μM DTPA.

[0141] In some embodiments, the compositions of the present disclosure include surfactants. The range of exemplary concentrations of surfactants in the compositions can be about 0.01% to about 0.1% (w / v), about 0.03% to about 0.6% (w / v), about 0.03% to about 0.08% (w / v), about 0.03% to about 0.1% (w / v), about 0.05% to about 0.1% (w / v), or about 0.06% to about 0.1% (w / v). In some embodiments, the compositions of the present disclosure include surfactants at a concentration of about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v) or about 0.1% (w / v). Exemplary surfactants include polysorbates (e.g., polysorbate 20 (PS20), polysorbate 40 (PS40), polysorbate 60 (PS60), and polysorbate 80 (PS80)), dicarboxylic acids, oxalic acid, succinic acid, fumaric acid, phthalic acid, polyoxyethylene sorbitan monooleate, poloxamers (e.g., P188), and polyethylene glycol. Other surfactants may also be used. In some embodiments, the surfactant is P188. In some embodiments, the surfactant is PS80. In some embodiments, the compositions of the present disclosure comprise about 0.01% to about 0.1% (w / v), about 0.03% to about 0.6% (w / v), about 0.03% to about 0.08% (w / v), about 0.03% to about 0.1% (w / v), about 0.05% to about 0.1% (w / v), about 0.06% to about 0.1% (w / v) of PS80. In some embodiments, the compositions of the present disclosure comprise about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), or about 0.1% (w / v) of PS80. In some embodiments, the compositions of the present disclosure comprise about 0.06% (w / v) of PS80.

[0142] The pH of the present disclosure composition can be about 6 to about 7.5, about 6 to about 7, about 6.5 to about 7.5, or about 6.5 to about 7.1. In some embodiments, the pH of the present disclosure composition is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4 or about 7.5. In some embodiments, the pH of the present disclosure composition is about 6.5-7.1. In some embodiments, the pH of the present disclosure composition is about 6.8. Studies described herein have shown that adjusting the pH of the composition comprising arginine is beneficial to reducing the D32 isomerization of the subject antibody. The pH of the composition can be measured by any means known to those skilled in the art. The means for measuring pH is to use a pH meter with a microelectrode. The pH of the composition can be adjusted using any means known in the art (e.g., by adding or adjusting the concentration of an acid, base or buffer).

[0143] In some embodiments, the present disclosure provides a composition comprising a humanized anti-C1s antibody, arginine or a salt thereof, and one or more of the following: a buffer, a stabilizer or a cryoprotectant, a chelating agent, and a surfactant.

[0144] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine HCl; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine HCl is L-arginine HCl.

[0145] In some embodiments, the present disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine citrate; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7.

[0146] In some embodiments, the present disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine oxalate; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7.

[0147] In some embodiments, the present disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine phosphate; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7.

[0148] In some embodiments, the present disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine succinate; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7.

[0149] In some embodiments, the present disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine tartrate; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7.

[0150] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM acetate; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0151] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM citrate; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0152] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM oxalate; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0153] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM phosphate; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0154] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM succinate; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0155] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM tartrate; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0156] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sorbitol; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0157] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) trehalose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0158] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM EDTA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0159] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 5 mM to about 10 mM methionine; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0160] In some embodiments, the disclosure provides a composition comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine hydrochloride; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) poloxamer 188 (P188), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0161] In some embodiments, the present disclosure provides a composition comprising (a) an antibody at about 150 mg / mL; (b) arginine HCl at about 150 mM; (c) histidine at about 10 mM; (d) sucrose at about 3% (w / v); (e) DTPA at about 10 μM; and (f) PS80 at about 0.06% (w / v), and wherein the pH of the composition is from about 6.5 to about 7.1, or about 6.8. In some embodiments, the composition comprises (a) an antibody at about 150 mg / mL; (b) arginine HCl at about 150 mM; (c) citrate at about 20 mM; and (f) PS80 at about 0.02% (w / v), and wherein the pH of the composition is about 6.5. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0162] The compositions of the present disclosure may include other excipients, including but not limited to water for injection, diluents, solubilizers, soothing agents, additional buffers, inorganic or organic salts, antioxidants, etc. In some embodiments, the compositions of the present disclosure do not include other excipients other than those described above. Other pharmaceutically acceptable carriers or excipients (such as those described in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)) may be included in the formulation, provided that they do not adversely affect the desired properties of the formulation. In some embodiments, preservatives may be added. In some embodiments, the composition is substantially free of preservatives. Cryoprotectants or lyoprotectants may be included in the lyophilized formulation.

[0163] The compositions of the present disclosure may be in liquid form, in lyophilized form where the lyophilized preparation is reconstituted with a sterile solution prior to administration, or in liquid form reconstituted from the lyophilized form. Standard procedures for reconstituting the lyophilized composition are to back-add a volume (typically equivalent to the volume removed during lyophilization) of pure water; however, solutions containing antibacterial agents may be used to produce pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54. In some embodiments, the compositions of the present disclosure are in liquid form. The liquid formulation may be ready for injection, or may be diluted prior to injection.

[0164] In some embodiments, the present disclosure provides a stable liquid antibody formulation comprising a humanized anti-C1s antibody, arginine or a salt thereof, and one or more of the following: a buffer, a stabilizer or cryoprotectant, a chelating agent, and a surfactant.

[0165] In some embodiments, the present disclosure provides a stable liquid antibody formulation comprising (a) about 50 mg / mL to about 250 mg / mL of a humanized anti-C1s antibody; (b) about 50 mM to about 200 mM arginine HCl; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 10% (w / v) sucrose; (e) about 1 μM to about 50 μM DTPA; and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is about 6 to about 7. In some embodiments, the arginine HCl is L-arginine HCl.

[0166] In some embodiments, the present disclosure provides a stable liquid antibody formulation comprising (a) about 150 mg / mL of antibody; (b) about 150 mM arginine HCl; (c) about 10 mM histidine; (d) about 3% (w / v) sucrose; (e) about 10 μM DTPA; and (f) about 0.06% (w / v) PS80, and wherein the pH of the composition is about 6.5 to about 7.1, or about 6.8. In some embodiments, the arginine hydrochloride is L-arginine hydrochloride.

[0167] The compositions contemplated are stable at 5°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. In some embodiments, the compositions are stable at 5°C for at least about 12, 18, 24, or 30 months or longer. In some embodiments, they are stable at 5°C for at least about 6 months or longer. In some embodiments, they are stable at 5°C for at least about 9 months. In some embodiments, they are stable at 5°C for at least about 1 year or longer, or for more than about 2 years.

[0168] The compositions disclosed herein show a high level of stability. As used herein, the term "stable" about compositions means that the antibody in the compositions still retains an acceptable degree of structure and / or function and / or biological activity after the storage time length defined. Compositions can be stable, even if the antibody contained therein does not maintain 100% of its structure and / or function and / or biological activity after the storage time length defined. In some cases, maintaining about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the antibody structure and / or function and / or biological activity after the storage time length defined can be considered as "stable".

[0169] In some embodiments, stability is measured by determining the percentage of natural antibodies remaining in the formulation after storing a defined length of time at a given temperature. The percentage of natural antibodies can be determined by, for example, size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]) or any other method known in the art characterizing monomer %, high molecular weight material % and low molecular weight material %. In some embodiments, stability is measured by determining thermal stability (e.g., by performing differential scanning calorimetry (DSC)). In some embodiments, stability is measured by determining mechanical stability (e.g., by performing controlled stirring). In some embodiments, stability is measured by determining solution turbidity (e.g., by measuring the optical density (OD) of 340nm to 360nm). In some embodiments, stability is determined by measuring sub-visible particles (e.g., by using the light obstruction method of a liquid particle counter or by microfluidic imaging (MFI)). In some embodiments, stability is determined by measuring chemical degradation (such as isomerization, oxidation and deamidation) using a tryptic peptide map. In some embodiments, stability is determined by measuring solution opalescence using microturbidimetry.

[0170] In some embodiments, stability can be assessed indirectly by measuring PS80 concentration. In some embodiments, PS80 concentration is measured by high performance liquid chromatography with a charged aerosol detector (HPLC-CAD).

[0171] Stability can also be assessed by measuring the biological activity of the antibody and / or its binding affinity to its target. For example, a formulation of the invention can be considered stable if, after storage for a defined length of time (e.g., 1 to 12 months) at, e.g., 5°C, 25°C, 45°C, etc., the humanized anti-C1s antibody contained in the composition binds to human C1s with an affinity that is at least 50%, 60%, 70%, 80%, 90%, 95% or greater of the binding affinity of the antibody before storage.

[0172] The compositions disclosed herein inhibit antibody isomerization. In some embodiments, isomerization is aspartic acid to isoaspartic acid. In some embodiments, isomerization occurs at D32 of the antibody light chain located at CDR1. As long as the function of the antibody is not impaired, a certain level of isomerization may be acceptable. Therefore, the composition may have an acceptable level of isomerization, even if the antibody contained therein does not maintain 100% of its structure and / or function and / or biological activity after storing a defined length of time. In some cases, maintaining about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the antibody structure and / or function and / or biological activity after storing a defined length of time may be considered as an acceptable level of isomerization. Isomerization may be determined by any suitable method known in the art. In some embodiments, isomerization is determined by performing a total peptide map analysis. In some embodiments, isomerization is measured by determining the isomerization percentage in the formulation after storing a defined length of time at a given temperature. In some embodiments, the isomerization rate of the antibody after storage for 12 weeks at 5° C. is less than about 10%, less than about 9%, less than about 8%, or less than about 7.5%. In some embodiments, the isomerization rate of the antibody after storage for 12 weeks at 25° C. is less than about 25%, less than about 24%, less than about 23%, less than about 21%, or less than about 20%. In some embodiments, the isomerization rate of the antibody after storage for 12 weeks at 40° C. is less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, or less than about 35%.

[0173] In some embodiments, the antibody has a lower isomerization rate (e.g., at D32 of light chain CDR1) than the antibody in the corresponding formulation without arginine or its salt. In some embodiments, the isomerization rate of the antibody at 40°C is at least about 2% or at least about 3% per week. In some embodiments, the antibody has an isomerization rate of at least about 1%-3% lower per month at 25°C or at least about 0.4%, 0.5% or 0.6% lower per month at 5°C compared to the antibody in the corresponding formulation without arginine or its salt. In some embodiments, the isomerization rate of the antibody after storage at 25°C for 12 weeks is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lower than the antibody in the corresponding formulation without arginine or its salt.

[0174] Route of administration

[0175] The antibodies of the present disclosure may be administered to a subject using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or local routes. Generally, routes of administration contemplated by the present disclosure include, but are not necessarily limited to, enteral, parenteral or inhalation routes.

[0176] Parenteral administration routes other than administration by inhalation include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intrathecal, and intravenous routes, i.e., any route of administration other than through the digestive tract. Parenteral administration may be performed to achieve systemic or local delivery of the subject antibody. Where systemic delivery is desired, administration typically involves topical or mucosal administration of the invasive or systemic absorption of the pharmaceutical preparation.

[0177] If desired, the administration routes can be combined or adjusted according to the desired effect. The composition can be administered in a single dose or multiple doses. In some embodiments, the composition of the present disclosure is administered intravenously. In some embodiments, the composition of the present disclosure is administered subcutaneously.

[0178] Dosage and dosage form

[0179] The appropriate dosage can be determined by the attending physician or other qualified medical personnel based on a variety of clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound to be administered, the patient's sex, the time and route of administration, the general health, and other drugs administered concurrently.

[0180] The skilled person will readily appreciate that dosage levels and administration schedules may vary depending on the specific antibody, severity of symptoms and susceptibility of the subject to side effects. The preferred dosage and administration schedule for a given compound may be determined by a skilled person in the art by a variety of means.

[0181] The present disclosure also provides a pharmaceutical unit dosage form, which contains a therapeutically effective amount of the composition of the present disclosure, for treating one or more complement-mediated diseases in a subject by administering the dosage form to the subject. In some embodiments, the subject is a human. The term "pharmaceutical unit dosage form" refers to physically discrete units suitable as unit doses for subjects to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic / preventive effect.

[0182] The unit dosage form can be a container containing the formulation. Suitable containers include, but are not limited to, sealed ampoules, vials (e.g., glass vials), bottles, syringes, and test tubes. The container can be made of a variety of materials (e.g., glass or plastic) and can have a sterile access port (e.g., the container can be a vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, the container is a vial. In some embodiments, the container is a prefilled syringe. Typically, the container should maintain the sterility and stability of the formulation.

[0183] In some embodiments, the composition is packaged in vials (10R) made of type I glass and closed with stoppers (flurotec coated chlorobutyl) sealed with flip-off caps with flanges (aluminum). In some embodiments, the vials are filled with 8 mL of the composition.

[0184] In some embodiments, the composition is packaged in a pre-filled syringe made of glass (OMPI) and closed with a rubber stopper (Novapure). In some embodiments, the pre-filled syringe is filled with 2 mL of the composition.

[0185] An exemplary drug delivery device may involve a needle-based injection system as described in Table 1 of Section 5.2 of ISO 11608-1:2014(E). As described in ISO 11608-1:2014(E), needle-based injection systems can be broadly divided into multi-dose container systems and single-dose (partially or completely emptied) container systems. The container may be a replaceable container or an integral non-replaceable container.

[0186] As further described in ISO 11608-1:2014(E), a multiple-dose container system may involve a needle-based injection device with a replaceable container. In such a system, each container contains multiple doses, the sizes of which may be fixed or variable (pre-set by the user). Another multiple-dose container system may involve a needle-based injection device with an integral non-replaceable container. In such a system, each container contains multiple doses, the sizes of which may be fixed or variable (pre-set by the user).

[0187] As further described in ISO 11608-1:2014 (E), a single dose container system can relate to an injection device based on a needle with a replaceable container. In an example of such a system, each container accommodates a single dose, thereby discharging the entire deliverable volume (completely emptying). In another example, each container accommodates a single dose, thereby discharging a portion of the deliverable volume (partially emptying). Also as described in ISO 11608-1:2014 (E), a single dose container system can relate to an injection device based on a needle with an integral non-replaceable container. In an example of such a system, each container accommodates a single dose, thereby discharging the entire deliverable volume (completely emptying). In another example, each container accommodates a single dose, thereby discharging a portion of the deliverable volume (partially emptying).

[0188] An exemplary sleeve-triggered autoinjector with manual needle inserter is described in International Publication WO 2015 / 004052. An exemplary audible end-of-dose feedback mechanism is described in International Publications WO 2016 / 193346 and WO 2016 / 193348. An exemplary needle safety mechanism after use of an autoinjector is described in International Publication WO 2016 / 193352. An exemplary needle sheath remover mechanism for a syringe autoinjector is described in International Publication WO 2016 / 193353. An exemplary support mechanism for supporting the axial position of a syringe is described in International Publication WO 2016 / 193355.

[0189] Kits and Products

[0190] The present disclosure provides a kind of test kit or article, and this test kit or article comprises the composition of the present disclosure.Test kit or article comprises the container containing the composition of the present disclosure.Test kit or article may further comprise one or more containers containing pharmaceutically acceptable excipients, and comprise other materials required from the viewpoint of business and user, including filter, needle and syringe.Related to the test kit may be instructions generally included in the commercial packaging of treatment, prevention or diagnosis product, these instructions contain information about, for example, indication, usage, dosage, manufacture, application, contraindication and / or warning (there is information about the use of such treatment, prevention or diagnosis product).Test kit may also be associated with a label, and this label may be any kind of data carrier (for example, leaflet, sticker, chip, printed matter or barcode) containing information.In certain embodiments, the instructions as listed above may be included in or on a label.Test kit may further comprise a device for applying a preparation and a device particularly containing a composition, i.e. a pre-filled device, such as, but not limited to, a pre-filled syringe or a pre-filled automatic syringe. The kit may also comprise a container containing the composition, ie a pre-filled container, such as a pre-filled vial, cartridge, sachet or ampoule.

[0191] Complement-mediated diseases

[0192] The compositions of the present disclosure can be used to treat complement-mediated diseases. Therefore, on the one hand, the present disclosure provides a method for treating complement-mediated diseases. The method generally involves administering an effective amount of the compositions of the present disclosure to a subject in need. In some cases, the compositions of the present disclosure are administered to regulate the activity of complement C1s in individual cells, tissues, fluids or organs, and to treat complement-mediated diseases or disorders. The present disclosure provides methods for inhibiting the activation of complement component C4 of an individual, which methods include administering an effective amount of the compositions of the present disclosure to the subject. The present disclosure provides methods for inhibiting the activity of complement C1s of a subject, which methods include administering an effective amount of the compositions of the present disclosure to the subject. The present disclosure provides methods for reducing the level of complement component cleavage products in a subject (e.g., in an individual's fluid, tissue or organ), which methods include administering an effective amount of the compositions of the present disclosure to the subject.

[0193] In some cases, an "effective amount" of a composition of the present disclosure is an amount that, when administered to a subject in need thereof in one or more doses, reduces the level of a complement component cleavage product in the subject (e.g., in a fluid, tissue, or organ of the individual). In some cases, an "effective amount" of a composition of the present disclosure is an amount that, when administered to a subject in need thereof in one or more doses, reduces the level of a complement component cleavage product in the subject (e.g., in a fluid, tissue, or organ of the individual) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the level of a complement component cleavage product in a fluid, tissue, or organ in the absence of treatment with the composition (e.g., prior to treatment with the composition). In some embodiments, the complement component cleavage product is a C4 cleavage product (e.g., C4b). In some embodiments, the complement component cleavage product is a C2 cleavage product (e.g., C2a). In some embodiments, the complement component cleavage product is a C3 cleavage product.

[0194] In some cases, an "effective amount" of a composition of the present disclosure is an amount that, when administered in one or more doses to a subject in need thereof, reduces the activity of the classical complement pathway in the subject (e.g., in a fluid, tissue, or organ of the individual). In some cases, an "effective amount" of a composition of the present disclosure is an amount that, when administered in one or more doses to a subject in need thereof, reduces the activity of the classical complement pathway in the subject (e.g., in a fluid, tissue, or organ of the individual) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% within about 48 hours, about 24 hours, about 12 hours, about 8 hours, or about 4 hours of administration of the humanized anti-C1s antibody, compared to the activity of the classical complement pathway in the fluid, tissue, or organ in the absence of treatment with the composition (e.g., prior to treatment with the composition). The activity level of the classical complement pathway can be determined using any of a variety of methods. As a non-limiting example, the activity of the classical complement pathway can be determined in vitro, such as by determining the activity level of the classical complement pathway in a blood, serum or plasma sample obtained from an individual. For example, the classical complement pathway in a blood, serum or plasma sample can be activated in vitro, and the amount of complement component cleavage products (such as C5b-9) generated by such activation can be determined.

[0195] In some embodiments, an "effective amount" of a composition of the present disclosure is an amount that, when administered in one or more doses to a subject in need thereof, maintains a reduction in the level of activity of the classical complement pathway in the subject (e.g., in a fluid, tissue or organ of the individual) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the level of activity of the classical complement pathway in the fluid, tissue or organ in the absence of treatment with the composition (e.g., prior to treatment with the composition), wherein the reduction is maintained for a period of about 4 hours to about 30 days (e.g., 4 hours to 8 hours, 8 hours to 24 hours, 2 days to 4 days, 4 days to 7 days, 7 days to 14 days, 14 days to 21 days, or 21 days to 30 days).

[0196] In some embodiments, an "effective amount" of a composition of the present disclosure is an amount that, when administered in one or more doses to a subject in need thereof, maintains a reduction in the level of a complement component cleavage product in the subject (e.g., in a fluid, tissue, or organ of the individual) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the level of the complement component cleavage product in the fluid, tissue, or organ in the absence of treatment with the composition (e.g., prior to treatment with the composition), wherein the reduction is maintained for a period of time of about 4 hours to about 30 days (e.g., 4 hours to 8 hours, 8 hours to 24 hours, 2 days to 4 days, 4 days to 7 days, 7 days to 14 days, 14 days to 21 days, or 21 days to 30 days).

[0197] In some cases, an "effective amount" of a composition of the present disclosure is an amount that, when administered in one or more doses to a subject in need thereof, reduces the production of C4b2a (i.e., a complex of complement C4b and C2a; also known as "C3 convertase") in a subject (or in a fluid, tissue, or organ of an individual) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the amount of C4b2a produced in the subject or fluid, tissue, or organ in the absence of treatment with the composition (e.g., prior to treatment with the composition).

[0198] The present disclosure provides a method for regulating complement activation. In some embodiments, the method inhibits complement activation, for example to reduce the production of C4b2a. In some embodiments, the present disclosure provides a method for regulating complement activation in an individual suffering from a complement-mediated disease, the method comprising administering a composition of the present disclosure to the individual. In some embodiments, the method inhibits complement activation.

[0199] In some embodiments, a complement-mediated disease is characterized by the presence of an elevated (higher than normal) amount of C1s or an elevated level of complement C1s activity in a cell, tissue, or fluid. For example, in some embodiments, a complement-mediated disease is characterized by the presence of an elevated amount of C1s and / or elevated activity of C1s in brain tissue and / or cerebrospinal fluid. An "above normal" amount of C1s in a cell, tissue, or fluid indicates that the amount of C1s in the cell, tissue, or fluid is higher than a normal control level, such as higher than the normal control level for an individual or population of individuals in the same age group. An "above normal" level of C1s activity in a cell, tissue, or fluid indicates that proteolytic cleavage caused by C1s in the cell, tissue, or fluid is higher than a normal control level, such as higher than the normal control level for an individual or population of individuals in the same age group. In some embodiments, an individual suffering from a complement-mediated disease exhibits one or more additional symptoms of the disease. It should be understood that the term "disease" encompasses "disorder". The two terms can be used interchangeably. In some embodiments, a complement-mediated disease is a classical complement-mediated disease.

[0200] In some embodiments, a complement-mediated disease is characterized by the presence of a lower than normal amount of C1s or a lower level of complement C1s activity in a cell, tissue, or fluid. For example, in some embodiments, a complement-mediated disease is characterized by the presence of a lower amount of C1s and / or lower activity of C1s in brain tissue and / or cerebrospinal fluid. A "lower than normal" amount of C1s in a cell, tissue, or fluid indicates that the amount of C1s in the cell, tissue, or fluid is lower than a normal control level, such as lower than a normal control level for an individual or population of individuals in the same age group. A "lower than normal" level of C1s activity in a cell, tissue, or fluid indicates that proteolytic cleavage caused by C1s in the cell, tissue, or fluid is lower than a normal control level, such as lower than a normal control level for an individual or population of individuals in the same age group. In some embodiments, an individual suffering from a complement-mediated disease exhibits one or more additional symptoms of the disease.

[0201] A complement-mediated disease is a disease in which the amount or activity of complement C1s causes an individual to become ill. In some embodiments, the complement-mediated disease is selected from the group consisting of alloimmune diseases, autoimmune diseases, cancer, blood diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative disorders, eye diseases, kidney diseases, transplant rejection, vascular diseases, and vasculitis diseases. In some embodiments, the complement-mediated disease is an autoimmune disease. In some embodiments, the complement-mediated disease is an alloimmune disease. In some embodiments, the complement-mediated disease is cancer. In some embodiments, the complement-mediated disease is an infectious disease. In some embodiments, the complement-mediated disease is an inflammatory disease. In some embodiments, the complement-mediated disease is a blood disease. In some embodiments, the complement-mediated disease is ischemia-reperfusion injury. In some embodiments, the complement-mediated disease is an eye disease. In some embodiments, the complement-mediated disease is kidney disease. In some embodiments, the complement-mediated disease is transplant rejection. In some embodiments, the complement-mediated disease is antibody-mediated transplant rejection. In some embodiments, the complement-mediated disease is a vascular disease. In some embodiments, the complement-mediated disease is a vasculitic disorder. In some embodiments, the complement-mediated disease is a neurodegenerative disease.

[0202] Examples of complement-mediated diseases or disorders include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, allergic reactions, antibody-mediated rejection (AMR), argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases (including, e.g., autoimmune hemolytic anemia (AIHA); warm antibody AIHA; mixed AIHA;etc.), Barak-Simons syndrome, Behcet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, catastrophic antiphospholipid syndrome, cerebral amyloid angiopathy, chronic inflammatory demyelinating polyneuropathy (CIDP), cold agglutinin disease (CAD), corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemic vasculitis, dementia pugilistica, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangles with calcifications, discoid lupus erythematosus, Down syndrome, I Wencke syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), chromosome 17-related frontotemporal dementia with parkinsonism, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, hemolytic uremic syndrome, hereditary angioedema, hypophosphatasia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infectious diseases (e.g., caused by bacteria (e.g., Neisseria meningitidis meningitidis or Streptococcus), diseases caused by viruses (e.g., human immunodeficiency virus [HIV]) or other infectious agents), inflammatory diseases, ischemia / reperfusion injury, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency type A (MoCD), membranoproliferative glomerulonephritis type I (MPGN), membranoproliferative glomerulonephritis type II (MPGN) (dense deposit disease), membranous nephritis, multi-infarct dementia, lupus (e.g., systemic lupus erythematosus (SLE), LE), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, postencephalitis parkinsonism, polymyositis, prion protein amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (E coli)(STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, tangled dementia only, transplant rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegener's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, mixed cryoglobulinemia type II, mixed cryoglobulinemia type III, nephritis, drug-induced thrombocytopenia, lupus nephritis, acquired epidermolysis bullosa, delayed hemolytic transfusion reaction, hypocomplementemic urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion failure.;

[0203] In some embodiments, the complement-mediated disease is CAD. In some embodiments, the complement-mediated disease is ITP. In some embodiments, the complement-mediated disease is CIDP. In some embodiments, the complement-mediated disease is AMR.

[0204] In some embodiments, the compositions of the present disclosure prevent or delay the onset of at least one symptom of a complement-mediated disease in a subject. In some embodiments, the compositions of the present disclosure reduce or eliminate at least one symptom of a complement-mediated disease in a subject. Examples of symptoms include, but are not limited to, symptoms associated with autoimmune diseases, cancer, blood diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative disorders, kidney disease, transplant rejection, eye diseases, vascular diseases, or vasculitic disorders. Symptoms can be neurological symptoms, such as impaired cognitive function, impaired memory, loss of motor function, etc. Symptoms can also be the activity of C1s protein in cells, tissues, or fluids of an individual. Symptoms can also be the degree of complement activation in cells, tissues, or fluids of an individual.

[0205] "Treatment" means at least alleviating the symptoms associated with the pathological condition from which the host suffers, wherein alleviating is used in a broad sense to refer to at least reducing the magnitude of a parameter (e.g., symptom) associated with the pathological condition being treated (e.g., a complement-mediated disease). Thus, treatment also includes situations in which the pathological condition or at least the symptoms associated therewith are completely inhibited, e.g., prevented from occurring, or stopped, e.g., terminated, such that the host no longer suffers from the pathological condition or at least no longer suffers from the symptoms characterizing the pathological condition.

[0206] According to the subject method, a variety of hosts can be treated (wherein the term "host" is used interchangeably with the terms "subject", "individual" and "patient" herein). Typically, such a host is a "mammal" or "mammals", wherein these terms are widely used to describe organisms in the class Mammalia, including carnivores (e.g., cats), herbivores (e.g., cattle, horses and sheep), omnivores (e.g., dogs, goats and pigs), rodents (e.g., mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees and monkeys). In some embodiments, the host is an individual with a complement system, such as a mammal, fish or invertebrate. In some embodiments, the host is a mammal, fish or invertebrate companion animal, agricultural animal, work animal, zoo animal or laboratory animal containing a complement system. In some embodiments, the host is a human.

[0207] Examples

[0208] Example 1. Design of formulation development study

[0209] SAR445088 is a humanized immunoglobulin G (IgG) subclass 4 (IgG4) monoclonal antibody (mAb) produced in Chinese hamster ovary (CHO) cells that binds and inhibits the activation form of C1s in the classical pathway (CP). The mechanism of SAR445088 is specific to CP, so that the lectin pathway and the bypass pathway remain functionally intact. The LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, HC CDR3, VH, VL, and heavy and light chain sequences of SAR445088 are described elsewhere in this application as SEQ ID NO.1-10. These sequences are also described in U.S. Patent No. 9,512,233, U.S. Patent No. 10,729,767, and U.S. Patent Application Publication No. US2020 / 0048332, each of which is incorporated herein by reference in its entirety.

[0210] Early development studies have demonstrated that the antibody has a higher tendency to self-associate (kD of -22.7 mL / g in 10 mM histidine at pH 6.0 and kD of -12.4 mL / g in 10 mM histidine + 150 mM NaCl at pH 6.0) and has a tendency to isomerize at the aspartic acid of the light chain CDR1 (LC D32 isomerization). Formulation development studies were conducted to evaluate the feasibility and liabilities associated with an optimized histidine-based liquid drug product (DP). A comprehensive set of studies was conducted to understand the effects of formulation composition and process conditions on the biophysical and chemical stability of SAR445088. For the optimized development of liquid SAR445088 DP, the effects of buffer type and pH, polysorbate 80 concentration, and metal chelators were all rigorously evaluated. The specific evaluations and their objectives are as follows:

[0211] Managing D32 isomerization

[0212] The objectives were to identify the edge of failure in preventing D32 isomerization and to evaluate the effects of pH and stabilizers on D32 isomerization.

[0213] Polysorbate 80 Screening

[0214] The objective was to evaluate the optimal concentration of polysorbate 80 (PS80) required to stabilize a SAR445088 liquid formulation upon agitation-induced stress and exposure to light. In addition, the optimal PS80 concentration in liquid DP was determined by testing the ability of various PS80 concentrations to mitigate the increase in turbidity and / or subvisible particle formation of SAR445088 samples after dilution 50-fold to 3 mg / mL with 0.9% saline.

[0215] Comparative study of metal incorporation and chelation

[0216] The objective was to examine the effects of transition metals that may leach into the drug substance (DS) during manufacturing on the chemical and physical stability of SAR445088 and PS80. The ability of the chelating agents EDTA and DTPA to protect the proteins from degradation was also evaluated during a set of worst-case stability experiments.

[0217] pH / buffer screening and DP stability

[0218] The objective was to evaluate the effects of buffer characteristics and pH on the physical and chemical stability of SAR445088 liquid formulations during refrigerated storage, ambient storage, and accelerated storage conditions. Histidine and phosphate buffer systems were selected for this study. The proposed target formulation matrix was tested for its ability to stabilize high concentration DP solutions during frozen and liquid storage. The effect of the addition of 3% sucrose on DP stability was also evaluated.

[0219] DP robustness study

[0220] The goal is to ensure that the final composition of the proposed target formulation is robust enough to withstand agitation-induced stress, freeze-thaw, and light exposure during storage or manufacturing without loss of formulation stability.

[0221] Example 2. Materials and methods

[0222] Material

[0223] Drug substance. Formulated drug substance (FDS) of SAR445088 at concentrations >150 mg / mL was prepared. For a given study conducted during formulation development, a specific batch of FDS was used. After the FDS was formulated to the target protein and excipient concentrations, the formulation was then filtered through 0.22 μm under laminar flow and then aseptically filled to prepare the liquid drug product (DP).

[0224] Excipients. All excipients used during formulation development studies were ACS, USP, and Eur.Ph. grade raw materials. Excipients used in the study included L-histidine, L-histidine monohydrochloride, L-arginine monohydrochloride, sucrose, PS80, sodium dihydrogen phosphate, sodium phosphate dibasic 7-hydrate, EDTA, and DTPA. All excipients were obtained from JTBaker except DTPA, which was obtained from Sigma Aldrich.

[0225] Analytical methods

[0226] pH. The pH of buffer and formulated antibody solutions was measured using a pH probe and pH meter from Mettler-Toledo. Results were considered comparable when the difference between duplicate measurements was within 0.1 pH unit.

[0227] Lunatic for protein concentration. Total protein concentration was determined by measuring UV absorbance at 280 nm on a microfluidic chip on a Big Lunatic spectrometer (Unchained Labs). Sample volumes of 2 to 5 μL were measured in duplicate (ε1%: 14.6 (g / 100 mL)-1 cm-1).

[0228] UV plate reader for turbidity and optical density. Sample turbidity was quantified by measuring the optical density (OD) from 340nm to 360nm on a SpectraMax i3 microplate reader (Molecular Devices). For each sample, 200 μL was loaded onto a UV-Vis transparent 96-well plate (Corning). OD was determined as the average value of the absorbance values ​​at 340nm, 345nm, 350nm, 355nm, and 360nm.

[0229] The size exclusion HPLC for high molecular weight (HMW) materials is used. Aggregation analysis is carried out by size exclusion chromatography (SEC). Sample is split 30 minutes with the flow rate of 0.5mL / min in 20mM sodium phosphate, 300mM sodium chloride (pH 6.5 ± 0.1) on 1260 series HPLC (Agilent, Santa Clara, California) equipped with TSK-GEL G3000SWXL (Tosoh Bioscience, Tokyo, Japan) analytical column and matching guard column. Each sample is injected twice. Detected by the UV absorbance at 280nm, and the chromatographic peak is integrated to determine the relative percentage of each eluted material.

[0230] The size exclusion ultra-high pressure liquid chromatography (UPLC) for HMW and low molecular weight (LMW) materials was used. Aggregation analysis was performed by size exclusion chromatography (SEC). The sample was split 20 minutes at a flow rate of 0.3 mL / min in 50 mM sodium phosphate, 300 mM sodium chloride (pH 7.0) on a Waters UPLC (Waters Corporation, Milford, Massachusetts) equipped with an Acquity BEH200 UPLC (Waters Corporation, Milford, Massachusetts) analytical column. Each sample was injected three times. The UV absorbance at 280 nm was detected, and the chromatographic peak was integrated to determine the relative percentage of each eluted material.

[0231] Subvisible particles. Subvisible particles were also measured by light obscuration on a high-precision liquid particle counter (HIAC) model 9703+ (Beckman Coulter). The counter was rinsed with MilliQ water filtered and degassed at 0.22 μm until the 10 μm particle count was ≤1 particle / mL. Standards of 2 μm, 10 μm, and 25 μm were measured to ensure accurate particle counts, and then the counter was fully washed to remove background signals. Using the 1 mL protocol, four separate injections of 0.2 mL volume samples were used to analyze the sample. The first sample measurement was ignored, and the subsequent three measurements were averaged.

[0232] cIEF for charge variants. Protein charge heterogeneity was measured by capillary isoelectric focusing (cIEF) using a Maurice instrument (Protein Simple) to detect UV absorbance at 280nm. Samples (1mL) and standard solutions were first diluted to 15mg / mL in formulation buffer and then diluted to 5mg / mL with water. Prior to analysis, samples were mixed with MasterMix using Onboard mixing. The isoelectric focusing of the sample consisted of a pre-focusing period of 3 minutes at 1500V, followed by a focusing period of more than 11 minutes at 3000V. Results were considered comparable when the difference between samples was equal to or less than 10%.

[0233] PS80 determination by charged aerosol detection (CAD). PS80 is a nonionic surfactant consisting mainly of hydrophilic sorbitan polyoxyethylene groups connected by esters and fatty acids. The separation of PS80 with other sample components (such as proteins and formulation ingredients) was accomplished by mixed mode HPLC (using Oasis MAX columns (2.1 × 20 mm, 30 μm particle size, part number 186002052)) (Waters) and charged aerosol detection (CAD) (Agilent Technologies, California). The column resin contains a hydrophobic polymer backbone supporting positively charged quaternary amines. As a result, the column provides a combination of reverse phase capability and ion exchange capability. This allows retention of hydrophobic compounds such as PS80 and produces electrostatic repulsion of the positive charge on the protein / excipient. The sample was eluted with a mobile phase gradient of 2% formic acid (A) in water and 2% formic acid (B) in isopropanol. Each sample was injected and passed through the column with CAD for 8 minutes within a detection window of 2.5 to 8 minutes.

[0234] Post-translational modifications (PTMs) were quantified by LC-MS. Protein samples were diluted to 1 mg / mL and then reduced, alkylated, and digested using a fully automated method with a mixture of trypsin and Lys-C on a STAR liquid handling system (Hamilton). Peptide mapping was performed on a Q Exactive HF LC-MS system. Acquired LC-MS and LC-MS / MS data were processed using BioPharma Finder 4.1 for identification and relative quantification of modifications.

[0235] Efficacy. C1s was directly immobilized on the surface of the Biacore SPR sensor chip. A calibration curve for antibody binding to C1s was generated using the original sample of the formulated antibody without buffer exchange. All samples were tested after diluting 1000 times in running buffer. All samples were tested in triplicate using three different dilution schemes (100X10, 50X20, and 25X40). The control sample was the original sample diluted to 1 μg / mL. A control sample was run once for every 6 samples.

[0236] Example 3. Management of D32 heterogeneity

[0237] Isomerization of D32 in the light chain (LC) CDR1 region of SAR445088 was identified as the major chemical degradation mechanism that directly affects the Biacore binding of the SAR445088 molecule. Figure 1As shown, Biacore binding is adversely affected by isomerization of D32 in CDR1. When the isomerization rate exceeds 40%, Biacore binding is greatly reduced. Based on benchmarking exercises, the expected end of shelf-life (EOS) level of D32 isomerization is estimated to be about 10%-15%.

[0238] Next, a systematic study was performed to evaluate the effect of pH and the presence of stabilizers (such as arginine hydrochloride) on the isomerization rate of SAR445088. Figure 2A-2D As shown, under all test temperatures, as pH increases from 6.2 to 6.8, the D32 isomerization rate decreases. In addition, compared with the counterpart without arginine hydrochloride, adding arginine hydrochloride further helps to reduce the isomerization rate as a stabilizer. As expected, the lyophilized formulations all show negligible isomerization under all test temperatures. Based on these results, the inclusion of higher pH (pH 6.8) and arginine hydrochloride seems to be beneficial to further develop antibody formulations. The high liquid formulation of pH can allow degradation based on deamidation, and the preferred SAR445088 formulation has an optimal pH and stabilizer concentration to minimize both isomerization and deamidation.

[0239] Example 4. PS80 legitimacy study

[0240] The optimal amount of PS80 in the SAR445088 formulation was determined by observing the resistance of test formulations (150 mg / mL SAR445088, 150 mM arginine HCl, 3% sucrose, 10 μM EDTA, 10 mM histidine, pH 7.0) containing 0-0.1% PS80 to the various stresses described in Table 2. The purpose of this study was to determine the appropriate amount of PS80 for reducing shear during manufacturing, shipping, and handling using the following study conditions.

[0241] Table 2. Research conditions for PS80 legitimacy.

[0242]

[0243] Orbital Oscillation Research

[0244] As shown in Figure 3, the presence of PS80 does not affect the formation of protein HMW species during orbital oscillation ( Figure 3A ). Samples without PS80 showed an increase in optical density / turbidity over time when orbitally shaken. However, samples containing as little as 0.01% PS80 showed greater stability ( Figure 3BFinally, samples without PS80 showed an increase in 2 μm, 10 μm, and 25 μm subvisible particles over time during orbital oscillation. However, samples containing >0.01% PS80 showed little increase in subvisible particle formation ( Figure 3C-Figure 3E ). Therefore, the concentration of >0.01% PS80 can control the degradation of SAR445088 due to oscillation.

[0245] Wrist Oscillation

[0246] As shown in Figure 4, samples without PS80 showed the highest tendency for the protein to form aggregates (HMW species) upon shaking. Samples with >0.03% PS80 showed good resistance to the formation of HMW species ( Figure 4A ). Samples without PS80 showed an increase in turbidity as early as 1 h after wrist shaking. 0.01% PS80 was sufficient to prevent the increase in turbidity ( Figure 4B ). The samples without PS80 showed the highest number of 2μm, 10μm and 25μm subvisible particles as early as 1 h after shaking. Thereafter, an insoluble whitish precipitate appeared and the samples without PS80 were not further evaluated for subvisible particles. Samples with >0.03% PS80 showed resistance to subvisible particle formation ( Figure 4C-4E ).

[0247] Wrist Oscillation + Silicone Incorporation

[0248] As shown in Figure 5, the samples without PS80 showed an increase in turbidity as early as 1 h after wrist shaking. 0.01% PS80 was sufficient to prevent the increase in turbidity. An insoluble whitish precipitate was formed in the samples without PS80, and the particles of these samples were not evaluated after T0 ( Figure 5A ). Samples with >0.01% PS80 had a higher number of 2 μm sub-visible particles due to the presence of silicone oil. Samples with >0.03% PS80 showed resistance to the formation of 10 μm and 25 μm particles under wrist oscillation in the presence of silicone ( Figure 5B-5D SEC analysis was not performed to prevent the column from being damaged by silicone oil.

[0249] IV Bag Dilution Studies

[0250] As shown in Figure 6, PS80 had no effect on the formation of protein aggregates in the samples when diluted and kept in the IV bag ( Fig. 6A All PS80 concentrations had little effect on turbidity in the test IV bags over a 24-hour period ( Figure 6B ). The target protein concentration for all IV bags is 3 mg / mL ( Figure 6C). Samples containing <0.01% PS80 showed an increase in 2μm and 10μm subvisible particles within 24h in the IV bag. Samples containing >0.03% PS80 showed resistance to subvisible particle formation ( Figure 6D-6F ).

[0251] Short-term photostability studies

[0252] As shown in Figure 7, PS80 did not affect the ability of the protein to form HMW species when exposed to ambient light, and all levels of PS80 tested had similar effects on the formation of HMW species ( Fig. 7A After 3 days of exposure to ambient light, the turbidity of all samples increased significantly ( Figure 7B No PS80 degradation was observed over the entire study period for any of the conditions tested ( Figure 7C ). Samples containing <0.01% PS80 showed an increase in 2μm, 10μm, and 25μm particles over time. Samples with >0.01% PS80 showed resistance to particle formation ( Figure 7D-7F ).

[0253] Summarize

[0254] Considering all the mechanical, physical and chemical stresses that the formulation is expected to encounter during its life cycle and reflected in the stress tests performed in this study, at least 0.03% PS80 seems necessary to provide a minimum acceptable level of protection against stress-induced HMW species and subvisible particle formation. However, 0.06% PS80 (600ppm) provided more adequate protection for the antibody formulation against all tested stresses.

[0255] Example 5. Study on the legitimacy of chelating agents

[0256] The identity and concentration of chelating agents suitable for SAR445088 formulations were determined to prevent PS80 oxidation. Different FDS batches containing 150 mg / mL SAR445088, 150 mM arginine HCl, 3% sucrose, 0.06% PS80, 10 mM histidine, pH 7.0 were spiked with chelating agents (EDTA or DTPA) and metal ions (iron (Fe), copper (Cu) and tungsten (W)) and subjected to refrigerated, ambient temperature and accelerated (40°C) stability testing, as described in Table 3. These two chelating agents were selected based on their use in approved biological products. These metal ions were selected based on metals found in manufacturing water, possible stainless steel utensils or contact parts (part contact), and tungsten formed by needles for glass pre-filled syringes.

[0257] Table 3. Chelating agent and metal ion incorporation schedule

[0258]

[0259] Batch 1 Chelating Agent Study

[0260] As shown in Figure 8, PS80 degradation was observed in all samples with 10 μM EDTA (with / without metal ions) as early as 2 weeks at 40°C, while PS80 degradation was mitigated by the use of 10 μM DTPA (even in the presence of metal ions) up to 3 months at 40°C ( Fig. 8A 50μM EDTA or 50μM DTPA abolished PS80 degradation ( Figure 8B ). In addition, ester profile analysis revealed that oxidation was the root cause of PS80 degradation, and the presence of 10 μM DTPA prevented PS80 oxidation ( Figure 8C ). The tendency to form HMW species was greatest with 10 μM EDTA. Less HMW species were formed with 10 μM DTPA than with 10 μM EDTA. 50 μM EDTA and DTPA prevented the formation of HMW species to a greater extent than 10 μM EDTA, but the difference was less pronounced compared to 10 μM DTPA ( Fig.8D ). Coloration of the solution was also observed at 10 μM EDTA compared to any other tested groups ( Fig. 9 ). Biacore analysis revealed that EDTA and DTPA had no direct effect on the potency of the molecule. A slight decrease in potency over time was observed at 40 °C ( Fig. 8E The chelators had no major effect on the propensity of the molecule to undergo LC D32 isomerization, heavy chain (HC) M251 oxidation, HCPENNYK deamidation, or HC VSNK deamidation.

[0261] Batch 2 Chelating Agent Study

[0262] As shown in Figure 10, PS80 degradation was observed in all samples with 10 μM EDTA (with / without ions) as early as 2 weeks at 40°C, while PS80 degradation was mitigated by the use of 10 μM DTPA (even in the presence of metal ions) up to 3 months at 40°C ( Fig. 10A 50μM EDTA or 50μM DTPA abolished PS80 degradation ( Fig. 10B). HMW species formation was greatest at 10 μM EDTA. Less HMW species were formed at 10 μM DTPA than at 10 μM EDTA. 50 μM EDTA and DTPA prevented HMW species formation to a greater extent than 10 μM EDTA, but 50 μM EDTA prevented HMW species formation to a similar extent as 10 μM DTPA ( Fig. 10C No significant trend in LMW species formation as a function of chelating agent was observed ( Fig. 10D ). Coloration of the solution was also observed with 10 μM EDTA compared to any other test group. Biacore analysis revealed that EDTA and DTPA had no direct effect on the potency of the molecule as no loss of potency was observed in the presence or absence of chelating agents after the samples were subjected to 25°C for 6 months. A slight decrease in potency over time was observed at 40°C ( Fig.10E ). The chelators had no significant effect on the propensity of the molecule to undergo LC D32 isomerization, HC M251 oxidation, HC PENNYK deamidation, or HC VSNK deamidation.

[0263] Batch 3 Chelating Agent Study

[0264] As shown in Figure 11, less PS80 degradation was observed for all samples compared to previous batches. After 6 months storage at 25°C, some PS80 degradation was observed for formulations without chelating agent or containing 10 μM EDTA compared to 10 μM DTPA ( Figure 11A-11B ). For formulations without chelating agent or with 10 μM EDTA, HMW species were formed more. Significantly less HMW species were formed at 10 μM DTPA than at 10 μM EDTA ( Fig. 11C No significant trend in LMW species formation as a function of chelating agent was observed. An overall increase in LMW species formation as a function of storage time and temperature was observed for all groups tested ( Fig.11D ). No change in solution color was observed for any of the samples at 5°C or 25°C, but a change was observed at 40°C. Biacore analysis revealed that EDTA and DTPA had no direct effect on the potency of the molecule, which retained substantial potency in either chelator after being subjected to 25°C for 3 months. A slight decrease in potency over time was observed at 40°C ( Fig.11E ). The chelators had no significant effect on the propensity of the molecule to undergo LC D32 isomerization, HC M251 oxidation, HCPENNYK deamidation, or HC VSNK deamidation.

[0265] Batch 4 Chelating Agent Study

[0266] As shown in Figure 12, less PS80 degradation was observed for all samples compared to previous batches. All concentrations of EDTA and DTPA appeared to mitigate PS80 degradation to a similar extent ( Figure 12A-12B ). Similar HMW species formation profiles were observed for EDTA- and DTPA-containing formulations maintained at 5°C and 25°C. DTPA-containing formulations showed less HMW species formation at 40°C compared to chelating agent-free or EDTA-containing formulations ( Fig. 12C ). The formulations containing DTPA showed less LMW species formation compared to the formulations without chelating agent or containing EDTA. For all groups tested, an overall increase in LMW species formation with storage time and temperature was observed ( Fig.12D ). No change in solution coloration was observed for any sample at 5°C or 25°C. In the presence of DTPA as a chelating agent, solution coloration at 40°C was not evident. Biacore analysis revealed that EDTA and DTPA had no direct effect on the potency of the molecule, which retained substantial potency after being subjected to 25°C for 1 month. A slight decrease in potency over time was observed at 40°C ( Fig.12E ). The chelators had no significant effect on the propensity of the molecule to undergo LC D32 isomerization, HC M251 oxidation, HC PENNYK deamidation, or HC VSNK deamidation.

[0267] Summarize

[0268] Based on comparative stability testing of SAR445088 formulation samples containing either DTPA or EDTA, it was determined that a concentration of 10 μM DTPA optimally mitigated PS80 degradation between batches, prevented HMW species formation and solution coloration, and had minimal impact on the molecule's ability to maintain potency.

[0269] Example 6. pH Screening / DP Stability Study

[0270] The optimal buffer system and target pH for the SAR445088 formulation were identified by subjecting different batches of SAR445088 to refrigerated, ambient, accelerated and frozen storage stability testing. The testing was performed using different primary containers (i.e., vials and prefilled syringes (PFS) as described in Table 4) to test the stability of SAR445088 liquid DP in these containers in parallel.

[0271] Table 4. pH Screening / DP Stability Study Plan

[0272]

[0273] Batch 1 Formulation Study

[0274] PFS stability study

[0275] As shown in Figure 13, under accelerated conditions at 40°C, SAR445088 had a higher tendency to form HMW species in potassium phosphate buffer than in histidine buffer. Overall, SAR445088 maintained substantial stability during ambient storage (25°C) and refrigerated storage (5°C) under all pH and buffer conditions tested. No significant differences in HMW species formation were observed between storage in BD neopak low, BD neopak high, and OMPI syringes. A slight decrease in HMW species formation at T0 and subsequent time points was observed for histidine buffer at pH 7.2 compared to histidine buffer at 6.7 or 6.8 ( Fig.13A ). Effect of buffer, pH or PFS on antibody concentration measured at the end of the stability testing period ( Fig. 13B ) or pH( Fig. 13C ) had no apparent effect. A general increase in turbidity as a function of time and temperature was observed for all buffers and target pH. However, no significant differences in turbidity were observed between the different pH, buffers, or syringe types tested ( Fig.13D ). For both 25°C and 40°C storage conditions, all formulations showed some degree of PS80 degradation over time. At all pH values, formulations containing histidine showed more PS80 degradation than formulations containing potassium phosphate ( Fig.13E ).

[0276] As shown in Figure 14, at all pH values ​​and buffers tested, there was no significant increase in subvisible particle formation upon storage at different temperatures, except for the potassium phosphate formulation at pH 7.0 (K 7.0) at 40°C for 3 months. Compared to the BD counterpart of OMPIPFS, more 2 μm particles were formed in OMPIPFS ( Fig.14A The formation of subvisible particles with sizes ≤10 μm and ≤25 μm was generally consistent, with a few outliers attributed to assay variability ( Figure 14B-Figure 14C ).

[0277] Vial Stability Studies

[0278] As shown in Figure 15, under accelerated conditions at 40°C, SAR445088 had a higher tendency to form HMW species in potassium phosphate buffer than in histidine buffer. Overall, SAR445088 retained substantial stability under all pH and buffer conditions during both ambient storage (25°C) and refrigerated storage (5°C). A slight decrease in HMW species formation at T0 and subsequent time points was observed when using H7.2 buffer compared to H6.7 and H6.8 ( Fig.15A). The buffer or pH has an effect on the antibody concentration measured at the end of the test period ( Fig. 15B ) or pH( Fig. 15C ) had no significant effect. A general increase in turbidity as a function of time and temperature was observed for all buffers and target pH values. However, the increase in turbidity was significantly greater for formulations containing potassium phosphate buffer than for formulations containing histidine ( Fig.15D ). PS80 degradation was evident over time in all formulations under storage conditions at 25°C and 40°C. Formulations containing histidine showed more PS80 degradation than formulations containing potassium phosphate at all pH values ​​( Fig.15E ). PS80 degraded more when the same formulation was placed in vials compared to PFS, likely due to increased headspace in the vials.

[0279] As shown in Figure 16, for all pH values ​​and buffers tested, no significant increase in subvisible particle formation was observed upon storage at different temperatures. The formation of subvisible particles ≤2 μm, ≤10 μm, and ≤25 μm was generally consistent, with a few outliers attributed to assay variability ( Figure 16A-16C ).

[0280] Frozen storage stability study

[0281] As shown in Figure 17, a slight decrease in the formation of HMW species was observed when frozen storage was performed at -80°C and -30°C ( Fig.17A Effect of frozen storage on antibody concentration measured at the end of the storage period ( Fig. 17B )、pH( Fig. 17C ), turbidity( Fig.17D ) and subvisible particles ( Fig.17E ) No observable effects. SAR445088 is stable for up to 12 months under refrigerated storage.

[0282] Peptide mapping and Biacore binding analysis

[0283] As shown in Figure 18, a general increase in hotspot modification levels as a function of temperature and time was observed in all buffers and pH values ​​tested. In particular, for D32 isomerization, a modification level of approximately 6% per week was observed in the histidine buffer formulation at pH 6.8 after 3 months of storage at 40°C, while a modification level of approximately 3% was observed after 12 months of storage at 5°C. No significant differences in modification levels were observed between different pH values ​​and buffer types ( Figures 18A-18D). K7.0 buffer showed a non-trending degree of HC M251 oxidation when stored at 40°C for 2 months (2M-40C), which may be due to operator error. Biacore analysis showed that SAR445088 retained relative activity after 6 months storage at 25°C and 12 months storage at 5°C for samples in histidine buffer at pH 6.8. A slight decrease in potency was observed for samples exposed to 40°C for 3 months.

[0284] Summarize

[0285] The SAR445088 formulation showed a higher propensity to form HMW species in potassium phosphate buffer than in histidine buffer. Potassium phosphate buffer had lower levels of PS80 degradation compared to histidine buffer. However, the propensity for molecular aggregation was the primary driver for the selection of histidine as a buffer, as the use of 10 μM DTPA was observed to effectively mitigate PS80 degradation in the presence of histidine. In histidine buffer, there were no significant differences in the critical quality attributes (CQAs) of the formulations over the pH range of 6.7-7.2. When comparing the BD Neopak Low, BD Neopak High, and OMPI syringes, there were no other significant differences in the CQAs, except for the relative increase in 2 μm particle formation in the OMPIPFS. The OMPI syringe also had high ≤10 μm and ≤25 μm subvisible particle counts, but the particle counts were below the USP specification limits (<6000 10 μm particles per small volume container and <600 25 μm particles per small volume container).

[0286] Batch 2 Formulation Study

[0287] PFS stability study

[0288] As shown in Figure 19, an overall increase in HMW species formation was observed over time and at 40°C. There was no significant change in the level of HMW species formation between pH 6.4-7.1 at 5°C or 25°C. A slight decrease in HMW species formation at T0 and subsequent time points at 5°C and 25°C was observed for H7.1 compared to H6.4 and H6.8 formulations ( Fig.19A An increase in LMW species formation was observed over time and at 40°C. No trend in LMW species formation as a function of formulation pH was observed at 5°C and 25°C ( Fig.19B No effect of formulation pH on antibody concentration at the end of the storage period was seen ( Fig.19C ) and pH( Fig.19D A general increase in turbidity with time and temperature was observed for all formulations tested, but no significant differences were observed between the different pH values ​​( Fig.19E Negligible PS80 degradation was observed in any of the tested formulations under all storage stability conditions ( Fig.19F ). Charge variant analysis showed an overall increase in the appearance of acidic variants at 25°C and 40°C for all formulations tested, with a corresponding decrease in the appearance of monomeric and basic variants. Charge variant analysis was only performed for storage up to 1 month at 40°C due to loss of peak resolution. At 5°C, charge variants remained relatively consistent for all conditions tested ( Figure 19G ).

[0289] As shown in Figure 20, 2 μm subvisible particles were more abundant than larger particles in all formulations tested, presumably due to the presence of silicone oil. However, it was evident that the appearance of subvisible particles did not increase significantly upon storage at different temperatures. The appearance of subvisible particles of size ≤2 μm, ≤10 μm, and ≤25 μm was generally consistent between formulation test samples, with a few outliers attributed to assay variability ( Figures 20A-20C ).

[0290] Vial Stability Studies

[0291] As shown in Figure 21, an overall increase in HMW species formation was observed over time and at 40°C. There was no significant change in HMW species formation between pH 6.4-7.1 at 5°C or 25°C. A slight decrease in HMW species formation at T0 and subsequent time points at 5°C and 25°C was observed for H7.1 compared to H6.4 and H6.8 formulations ( Fig.21A An increase in LMW species formation was observed over time and at 40°C. No trend in LMW species formation as a function of formulation pH was observed at 5°C and 25°C ( Fig.21B No effect of formulation pH on antibody concentration at the end of the storage period was seen ( Fig. 21C ) and pH( Fig.21D A general increase in turbidity with time and temperature was observed for all formulations tested, but no significant differences were observed between the different pH values ​​( Fig.21E No significant PS80 degradation was observed in any of the tested formulations under all storage stability conditions ( Fig.21F ). Charge variant analysis by cIEF showed an overall increase in the appearance of acidic variants at 25°C and 40°C for all formulations tested, with a corresponding decrease in the appearance of monomeric and basic variants. Charge variant analysis was only performed for storage up to 1 month at 40°C due to loss of peak resolution. At 5°C, charge variants remained relatively consistent for all conditions tested ( Figure 21G ).

[0292] As shown in Figure 22, it is evident that there was no significant increase in the formation of subvisible particles upon storage at different temperatures in any of the formulations tested. The appearance of subvisible particles with sizes ≤2 μm, ≤10 μm, and ≤25 μm was generally consistent between formulation test samples, with a few outliers attributed to assay variability ( Figures 22A-22C ).

[0293] Frozen storage stability study

[0294] As shown in Figure 23, there was no significant change in HMW species formation when frozen at -80°C and -30°C between the different formulations tested ( Fig.23A ). LMW species formation remained similar over time when stored frozen at -80°C and -30°C. Formulation samples using histidine buffer at pH 7.1 (H7.1) stored at -30°C showed non-trending levels of LMW species formation. This is attributed to the variability of the assay ( Fig. 23B Effect of frozen storage on antibody concentration at the end of storage period ( Fig.23C )、pH( Fig.23D ), turbidity( Fig.23E ) or sub-visible particle formation level ( Fig.23F ) had no significant effect.

[0295] Peptide mapping and Biacore analysis

[0296] As shown in Figure 24, a general increase in hotspot modification levels as a function of temperature and time was observed for all tested buffers and pHs in the vials and in the PFS. In particular, for D32 isomerization, a modification level of approximately 6% per week was observed in the histidine buffer formulation at pH 6.8 (H6.8) at 40°C. No significant differences in modification levels were observed between different pH values ​​and buffer types ( Figure 24A-Figure 24D ). Biacore analysis showed that SAR445088 retained relative activity after 6 months storage at 25°C and 5°C for samples in histidine buffer at pH 6.8. A slight decrease in potency was observed for all samples exposed to 40°C for 3 months ( Fig.24E ).

[0297] Summarize

[0298] The SAR45088 test formulations at pH 6.4 to 7.1 showed no significant differences for all CQAs tested.Formulation samples placed in PFS and vials and subjected to freezer stability maintained substantial stability over the course of the study (12 months).

[0299] Batch 3 Formulation Study

[0300] PFS stability study

[0301] As shown in Figure 25, an overall increase in HMW species formation was observed over time and at 40°C. No significant changes in the level of HMW species formation were observed between pH 6.4-7.1 at 5°C or 25°C. A slight decrease in HMW species formation at T0 and subsequent time points at 5°C and 25°C was observed for H7.1 compared to H6.4 and H6.8 formulations ( Fig.25A No significant differences in the levels of LMW species formation were observed between the pH values ​​tested ( Fig.25B The effect of formulation pH on the concentration measured at the end of the storage period ( Fig.25C ) or pH( Fig.25D ) had no observable effect. A general increase in turbidity with time and temperature was observed for all tested formulations, but no significant differences were observed between the different pH values ​​( Fig.25E Negligible PS80 degradation was observed in any of the tested formulations under all storage stability conditions ( Fig.25F Finally, the number of 2 μm subvisible particles was higher than the number of particles of other sizes for all tested formulations. However, there was no significant increase in the level of subvisible particles when stored at 5°C and 25°C. An increase in subvisible particles over time was observed in all formulations at 40°C ( Figure 25G ). There were no major differences in the trends of subvisible particles between the pH values ​​studied.

[0302] Vial Stability Studies

[0303] As shown in Figure 26, an overall increase in HMW species formation was observed over time and at 40°C. No significant changes in the level of HMW species formation were observed between pH 6.4-7.1 at 5°C or 25°C. A slight decrease in HMW species formation at T0 and subsequent time points at 5°C and 25°C was observed for H7.1 compared to H6.4 and H6.8 formulations ( Fig.26A No significant differences in the levels of LMW species formation were observed between the pH values ​​tested ( Fig.26B The effect of formulation pH on the concentration measured at the end of the storage period ( Fig.26C ) or pH( Fig.26D ) had no observable effect. A general increase in turbidity with time and temperature was observed for all tested formulations, but no significant differences were observed between the different pH values ​​( Fig.26E Negligible PS80 degradation was observed in any of the tested formulations under all storage stability conditions ( Fig.26FFinally, there was no significant increase in the level of subvisible particle formation when stored at different temperatures in any of the tested formulations. The levels of subvisible particle formation of sizes ≤2 μm, ≤10 μm, and ≤25 μm were generally consistent, with a few outliers attributed to operator error ( Figure 26G ).

[0304] Peptide mapping and Biacore analysis

[0305] As shown in Figure 27, a general increase in hotspot modification levels as a function of temperature and time was observed in all buffers and pHs tested. In particular, for D32 isomerization, a modification level of approximately 6% per week was observed in the histidine buffer formulation at pH 6.8 (H6.8) at 40°C. No significant differences in modification levels were observed between different pH values ​​and buffer types ( Figure 27A-Figure 27D ). Biacore analysis showed that SAR445088 retained relative activity after 3 months storage at 25°C and 5°C for samples in histidine buffer at pH 6.8. A slight decrease in potency was observed for all samples exposed to 40°C for 3 months ( Fig.27E ).

[0306] Summarize

[0307] The SAR445088 test formulations at pH 6.4 to 7.1 showed no significant differences for all CQAs tested. The formulation samples placed in PFS and vials maintained substantial stability over the course of the study (6 months).

[0308] Batch 4 Formulation Study

[0309] Vial Stability Studies

[0310] As shown in Figure 28, an overall increase in HMW species formation was observed over time and at 40°C, but to a lesser extent compared to previous batches. No significant changes in the level of HMW species formation were observed between pH 6.4-7.1 at 5°C or 25°C. A slight decrease in HMW species formation at T0 and subsequent time points was observed for pH 7.1 at 5°C and 25°C compared to the pH 6.4 and pH 6.8 formulations ( Fig.28A No clear differences in the levels of LMW species formation were observed between the tested pHs at 5°C and 25°C, but a slight increase over time was observed at 40°C ( Fig.28B The effect of formulation pH on the concentration measured at the end of the storage period ( Fig.28C ) or pH( Fig.28D) had no observable effect. A general increase in turbidity with time and temperature was observed for all tested formulations, but no significant differences were observed between the different pH values ​​( Fig.28E Negligible PS80 degradation was observed in any of the tested formulations under all storage stability conditions ( Fig.28F Finally, there was no significant increase in subvisible particle levels upon storage at different temperatures in any of the tested formulations. The formation levels of subvisible particles ≤2 μm, ≤10 μm, and ≤25 μm in size were generally consistent, with a few outliers attributed to assay variability ( Figure 28G ).

[0311] Peptide mapping and Biacore binding analysis

[0312] As shown in Figure 29, a general increase in hotspot modification levels as a function of temperature and time was observed in all buffers and pHs tested. In particular, for D32 isomerization, a modification level of approximately 10% per week was observed in the histidine buffer formulation at pH 6.8 (H6.8) at 40°C. No significant differences in modification levels were observed between different pH values ​​and buffer types ( Figure 29A-Figure 29D Biacore analysis showed that for samples in histidine buffer at pH 6.8, SAR445088 retained relative activity after 1 month of storage ( Fig.29E ).

[0313] Summarize

[0314] Over the course of the study (3 months), no significant differences were shown for the SAR445088 test formulations at pH 6.4 to 7.1 for all CQAs tested.

[0315] Batch 5 Formulation Study

[0316] As shown in Figure 30, a slight increase in the level of HMW species formation was observed over time and at 40°C, but to a lesser extent than in previous batches. Fig. 30A A slight increase in LMW species formation over time was observed at 40 °C ( Fig. 30B The effect of formulation pH on the concentration measured at the end of the storage period ( Fig. 30C ) or pH( Fig.30D ) No observable effect. A general increase in turbidity with time and temperature was observed for all formulations tested. ( Fig.30E Negligible PS80 degradation was observed under any storage stability conditions ( Fig.30FFinally, there was no significant increase in subvisible particle levels when stored at different temperatures. The levels of subvisible particles ≤2 μm, ≤10 μm, and ≤25 μm were generally consistent ( Figure 30G ).

[0317] Example 7. Robustness study

[0318] SAR445088 formulations using different batches of SAR445088 were subjected to various physicochemical stresses as described in Table 5.

[0319] Table 5. Study conditions for robustness study.

[0320]

[0321] Wrist Oscillator (small bottle)

[0322] As shown in Figure 31, the starting HMW species values ​​varied between batches, but wrist shaking in the vials had no significant effect on the level of HMW species formation ( Fig.31A ). Wrist oscillation affects the formation of LMW substances ( Fig.31B ), turbidity( Fig.31C ) or charge variant formation ( Fig.31D ) had no observable effect. Subvisible particle analysis showed that while the starting subvisible particles varied between batches, there was no increased trend toward formation of subvisible particles ≤2 μm, ≤10 μm, and ≤25 μm when wrist shaking was performed in the vials ( Fig.31E ).

[0323] PFS

[0324] As shown in Figure 32, the starting HMW species values ​​were different for different batches, but wrist shaking during PFS had no significant effect on HMW species formation ( Fig.32A ). Wrist oscillation affects the formation of LMW substances ( Fig.32B ), turbidity( Fig.32C ) or charge variant formation ( Fig.32D ) had no observable effect. Subvisible particle analysis showed that while the starting subvisible particles varied between batches and were larger compared to vials, there was no increased trend toward formation of subvisible particles ≤2 μm, ≤10 μm, and ≤25 μm when wrist shaking was performed in PFS ( Fig.32E ).

[0325] Freeze-thaw cycles

[0326] As shown in Figure 33, the starting HMW material values ​​of different batches were different, but freeze-thaw had no significant effect on the formation of HMW material ( Fig.33AThe effect of freeze-thaw on the LMW mass formation of the formulations measured at the end of the storage period ( Fig.33B ), turbidity( Fig.33C ),concentration( Fig.33D ) or pH( Fig.33E ) had no observable effect. Subvisible particle analysis showed a slightly increased trend toward the formation of ≤2μm, ≤10μm, and ≤25μm particles upon repeated freeze-thaw cycles, however, particle counts per container remained well within USP specifications (<6000 10μm particles per small volume container and <600 25μm particles per small volume container) ( Fig.33F ).

[0327] Photostability studies

[0328] As shown in FIG34 , a slight increase in HMW species formation was observed upon increased exposure to ambient temperature and light compared to the control sample covered in aluminum foil ( Fig.34A The effect of exposure to light on the LMW species formation of the formulations measured at the end of the storage period ( Fig.34B ), turbidity( Fig.34C ),concentration( Fig.34D ) or pH( Fig.34E ) had no observable effect. No PS80 degradation was observed in any sample ( Fig.34F No significant changes in charge variant formation were observed after exposure to ambient light for up to 14 days ( Figure 34G ). Total peptide mapping analysis showed an overall increase in D32 isomerization, VSNK deamidation, and PENNYK deamidation (including in the overlaid control sample), suggesting that these modifications are not a direct result of exposure to light. However, M251 oxidation increased upon increased exposure to light compared to the overlaid control. This is an expected effect ( Fig.34H ). Analysis of subvisible particle formation showed that for subvisible particles ≤2 μm, ≤10 μm, and ≤25 μm, the effect of exposure to ambient light was negligible ( Fig.34I ).

[0329] Summarize

[0330] The SAR445088 formulation demonstrated robustness against physicochemical stresses of wrist shaking, freeze-thaw cycles, and exposure to ambient light for 14 days in vials and PFS. Robustness was observed across batches and across pH values ​​from 6.4 to 7.1.

[0331] Example 8. Exemplary Liquid Formulations

[0332] The formulation characterization studies described herein, in addition to managing D32 isomerization, identified the appropriate concentration of PS80, identified the optimal type and concentration of chelator, determined the appropriate buffer and pH to provide DP stability (including optimal pH) and aid in DP robustness against physicochemical stress, and evaluated the effect of adding 3% sucrose.

[0333] In particular, the higher pH 6.8 and inclusion of arginine hydrochloride in the formulation reduced the rate of isomerization of D32 in the LCCDR1 of SAR445088. Such an optimized formulation provided resistance to isomerization when tested within different SAR445088 batches under refrigerated, ambient and accelerated storage conditions.

[0334] The optimal PS80 concentration was determined by evaluating formulations containing different PS80 concentrations for resistance to mechanical stress, simulated clinical dilution, and short-term exposure to light. These tests identified 600 ppm (0.06%) PS80 as the optimal surfactant concentration to protect SAR445088 from shear, IV dilution, and light exposure.

[0335] Due to the batch-to-batch variability in metal ion content of different drug substance batches and the inability of formulations containing 10 μM EDTA to mitigate PS80 degradation between different batches, DTPA at a level of 10 μM was selected as the optimal chelator for mitigating the risk of PS80 degradation. DTPA was more effective in preventing PS80 degradation (oxidation), reducing HMW species formation, and avoiding product solution discoloration, which was observed in formulations with 10 μM EDTA or when no chelator was used in the formulation (see Fig. 8A , Figure 8B , Fig.8D , Fig. 9 , Fig. 10A , Fig. 10B and Fig. 10C ).

[0336] A head-to-head comparison of SAR445088 formulations in histidine and phosphate buffers showed that histidine buffer, pH 6.8, was the best at reducing aggregation propensity under accelerated stability testing conditions.

[0337] Stability studies demonstrated that the addition of sucrose did not negatively affect protein stability and provided resistance to aggregation that could occur during repeated freeze-thaw cycles.

[0338] Different PFS were compared and they showed similar product properties. DP stability studies in vials and PFS were conducted at pH values ​​between 6.4 and 7.1 under refrigerated, ambient, accelerated and frozen conditions. Within this pH range between different batches, the PFS showed acceptable product properties, namely overall control of HMW species propensity, mitigation of PS80 oxidation and control of molecule-specific D32 isomerization in each case.

[0339] The analysis described herein showed that an exemplary SAR445088 liquid formulation that was optimal for stabilizing 150 mg / mL liquid DP as well as 150 mg / mL frozen FDS was 10 mM histidine, 150 mM arginine HCl, 3% (w / v) sucrose, 10 μM DTPA, 0.06% (w / v) PS80, pH 6.8 + 0.3.

[0340] Example 9. Components of a drug product

[0341] SAR445088DP can be composed of SAR445088, L-histidine, L-histidine hydrochloride monohydrate, L-arginine hydrochloride, sucrose, DTPA, and polysorbate 80. These excipients are known to be well tolerated after parenteral administration and are water soluble. Exemplary DP compositions and container closure details are summarized in Table 6 below.

[0342] Table 6. Components of exemplary SAR445088DP.

[0343]

[0344]

[0345] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each document is cited, which in some cases may be incorporated in its entirety.

[0346] As used herein, the indefinite articles "a" and "an" in the specification and in the claims should be understood to mean "at least one", unless explicitly stated to the contrary.

[0347] It should also be understood that, in any method claimed herein that includes more than one step or operation, the order of the steps or operations of the method is not necessarily limited to the order in which the steps or operations of the method are described, unless explicitly stated to the contrary.

[0348] In the claims and the foregoing description, all transitional phrases such as "comprising," "including," "with," "having," "containing," "involving," "having," "consisting of," and the like shall be construed as open-ended, i.e., meaning including, but not limited to, including. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures.

[0349] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0350] Where a range of values ​​is provided, every value between and including the upper and lower end points of that range is specifically contemplated and described herein.

Claims

1. A composition comprising: (a) a humanized antibody that specifically binds to complement component C1s, wherein the antibody comprises a light chain (LC) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and (b) about 50 mM to about 200 mM arginine or a salt thereof.

2. The composition of claim 1, wherein the composition comprises about 100 mM to about 200 mM or about 100 mM to about 150 mM arginine or a salt thereof.

3. The composition of claim 1 or claim 2, wherein the arginine salt is arginine hydrochloride, arginine citrate, arginine oxalate, arginine phosphate, arginine succinate or arginine tartrate.

4. The composition of any one of claims 1-3, wherein the arginine salt is arginine hydrochloride, optionally wherein the composition comprises about 150 mM arginine hydrochloride.

5. The composition of any one of claims 1-4, wherein the composition comprises a buffer at a concentration of about 1 mM to about 50 mM, about 5 mM to about 25 mM, or about 10 mM to about 20 mM.

6. The composition of claim 5, wherein the buffer is histidine, acetate, citrate, oxalate, phosphate, succinate or tartrate.

7. The composition of any one of claims 1-6, wherein the composition comprises about 10 mM histidine.

8. The composition of any one of claims 1-7, wherein the composition further comprises a stabilizer at a concentration of about 1% to about 8% (w / v) or about 1% to about 5% (w / v).

9. The composition of claim 8, wherein the stabilizer is sucrose, sorbitol or trehalose.

10. The composition of any one of claims 1-9, wherein the stabilizer is sucrose, optionally wherein the composition comprises about 3% (w / v) sucrose.

11. The composition of any one of claims 1-10, wherein the composition further comprises a chelating agent, optionally wherein the concentration of the chelating agent is about 1 μM to about 50 μM, or about 5 μM to about 25 μM, or about 10 μM to about 25 μM.

12. The composition of claim 11, wherein the chelating agent is diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA) or methionine.

13. The composition of any one of claims 1-12, wherein the composition comprises about 10 μM diethylenetriaminepentaacetic acid (DTPA).

14. The composition of any one of claims 1-13, wherein the composition further comprises a surfactant at a concentration of about 0.01% to about 0.1% (w / v), or about 0.03% to about 0.06% (w / v).

15. The composition of claim 14, wherein the surfactant is polysorbate 80 (PS80) or poloxamer 188 (P188).

16. The composition of any one of claims 1-15, wherein the surfactant is PS80, optionally wherein the composition comprises about 0.06% (w / v) polysorbate 80.

17. The composition of any one of claims 1-16, wherein the pH of the composition is from about 6 to about 7.5, from about 6 to about 7, from about 6.5 to about 7.5, or from about 6.5 to about 7.

1.

18. The composition of any one of claims 1-17, wherein the pH of the composition is about 6.

8.

19. The composition of any one of claims 1-18, wherein the composition comprises about 50 mg / mL to about 250 mg / mL, or about 100 mg / mL to about 200 mg / mL of the anti-C1s antibody.

20. The composition of any one of claims 1-19, wherein the composition comprises about 150 mg / mL of anti-C1s antibody.

21. The composition of any one of claims 1-20, wherein the composition comprises: (a) about 50 mg / mL to about 250 mg / mL of the antibody; (b) about 50 mM to about 200 mM arginine HCl; (c) about 1 mM to about 50 mM histidine; (d) about 1% to about 8% (w / v) sucrose; (e) about 1 μM to about 50 μM diethylenetriaminepentaacetic acid (DTPA); and (f) about 0.01% to about 0.1% (w / v) polysorbate 80 (PS80), and wherein the pH of the composition is from about 6 to about 7.

5.

22. The composition of claim 21, wherein the composition comprises: (a) about 150 mg / mL of the antibody; (b) about 150 mM L-arginine HCl; (c) about 10 mM histidine; (d) about 3% (w / v) sucrose; (e) about 10 μM DTPA; and (f) about 0.06% (w / v) PS80, and The pH of the composition is from about 6.5 to about 7.1, or about 6.

8.

23. The composition of any one of claims 1-22, wherein the antibody comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:7 and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:

8.

24. The composition of any one of claims 1-23, wherein the antibody is a Fab fragment, a F(ab')2 fragment, a scFv or a Fv.

25. The composition of any one of claims 1-23, wherein the antibody comprises a heavy chain constant region of isotype IgG4.

26. The composition of claim 25, wherein relative to the IgG4 constant region sequence of SEQ ID NO: 11, the IgG4 constant region comprises proline, glutamic acid, leucine and serine substitutions at amino acid residues 108, 115, 308 and 314, respectively.

27. The composition of any one of claims 1-23 and 25-26, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

10.

28. The composition of any one of claims 1-27, wherein the antibody has a lower isomerization rate at D32 of the light chain CDR1 compared to the antibody in a corresponding formulation without arginine or a salt thereof, optionally wherein the D32 isomerization is determined by total peptide mapping analysis.

29. The composition of any one of claims 1-28, wherein the antibody (i) has an isomerization rate that is at least about 2%-3% lower per week at 40°C, at least about 1%-3% lower per month at 25°C, or at least about 0.4%-0.6% lower per month at 5°C, compared to the antibody in a corresponding formulation without arginine or a salt thereof; or (ii) The isomerization rate after storage at 25°C for 12 weeks is approximately 10% lower compared to the antibody in a corresponding formulation without arginine or its salt.

30. The composition of any one of claims 1-28, wherein the antibody has an isomerization rate of less than 7.5% after storage at 5°C for 12 weeks, less than 20% after storage at 25°C for 12 weeks, or less than 35% after storage at 40°C for 12 weeks.

31. The composition of any one of claims 1-30, wherein the composition is in liquid, lyophilized, or reconstituted lyophilized form.

32. The composition of claim 31 , wherein the composition is in liquid form.

33. A container comprising the composition of any one of claims 1-32.

34. The container of claim 33, wherein the container is a vial or a syringe.

35. The container of claim 34, wherein the syringe is a pre-filled syringe.

36. A kit or article of manufacture comprising the container of any one of claims 33-35.

37. A pharmaceutical unit dosage form suitable for parenteral administration to humans, comprising a composition as claimed in any one of claims 1 to 32 in a container.

38. A method comprising administering to a human a composition as claimed in any one of claims 1 to 32.

39. A method of reducing the level of complement component cleavage products in a human, the method comprising administering to the human a composition as described in any one of claims 1-32.

40. A method of inhibiting C1s-mediated cleavage of complement components in a human, the method comprising administering to the human a composition of any one of claims 1-32.

41. The method of any one of claims 38-40, wherein the human suffers from cold agglutinin disease (CAD), immune thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), or antibody-mediated rejection (AMR).

42. A method of treating a complement-mediated disease in a human in need thereof, the method comprising administering to the human a composition of any one of claims 1-32.

43. The method of claim 42, wherein the complement-mediated disease is cold agglutinin disease (CAD), immune thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), or antibody-mediated rejection (AMR).

44. The method of any one of claims 38-43, wherein the administration is intravenous or subcutaneous.

45. A drug delivery device comprising a primary container containing a composition as claimed in any one of claims 1 to 32, wherein the drug delivery device is a sleeve-activated autoinjector with a manual needle inserter.

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