TGF-beta antibody-based compositions and their application

Abstract

FIELD: biotechnology.SUBSTANCE: pharmaceutical composition containing an antibody to TGF-β is proposed β, where the composition is an aqueous liquid solution. A finished product containing the said composition is also proposed. Furthermore, the invention relates to the use of said composition for the manufacture of a medicinal product for the treatment of cancer in a patient in need thereof.EFFECT: improved stability of a composition based on an antibody to TGF-β.14 cl, 15 dwg, 8 tbl, 7 ex

Description

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 212,473, filed June 18, 2021. The disclosure of this priority application is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on June 17, 2022, is named 022548_WO064_SL.txt and is 19,437 bytes in size.BACKGROUND OF THE INVENTION

[0003] Transforming growth factor beta (TGF-β) is a cytokine that controls many key cellular functions, including proliferation, differentiation, survival, migration, and epithelial-mesenchymal transition. It regulates various biological processes, such as extracellular matrix formation, wound healing, embryonic development, bone development, hematopoiesis, immune and inflammatory responses, and malignant transformation. Dysregulation of TGF-β leads to pathological conditions, such as developmental defects, cancer, chronic inflammation, and autoimmune and fibrotic diseases.

[0004] There are three known isoforms of TGF-β—TGF-β1, 2, and 3. All three isoforms are initially translated as a propeptide. After cleavage, the mature C-terminal end remains associated with the N-terminus (termed the latency-associated peptide, or LAP) to form the small latent complex (SLC), which is secreted from the cell. Inability of SLC to bind to TGF-β receptor II (TGFβRII) prevents receptor engagement. Activation by dissociation of the N- and C-termini occurs through one of several mechanisms, including proteolytic cleavage, acidic pH, or integrin structural changes (Connolly et al., Int J Biol Sci. (2012) 8(7):964–78).

[0005] TGF-β1, 2, and 3 are pleiotropic in function and are expressed in distinct patterns in different cell types and tissues. They exhibit similar activities in vitro, but individual knockouts in specific cell types reveal distinct roles in vivo despite their shared ability to bind the same receptor (Akhurst et al., Nat Rev Drug Discov. (2012) 11(10):790–811). Upon binding of TGF-β to TGFβRII, the constitutive kinase activity of the receptor phosphorylates and activates TGF-β receptor I (TGFβRI), which phosphorylates SMAD2 / 3, mediating association with SMAD4, nuclear localization, and transcription of TGF-β-responsive genes. Ibid. In addition to this canonical signaling cascade, the noncanonical pathway transmits signals through other factors, including p38 MAPK, PI3K, AKT, JUN, JNK, and NF-κB. TGF-β signaling is also modulated by other pathways, including WNT, Hedgehog, Notch, INF, TNF, and RAS.Thus, the end result of TGF-β signaling is a cross-talk between all of these signaling pathways that integrates the cell's state and environment into a single whole.Ibid.

[0006] Considering the diverse functions of TGF-β, there is a need for effective therapy with pan-TGF-β-specific antibodies. SUMMARY OF THE INVENTION

[0007] The present invention provides pharmaceutical compositions that comprise an antibody to TGF-β. In one aspect, the present invention provides a pharmaceutical composition, wherein the composition is an aqueous liquid solution comprising: 20-200 mg / ml of an antibody to TGFβ, wherein the antibody comprises the amino acid sequence of a heavy chain variable domain (V H ), corresponding to residues 1-120 of SEQ ID NO:1, and the amino acid sequence of the light chain variable domain (V L), corresponding to residues 1-108 of SEQ ID NO:2, 10-50 mM acetate, optionally 25 mM acetate, and 5-15% w / v sucrose, optionally 8% w / v sucrose, wherein the solution has a pH of 5.0 ± 0.2 or 5.0 ± 0.3. In some embodiments, the composition is an aqueous liquid solution at a pH of 4.7-5.3.

[0008] In some embodiments, the antibody comprises the heavy chain amino acid sequence set forth under SEQ ID NO:1 (with or without a C-terminal lysine) and the light chain amino acid sequence set forth under SEQ ID NO:2.

[0009] In some embodiments, the TGFβ antibody is present at a concentration of 40-180 mg / mL, optionally 50 mg / mL or 150 mg / mL.

[0010] In some embodiments, the composition comprises a surfactant, such as a polysorbate (e.g., polysorbate 80 (PS80)). In particular embodiments, the composition comprises PS80 at a concentration of 0.01-0.10% w / v, optionally 0.06% w / v.

[0011] In some embodiments, the composition comprises a chelating agent, optionally selected from EDTA and DPTA. In certain embodiments, the chelating agent is present at a concentration of 0-20 μM, optionally 10 μM.

[0012] In specific embodiments, the composition comprises 50 mg / mL, 75 mg / mL, or 150 mg / mL of an anti-TGFβ antibody, 25 mM acetate, 10 μM EDTA, 0.06% PS80, and 8% w / v sucrose at a pH of 5.0+0.3. In certain embodiments, the antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO:1 (with or without a C-terminal lysine) and the light chain amino acid sequence set forth in SEQ ID NO:2.

[0013] The present invention also provides a finished article comprising a vial and instructions for use, wherein the vial contains approximately 16 ml of the composition of the present invention.

[0014] Also provided herein are methods for treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a composition of the present invention. In some embodiments, the method further comprises administering an additional anti-cancer therapeutic agent. In specific embodiments, the composition is administered intravenously at a dose of 5 mg / kg or 15 mg / kg, optionally every two weeks. The present invention also provides a composition of the present invention for use in treating a patient in need thereof in these methods, as well as the use of a composition of the present invention for the manufacture of a medicament for treating a patient in need thereof in a treatment method of the present invention.

[0015] Other features, objects, and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that the detailed description, while indicating embodiments and aspects of the present invention, is for illustrative purposes only and not limiting. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1 shows a photograph showing the degree of opalescence in formulations based on 80 mg / ml Ab1 with acetate or histidine buffer over a range of pH.

[0017] Figures 2A-C are bar graph panels showing the gradual formation of 2 μm (Figure 2A), 10 μm (Figure 2B), and 25 μm (Figure 2C) particles that are not visible to the naked eye in acetate and histidine formulations over 4 weeks of storage at 5°C, 25°C, or 40°C.

[0018] Fig. 3 shows a bar graph showing the viscosity values ​​(in centipoise (cP)) of acetate and histidine formulations immediately after receipt (T0) and after four weeks of storage at 40°C.

[0019] Fig. 4A and 4B are bar graph panels showing the pH values ​​of acetate and histidine formulations during 4 weeks of storage at 40°C, 25°C, or 5°C.

[0020] Fig. 5 shows a scatter diagram graph showing the optical density values ​​(340-360 nm) of acetate (pH 4.7, 5, and 5.5) and histidine (pH 5.5, 6, and 6.5) at T0 and after 4 weeks of storage at 5°C, 25°C, and 40°C.

[0021] Fig. 6 is a panel of graphs showing the gradual formation of HMWS in formulations at different polysorbate (PS80) concentrations over 2 weeks of storage at 5°C (upper left panel), 2 weeks of storage at 40°C (upper right panel), 48 hours of vigorous agitation (lower left panel), and freeze / thaw (FT) cycles at -30°C to room temperature (lower right panel). SR_# or Ch_#: # represents the % concentration of PS80. SR and Ch represent two different PS80 suppliers.

[0022] Fig. 7A shows a pair of bar graphs showing Ab1 concentrations after dilution in saline or dextrose in polyolefin (PO) or polyvinyl chloride (PVC) IV bags.

[0023] Fig. 7B shows a pair of bar graphs showing particles not visible to the naked eye (≥10 μm) after dilution in saline (S) or dextrose (D) in PO or PVC IV bags. T0: time zero. T24: 24 hours. T48: 48 hours.

[0024] Fig. 8A and 8B are graphs showing the gradual formation of HMWS in formulations with different concentrations of Ab1 over 12 weeks of storage at 5°C, 25°C, and 40°C (Fig. 8A) and over 6 months of storage at -20°C (Fig. 8B).

[0025] Fig. 9 shows a pair of graphs showing the oxidation of M252 (left panel) and the gradual formation of HMWS in % (right panel) in Ab1-based formulations with added metals.

[0026] Fig. 10 shows a pair of bar graphs showing the gradual formation of HMWS and subspecies in different formulations after one month of storage at 40°C (left panel) or three months of storage at 25°C. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides stable pharmaceutical compositions comprising a pan-TGFβ-specific monoclonal antibody in an aqueous liquid solution. One such antibody is Ab1. Ab1 is an IgG4 monoclonal antibody that targets all three human TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) and has the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2.

[0028] The therapeutic success of monoclonal antibodies depends in part on the processability, stability, and delivery characteristics of antibody-based drug candidates. Unsatisfactory solution properties, such as high solution viscosity or opalescence, have a significant impact on the feasibility of developing antibody-based drugs. Studies of Ab1-based formulations have shown that this antibody is surface-active and has a high tendency to form both invisible and visible particles upon solution agitation or other interfacial stress conditions. The present inventors have found that the formulations of the present invention, which are based on an acetate buffer characterized by an acidic pH of approximately 5.0, significantly improve the stability of the formulation during storage and transportation, including reducing particle formation.The present inventors found that Ab1 exhibits undesirable solution properties, such as opalescence and poor colloid stability at higher pH (e.g., pH 6.0). The present inventors also found that particle formation in solution can be further reduced by adding a surfactant and that the inclusion of a chelating agent also helps improve the formulation. Panspecific monoclonal antibodies to TGF β

[0029] The monoclonal antibody constituted herein comprises the complementarity determining regions (CDRs) of Ab1. Such antibodies are collectively referred to herein as "Ab1-related antibodies," which include Ab1 itself. In some embodiments, the antibody is a fully human antibody comprising a human IgG4 constant region and a human κ light chain constant region. In additional embodiments (e.g., Ab1), the human IgG4 constant region is characterized by a mutation at position 228 (Eu numbering). In some embodiments (e.g., Ab1), the mutation is a serine-to-proline mutation (S228P).

[0030] The amino acid sequences of the heavy and light chains of Ab1 are shown below as SEQ ID NOs: 1 and 2, respectively. The S228P site is boxed and shown in bold in SEQ ID NO: 1. The variable domains are shown in italics. The CDRs are shown in boxes. The glycosylation site in the heavy chain constant domain is shown in bold (N297).

[0031] In some embodiments, the antibody herein provides an antibody (e.g., a human antibody) having the CDRs shown above. That is, the antibodies have the following amino acid sequences of the heavy chain and light chain CDRs: HCDR1 SNVIS (SEQ ID NO: 3) HCDR2 GVIPIVDIANYAQRFKG (SEQ ID NO: 4) HCDR3 TLGLVLDAMDY (SEQ ID NO: 5) LCDR1 RASQSLG SSYLA (SEQ ID NO: 6) LCDR2 GASSRAP (SEQ ID NO: 7) LCDR3 QQYADSPIT (SEQ ID NO: 8) Thus, the antibody may comprise SEQ ID NOs: 3, 4, 5, 6, 7, and 8.

[0032] In additional embodiments, the antibody has a heavy chain variable domain (amino acids 1-120 of SEQ ID NO:1) and a light chain variable domain (amino acids 1-108 of SEQ ID NO:2) as shown above. In some embodiments, the antibody constituted herein lacks a C-terminal lysine in the heavy chain.

[0033] In specific embodiments, the antibody constituted herein is Ab1. Ab1 has a calculated molecular weight of 144 kDa in non-glycosylated form. Ab1 has a molecular weight of 147.011 kDa as determined by mass spectrometry, a theoretical experimental isoelectric point (pI) of 6.78, and an experimental pI of approximately 5.9-7.1. II. Methods for producing antibodies

[0034] Antibodies related to Ab1 can be produced by methods well known in the art. DNA sequences encoding the heavy and light chains of the antibodies can be inserted into expression vectors such that the genes are operably linked to the necessary expression control sequences, such as transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, etc. The coding sequence of the antibody light chain and the coding sequence of the antibody heavy chain can be inserted into separate vectors and can be operably linked to the same or different expression control sequences (e.g., promoters).In one embodiment, both coding sequences are inserted into the same expression vector and can be operably linked to the same expression control sequences (e.g., a common promoter), to separate identical expression control sequences (e.g., promoters), or to different expression control sequences (e.g., promoters). The antibody coding sequences can be inserted into the expression vector using standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if restriction sites are absent).

[0035] In addition to the antibody chain genes, recombinant expression vectors may carry regulatory sequences that control the expression of the antibody chain genes in the host cell. Examples of regulatory sequences for expression in mammalian host cells include viral elements that drive high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian gene promoters such as the native immunoglobulin and actin gene promoters.

[0036] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present invention may carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selectable marker genes. For example, a selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate to a host cell into which the vector has been introduced. Selectable marker genes may include the dihydrofolate reductase (DHFR) gene (for use in DHFR-containing host cells with methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0037] Expression vectors encoding the antibodies of the present invention are introduced into host cells for expression. The host cells are cultured under conditions suitable for expression of the antibody, which is then harvested and isolated. Host cells include mammalian, plant, bacterial, or yeast cells. Mammalian cell lines available as expression hosts are known in the art and include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a variety of other cell lines. Cell lines can be selected based on their expression levels.Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells.

[0038] Furthermore, antibody expression can be enhanced using a variety of well-known techniques. For example, the glutamine synthetase gene expression system (GS system) is a general approach for enhancing expression under specific conditions.

[0039] The tissue culture medium used for the host cells may or may not contain animal-derived components (ADCs), such as bovine serum albumin. In some embodiments, ADC-free culture media are preferred for human safety. Tissue culture can be performed using a fed-batch culture method, a continuous perfusion method, or any other method suitable for the host cells and the desired yield.III. Antibody-based formulations

[0040] The formulations of the present invention provide excellent stability for Ab1-related antibodies to TGF-β, including Ab1. "Stable" or "stability" refer to the ability of an antibody in a composition to maintain its physical stability, chemical stability, and / or biological activity during storage and / or when exposed to physical or chemical stress. Stability can be in the context of a selected temperature, such as under frozen conditions (e.g., from -70°C to -30°C), under refrigerated conditions (e.g., 2-8°C), or at room temperature (e.g., 23-25°C) for a selected period of time, such as 16 weeks, 24 weeks, 36 weeks, four months, six months, one year, two years, three years, or longer. Protein stability can be measured using assays that are conducted over a shorter period of time, but the results of which indicate stability under clinical conditions.Such assays include freeze / thaw cycling assays, in which the protein composition is subjected to one or more freeze-thaw cycles; or agitated assays, in which the protein composition is subjected to mechanical agitation for a predetermined period.Protein stability can be determined by storing a protein-based composition at a specified storage temperature (e.g., 2-8°C) for a selected period of time and analyzing its structural and functional characteristics, such as the degree of dimerization or aggregation (e.g., measured by size-exclusion HPLC or a protein gel), protein degradation (e.g., measured by size-exclusion HPLC or a protein gel), composition color change, liquid composition clarity, enzymatic activity, glycan content and composition, receptor binding affinity, residual methionine oxidation, and composition biological activity. See also the examples below for more detailed illustrations of antibody formulation stability testing methods.

[0041] An antibody described herein "retains its chemical stability" in a pharmaceutical composition if the chemical stability at the time is such that the antibody is considered to retain its biological activity, as defined below. To assess chemical stability, chemically altered forms of the antibody can be detected and quantified. The chemical alteration can include size modifications and can be assessed using methods known in the art, such as size exclusion chromatography, capillary isoelectric focusing (cIEF), liquid chromatography / mass spectrometry (LCMS), SDS-PAGE, and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS).Other types of chemical change include charge changes, which can occur, for example, as a result of deamidation or oxidation, and can be assessed using ion-exchange chromatography, mass spectrometry, or size-exclusion chromatography. In some embodiments, the type of chemical change that occurs during accelerated storage of compositions containing the antibodies described herein involves oxidation of the antibodies. In some embodiments, residues M252 and M428 of SEQ ID NO: 1 are oxidized in samples with added metals under accelerated storage conditions.

[0042] The compositions of the present invention contain one or more pharmaceutically acceptable excipients. The term "excipient" or "carrier" is used herein to describe any ingredient other than the compound(s) of the present invention. An excipient can be an inert substance that serves as a diluent, carrier medium, vehicle, preservative, binder, or stabilizing agent for the active ingredient(s) of the drug. For example, the compositions can contain a buffering agent, an isotonic agent, and / or a stabilizing agent, such as an antioxidant. In some cases, a single agent can serve more than one of these purposes.In some embodiments, a composition of the present invention comprises an anti-TGF-β antibody described herein, a buffering agent such as acetate, a stabilizer such as sucrose, and a surfactant such as polysorbate 80 (PS80). The anti-TGF-β antibody described herein is characterized by improved stability due to the combination of specific components in the composition. The compositions of the present invention can be aqueous liquid solutions or lyophilized preparations. In preferred embodiments, the compositions of the present invention are aqueous liquid solutions.

[0043] In some embodiments, the composition comprises a stabilizing agent such as L-methionine. In specific embodiments, the composition is an aqueous liquid composition comprising 5-20 mM (e.g., 10 mM) L-methionine.

[0044] In some embodiments, the composition comprises a bulking agent such as mannitol. In specific embodiments, the composition is an aqueous liquid composition comprising 1-10% (e.g., 3.5%) mannitol (w / v).

[0045] In some embodiments, the composition is an aqueous liquid composition containing a buffer, such as an L-histidine buffer. In specific embodiments, the aqueous liquid composition comprises 5-20 mM (e.g., 10 mM) L-histidine.

[0046] In some embodiments, the pH of the buffer is in the range of about 4.0 to about 6.0. In some embodiments, the pH of the buffer is 6.0. In a preferred embodiment, the pH of the buffer is 5.0 + 0.3. In some embodiments, the pH of the buffer is adjusted using sodium hydroxide.

[0047] In some embodiments, the composition is an aqueous liquid composition comprising 40-180 mg / mL (e.g., 50-150 mg / mL) of an Ab1-related antibody (e.g., Ab1); 10-50 mM (e.g., 10-30 mM) acetate, and 1-10% (e.g., 6-8%) w / v sucrose. In some other embodiments, the composition is an aqueous liquid composition comprising 15-40 mg / mL of a monoclonal antibody to TGF-β, 5-20 mM (e.g., 10 mM) L-histidine, 1-10% (e.g., 6-8%) sucrose (w / v), 1-10% (e.g., 3.5%) mannitol (w / v), and 5-20 mM (10 mM) L-methionine. The pH of the aqueous liquid composition can be 4.0-6.0 (e.g., 4.7-5.5).

[0048] In some embodiments, the aqueous liquid composition comprises 0.01-0.07% w / v surfactant(s). Examples of surfactants include nonionic detergents such as polysorbates (e.g., polysorbates 20 and 80) and poloxamers (e.g., poloxamer 188). In some embodiments, the aqueous liquid composition comprises 0.01-0.07% polysorbate 80 (e.g., greater than 0.025% or 0.05-0.06% PS80). In some cases, the presence of surfactant(s) can help reduce turbidity / opalescence of the liquid composition.

[0049] In some embodiments, the aqueous liquid composition comprises 0-50 μM (e.g., 10 μM) of a chelating agent(s), such as EDTA or DPTA.

[0050] In a preferred embodiment, the composition is an aqueous liquid composition comprising 50 or 150 mg / ml of monoclonal antibodies to TGF-β, 25 mM acetate, 10 μM EDTA or DPTA, 0.06% PS80, and 8% w / v sucrose. In specific embodiments, the aqueous liquid composition has a pH of 5+0.3.

[0051] In some embodiments, the composition is an aqueous liquid composition comprising 25 mg / mL of monoclonal antibodies to TGF-β, 10 mM L-histidine, 2% (w / v) sucrose, 3.5% (w / v) mannitol, 10 mM L-methionine, 0.01% (w / v) polysorbate 80, wherein the aqueous liquid composition has a pH of 6.0. In specific embodiments, the composition is an aqueous liquid composition comprising 25 mg / ml of monoclonal antibodies to TGF-β, 1.18 mg / ml of L-histidine monohydrochloride, 0.68 mg / ml of L-histidine, 1.5 mg / ml of L-methionine, 0.1 mg / ml of polysorbate 80, 20 mg / ml of sucrose, and 35.3 mg / ml of mannitol at a pH of 6.0. Ingredients Conc. (mg / ml) Ab1 L-histidine monohydrochloride L-histidine L-methionine Polysorbate 80 Sucrose Mannitol Water for injection 25.0 1.18 0.68 1.50 0.10 20.0 35.3 qs

[0052] Aqueous liquid compositions can be prepared by mixing Ab1 produced using recombinant technology and subsequently purified from host cells with the excipients described herein in water and adjusting the resulting mixture to the desired pH. For example, monoclonal antibodies to TGF-β and the desired excipients can be added to acetate buffer at the desired pH or buffer exchanged with acetate buffer.

[0053] In some embodiments, an aqueous liquid composition can be obtained by diluting the lyophilized composition of the present invention. The dilution can be performed using a pharmaceutically acceptable liquid, such as sterile water, saline (e.g., 0.9% sodium chloride), or acetate-buffered saline.IV. Finished goods

[0054] The compositions of the present invention can be supplied in a finished article (e.g., a kit) that includes instructions for use and, optionally, other therapeutic agents for treating the disorder. The active pharmaceutical ingredient (API) in the article (e.g., Ab1) can be supplied in an amount that can be easily administered according to the dosage regimens described herein.

[0055] For example, the finished article may include a vial that contains 800 mg of Ab1 in 16 ml of an aqueous liquid solution containing 25 mM acetate, 8% sucrose, 10 μM EDTA or DTPA, 0.06% PS80 at a pH of 5.0+0.3. In some embodiments, the vial contains 800 mg of Ab1, 15 mg of acetate, 800 mg of sucrose, and 6 mg of PS80. In some embodiments, the vial contains 800 mg of Ab1, 24 mg of acetate, 1280 mg of sucrose, and 9.6 mg of PS80. In particular embodiments, the vial is a pre-treated glass vial containing a standard closure. For example, the vial may be a Type 1 ISO 20R glass tube vial with a 20 mm West stopper as a closure.

[0056] The composition of the present invention can be stored at a temperature of -3°C to 5°C for two years or more.V. Use of Ab1 and related antibodies

[0057] The TGF-β receptor is widely expressed on immune cells, resulting in a broad range of effects of TGF-β on both the innate and adaptive immune systems. TGF-β has been associated with many disease states, such as developmental defects, cancer, chronic inflammation, autoimmunity, and fibrotic diseases. A therapeutic amount of Ab1 or a related antibody can be used to treat these conditions. A "therapeutically effective" amount refers to the amount of Ab1, a related antibody, or other therapeutic agent mentioned herein that alleviates one or more symptoms of the condition being treated. This amount may vary depending on the condition or patient being treated and can be determined by a healthcare professional using well-known principles.

[0058] A suitable dosage level of the pharmaceutical composition described herein can be determined based on a variety of factors, including the age, weight, disease state, general health and medical history of the patient, as well as the route and frequency of administration of the drug, the pharmacodynamics and pharmacokinetics of the active ingredient Ab1 in the drug and any other drugs that the patient may be taking concomitantly. In some embodiments, Ab1 or a related antibody can be administered at a dose of 40, 20 or 15 mg / kg or less (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mg / kg). In some embodiments, Ab1 can be administered at a dose of 5 mg / kg and 15 mg / kg. The dosing frequency may be, for example, daily, every two, three, four, or five days, weekly, every two weeks, every three weeks, monthly, or every two months. In some embodiments, the dosing frequency is every two weeks.The intervals between successive doses may be two weeks, or less or more than two weeks, if the doctor deems it appropriate.

[0059] The antibody can be administered intravenously (such as by intravenous infusion for 0.5-8 hours), subcutaneously, locally, or by any other administration route that is suitable for the condition and dosage form.

[0060] Ab1 and related antibodies are derived from human antibody genes and thus have low immunogenicity in humans; however, patients may be monitored for adverse events when treated with Ab1 or a related antibody.

[0061] In some embodiments, the efficacy of the antibodies of the present invention may be indicated by one or more of the following in a patient (e.g., in diseased tissue, such as tumor tissue in a patient): (1) a decrease in the level or activity of TGF-β, (2) an increase in the levels of MIP2 and / or KC / GRO, (3) activation or infiltration into tumor tissue of CD8+ T cells, such as INF-γ-positive CD8+ T cells, and (4) an increase in clustering of natural killer (NK) cells.

[0062] Patients may be adults (e.g., patients aged 18 years or older, including elderly patients aged 65 years or older). Patients may be pediatric patients (patients under 18 years of age, such as patients aged newborns to 6 years old, patients aged 6 to 12 years old, or patients aged 12 to 18 years old).

[0063] In some embodiments, an Ab1-based pharmaceutical composition comprising Ab1 at a concentration of 50 mg / mL and containing 25 mM acetate, 8% sucrose, 10 μM EDTA or DPTA, and 0.06% PS80 (pH 5.0+0.3) (e.g., supplied in 10 mL vials) is administered intravenously to patients at a dose of 5 mg / kg or 15 mg / kg every two weeks until the desired therapeutic outcome is achieved. For IV administration, the Ab1-based composition can be diluted with saline or IV dextrose solution (typically containing 5% dextrose in water). For example, PO or PVC IV infusion bags can be used. In some embodiments, the Ab1-based formulation is diluted with saline in PVC bags before use. In some embodiments, the Ab1-based formulation is diluted with a dextrose solution for IV administration in PVC bags prior to use.In some embodiments, the Ab1-based formulation is diluted with saline in PO bags prior to administration. In some embodiments, the Ab1-based formulation is diluted with dextrose solution for IV administration in PO bags prior to administration. Non-oncologic disease states.

[0064] Conditions that can be treated with Ab1 and related antibodies may include, but are not limited to, bone defects (e.g., osteogenesis imperfecta), glomerulonephritis, scarring of nerve tissue or skin, pulmonary fibrosis or pneumofibrosis (e.g., idiopathic pneumofibrosis), radiation-induced fibrosis, liver fibrosis, myelofibrosis, scleroderma, immune-mediated diseases (including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Berger's disease, and transplant rejection), and Dupuytren's contracture.

[0065] They may also be applicable to the treatment, prevention, and reduction of the risk of forms of renal failure, including but not limited to focal segmental glomerulosclerosis (FSGS), diabetic (type I and type II) nephropathy, radiation nephropathy, obstructive nephropathy, diffuse systemic sclerosis, congenital kidney disease (such as polycystic kidney disease, medullary sponge kidney, horseshoe kidney), glomerulonephritis, nephrosclerosis, nephrocalcinosis, systemic or glomerular hypertension, tubulointerstitial nephropathy, renal tubular acidosis, renal tuberculosis, and renal infarction. In particular, they are useful in combination with antagonists of the renin-angiotensin-aldosterone system, including, but not limited to, renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, Ang II receptor antagonists (also known as "Ang II receptor blockers"), and aldosterone antagonists. See, for example, WO 2004 / 098637, the disclosure of which is incorporated herein by reference in its entirety.

[0066] Ab1 and related antibodies are applicable for the treatment of diseases and conditions associated with ECM deposition, such as systemic sclerosis, postoperative adhesions, keloid and hypertrophic scarring, proliferative vitreoretinopathy, surgical drainage of glaucoma, corneal injury, cataract, Peyronie's disease, adult acute respiratory distress syndrome, liver cirrhosis, post-myocardial infarction scarring, post-angioplasty restenosis, post-subarachnoid hemorrhage scarring, post-laminectomy fibrosis, post-tendon and other tissue repair fibrosis, biliary cirrhosis (including sclerosing cholangitis), pericarditis, pleurisy, tracheostomy, penetrating CNS injury, eosinophilic myalgic syndrome, restenosis vascular, veno-occlusive disease, pancreatitis and psoriatic arthropathy.

[0067] In addition, Ab1 and related antibodies are useful in conditions where stimulation of re-epithelialization is beneficial. Such conditions include, but are not limited to, skin diseases such as venous ulcers, venous pressure ulcers (bedsores), diabetic ulcers, graft acceptance sites, transplant donor sites, abrasions and burns, bronchial epithelial diseases such as asthma, ARDS, intestinal epithelial diseases such as mucositis associated with cytotoxic drug treatment, esophageal ulcers (reflux disease), gastroesophageal reflux disease, gastric ulcers, small intestinal and colonic lesions (inflammatory bowel disease).

[0068] In addition, Ab1 and related antibodies are additionally used for conditions requiring endothelial cell proliferation, such as stabilizing atherosclerotic plaques, promoting vascular anastomotic healing, or for conditions requiring suppression of smooth muscle cell proliferation, such as arterial disease, restenosis, and asthma.

[0069] Ab1 and related antibodies are also useful for enhancing the immune response to macrophage-mediated infections, such as those caused by Leishmania spp., Trypanosorna cruzi, Mycobacterium tuberculosis, and Mycobacterium leprae, as well as the protozoan Toxoplasma gondii, the fungi Histoplasma capsulatum, Candida albicans, Candida parapsilosis, and Cryptococcus neoformans. They are also useful for reducing immunosuppression caused by, for example, tumors, AIDS, and granulomatous diseases.

[0070] Ab1 and related antibodies are also useful for the prevention and / or treatment of ophthalmological conditions such as glaucoma and post-trabeculectomy scarring.B. Oncologic disease states

[0071] TGF-β regulates several biological processes, including cell proliferation, epithelial-mesenchymal transition (EMT), matrix remodeling, angiogenesis, and immune functions. Each of these processes contributes to tumor progression. The ubiquitous detrimental role of TGF-β in patients with cancer across a variety of indications is evidenced by its increased levels within the tumor microenvironment as well as systemically. See, e.g., Kadam et al., Mo Biomark Diagn. (2013) 4(3):1-8. Studies have shown that in the malignant setting, TGF-β can induce EMT, and the resulting mesenchymal phenotype leads to enhanced cell migration and invasion.

[0072] Compositions comprising Ab1 and related antibodies are useful in the treatment of hyperproliferative diseases such as cancers including, but not limited to, skin cancer (e.g., melanoma, including unresectable or metastatic melanoma, cutaneous squamous cell carcinoma, and keratoacanthoma), lung cancer (e.g., non-small cell lung cancer), esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), primary peritoneal cancer, bladder cancer, renal cancer or kidney cancer (e.g., renal cell carcinoma), urothelial carcinoma, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, prostate cancer, testicular cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), brain cancer, glioblastoma, glioma, mesothelioma, leukemia and lymphoma.

[0073] In some embodiments, compositions based on Ab1 and related antibodies are useful in the treatment of cancer in patients in whom previous therapy based on a therapeutic agent that is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody has failed or is not expected to work, i.e., patients who do not have a clinical response to a therapy that is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or do not have a response as predicted. In some embodiments, Ab1 and related antibodies are useful in the treatment of cancer in patients who have relapsed with a previous therapy that is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.As used in this document, the term "predictably" means that a medical professional can predict, without administration of the therapy, whether a patient will or will not respond to the therapy and whether the therapy will be ineffective or ineffective, based on his / her general medical knowledge and the specific conditions of the patient.

[0074] In some embodiments, the cancers are mesenchymal subtypes of solid tumors, including, but not limited to, mesenchymal colorectal cancer, mesenchymal ovarian cancer, mesenchymal lung cancer, mesenchymal head cancer, and mesenchymal neck cancer. Epithelial-mesenchymal transition (EMT) stimulates cellular migration and invasive properties by downregulating epithelial cell genes and upregulating mesenchymal gene expression. EMT is a hallmark of tumor progression and invasion. Up to a quarter of colorectal and ovarian cancer cases are mesenchymal. Thus, by downregulating TGF-β and inducing EMT through its action, Ab1 or a related antibody can be used to treat mesenchymal solid tumors. Mesenchymal subtypes of solid tumors can be identified by a variety of genetic markers and pathological tests.Markers include ACTA2, VIM, MGP, ZEB2, and ZWINT, which can be detected using qRT-PCR or immunohistochemistry. These markers can be used to select patients for anti-TGFβ antibody monotherapy or combination therapy according to the present invention.

[0075] In some embodiments, Ab1 and related antibodies are useful in the treatment of patients with advanced solid tumors.

[0076] Compositions containing Ab1 and related antibodies can also be used in the treatment of hematopoietic disorders or malignancies such as multiple myeloma, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, non-Hodgkin's lymphoma and leukemia, as well as various forms of sarcoma such as Kaposi's sarcoma.

[0077] Compositions comprising Ab1 and related antibodies may also be useful for suppressing cyclosporine-mediated malignancy or cancer progression (e.g., metastasis).

[0078] Of course, it should be understood that in the context of cancer therapy, "treatment" includes any medical intervention that results in slowing the growth of cancer, delaying the progression or recurrence of cancer, or reducing the metastasis of cancer, as well as in partial remission of cancer, with the aim of prolonging the life expectancy of the patient.C. Combination therapy in oncology

[0079] The level of cytotoxic T cell infiltration in cancer has been observed to correlate with favorable clinical outcome of treatment (Fridman et al., Nat Rev Cancer(2012) 12(4):298–306; and Galon et al., Immunity(2013) 39(1):11–26). Furthermore, the levels of T helper cells that assist cytotoxic T cells (CD4+ TH1) and the cytokines they produce (e.g., IFN-γ) also often correlate with positive patient outcomes. In contrast, the presence of Treg cells has been shown to correlate with poor patient prognosis (Fridman, supra).

[0080] TGF-β suppresses nearly all aspects of the antitumor immune response. This cytokine stimulates iTreg differentiation and reduces the proliferation and infiltration of cytotoxic (CD8+) cells. Suppressing TGFβ with Ab1 or a related antibody will attenuate the immunosuppressive tumor microenvironment described above, ensuring positive treatment outcomes in cancer patients.

[0081] Furthermore, the present inventors discovered that by weakening the immunosuppressive tumor microenvironment, Ab1 and related antibodies can enable checkpoint modulators, such as anti-PD-1 antibodies, to better induce immune responses. As a result, more patients may benefit from immunotherapy, such as treatment with anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies.

[0082] With or without therapeutic agents targeting immune checkpoint molecules, Ab1 and related antibodies can also be used in combination with other cancer therapies, such as chemotherapy (e.g., platinum-based or taxoid-based therapies), radiation therapy, and therapies targeting cancer antigens or oncogenic factors.

[0083] Cancers that can be treated with a combination comprising an Ab1 or related antibody and an immune checkpoint inhibitor such as an anti-PD-1 antibody include the cancers listed in the subsection above.

[0084] In some embodiments, the cancer forms are refractory to a previous therapy that is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, such as advanced or metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, squamous cell carcinoma of the head and neck, and Hodgkin's lymphoma. Refractory patients are patients whose disease progresses, as confirmed, for example, by radiography, within 12 weeks of starting treatment without any evidence of a response.

[0085] In some embodiments, Ab1 or a related antibody can be used in combination with another cancer therapy, such as anti-PD-1 antibody therapy, to treat mesenchymal cancers such as colorectal cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, head and neck squamous cell carcinoma, renal cell carcinoma, hepatocellular carcinoma, and cutaneous squamous cell carcinoma. See also the discussion above.

[0086] Examples of anti-PD-1 antibodies include nivolumab, pembrolizumab, pidilizumab, MEDI0608 (formerly AMP-514; see, e.g., WO 2012 / 145493 and U.S. Patent 9205148), PDR001 (see, e.g., WO 2015 / 112900), PF-06801591 (see, e.g., WO 2016 / 092419), and BGB-A317 (see, e.g., WO 2015 / 035606). In some embodiments, the anti-PD-1 antibodies include those disclosed in WO 2015 / 112800 (e.g., antibodies referred to as H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, H2M7798N, H4H9019P, H4xH9034P2, H4xH9035P2, H4xH9037P2, H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P and H4xH9008P in Table 1 of the PCT publication, and also referred to as H4H7798N, H4H7795N2, H4H9008P and H4H9048P2 in Table 3 of the PCT publication). The disclosure of WO 2015 / 112800 is incorporated herein by reference in its entirety.

[0087] For example, the antibodies disclosed in WO 2015 / 112800 and related antibodies, including antibodies and antigen-binding fragments having CDR, VH, and VL sequences or heavy and light chain sequences disclosed in such PCT publication, as well as antibodies and antigen-binding fragments binding to the same epitope of PD-1 as the antibodies disclosed in such PCT publication, can be used in combination with Ab1 or a related antibody of the present disclosure for the treatment of cancer. In related embodiments, a useful anti-PD-1 antibody may comprise the heavy and light chain amino acid sequences shown below by SEQ ID NOs: 9 and 10, respectively; the VH and VL sequences of SEQ ID NOs: 9 and 10 (shown in italics); or one or more (e.g., all six) CDRs of SEQ ID NOs: 9 and 10 (shown in boxes).

[0088] In other related embodiments, a useful anti-PD-1 antibody may comprise the heavy and light chain amino acid sequences shown below by SEQ ID NOs: 11 and 12, respectively; the VH and VL sequences of SEQ ID NOs: 11 and 12 (shown in italics), or one or more (e.g., all six) CDRs of SEQ ID NOs: 11 and 12 (shown in boxes). In related embodiments, a useful anti-PD-1 antibody may comprise the heavy and light chain amino acid sequences shown below by SEQ ID NOs: 11 and 12, respectively; the VH and VL sequences of SEQ ID NOs: 11 and 12 (shown in italics), or one or more (e.g., all six) CDRs of SEQ ID NOs: 9 and 10 (shown in boxes).

[0089] In some embodiments, the antibodies of the present invention, such as anti-PD-1 antibodies, lack a C-terminal lysine in the heavy chain. The C-terminal lysine can be removed during manufacturing or by recombinant technology (i.e., the coding sequence of the heavy chain does not include a codon for a terminal lysine at the C-terminus). Thus, the present invention also provides antibodies comprising the amino acid sequence of the heavy chain under SEQ ID NO: 3 without the C-terminal lysine. D. Biomarkers of treatment efficacy

[0090] The efficacy of Ab1 and related antibodies can be determined by biomarkers or target occupancy. For example, in tumor tissues, target occupancy can be analyzed by assessing active TGFβ levels in biopsies using the Meso Scale Discovery (MSD) assay. In blood, target engagement can be analyzed by assessing the effect of reduced circulating TGFβ levels on peripheral blood mononuclear cells, such as lymphocytes (T cells, B cells, NK cells) and monocytes. For example, increased proliferation of circulating CD8+ T cells can be assessed using CD45 + RO + CCR7 + CD28 + Ki67 + as markers in flow cytometry. Activation of circulating NK cells can be assessed using CD3-CD56 high / low CD16 + or CD137 + as markers in flow cytometry. In addition, Ki-67, PD-1, and ICOS can be used as markers of PD associated with T cell activation.

[0091] Immune modulation following treatment with Ab1 or a related antibody can be analyzed by assessing changes in the levels of infiltrating immune cells and immune markers using multiplex immunohistochemistry (IHC) assays, such as those available on the NeoGenomics platform. Specifically, NeoGenomic's MultiOmyx TIL panel provides staining for a panel of immune markers, allowing for quantification of the density and localization of various immune cells. Immune markers can indicate iTreg differentiation; CD8 infiltration and proliferation. + T cells; as well as the production of IFNγ in CD8 + T cells. Ab1 has been shown to suppress CD4 differentiation. + T cells in iTreg (see, e.g., Example 3 in US Patent Application Publication No. US2018 / 0244763), and also increases CD8 proliferation +T cells and their IFNγ production (as demonstrated by the mixed lymphocyte response assay; data not shown). Thus, the efficacy of treatment with Ab1 or a related antibody may be indicated by the suppression of iTreg, induction of CD8 proliferation + T cells and their infiltration into the tumor or other affected tissues, increased IFNγ production and / or increased CD8 ratio + T cells to Treg cells. Immune modulation by treatment with Ab1 or a related antibody can also be analyzed in peripheral blood by quantifying immune cells representing CD8 + T cells, Treg cells, NK cells, and other immune cells, based on methylation-sensitive PCR. Treatment efficacy may manifest clinically as a delay or reversal of disease progression, such as tumor progression.

[0092] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Illustrative methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. In case of conflict, the present description, including definitions, will prevail. In general, the nomenclature used in connection with the techniques of neurology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein is well known and widely used in the art. Enzymatic reactions and purification procedures are performed in accordance with the manufacturer's instructions, as generally practiced in the art, or as described herein.Furthermore, unless the context otherwise requires, singular terms will include the plural, and plural terms will include the singular. Throughout this description and embodiments, the words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," should be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other literature referenced herein are incorporated by reference in their entirety. Although a number of documents are cited herein, such citation does not constitute an admission that any of these documents form part of the common knowledge in the art.As used herein, the term "about" or "approximately" with respect to one or more values ​​of interest refers to a value that is similar to the stated reference value. In certain embodiments, the term refers to a range of values ​​that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or lesser) of the stated reference value, unless otherwise stated or otherwise apparent from the context.

[0093] For a better understanding of the present invention, the following examples are set forth. These examples are intended for illustrative purposes only and should in no way be construed as limiting the scope of the present invention. EXAMPLES

[0094] The following examples describe studies evaluating various Ab1 formulations to develop formulations with superior biological activity and long-term stability. The present inventors evaluated the effects of buffer type and pH on the physical and chemical stability of Ab1-based liquid formulations during refrigerated storage, accelerated storage, and storage under stress conditions. For this study, acetate and histidine buffer systems were selected, with and without the addition of sodium chloride.

[0095] The present inventors evaluated the optimal concentrations of polysorbate 80 (PS80) required to stabilize Ab1-based liquid formulations under various storage temperatures, freeze-thaw cycling, and agitation stress.

[0096] The present inventors also evaluated the physical stability of the Ab1-based drug formulation (DP) after dilution and incubation for up to 48 hours at room temperature in intravenous (IV) infusion bags. The DP was diluted to 0.5 mg / mL and 1.0 mg / mL. Both concentrations were evaluated in the following bag combinations: saline in polyvinyl chloride (PVC) bags, saline in polyolefin (PO) bags, dextrose in PVC bags, and dextrose in PO bags. The optimal concentration of PS80 in liquid DP was also examined for its ability to protect Ab1 after dilution.

[0097] The present inventors further investigated the effect of transition metals on the chemical and physical stability of the protein. Transition metals sometimes leach into the drug substance (DS) during manufacturing. EDTA and DTPA were evaluated for their ability to chelate and protect the protein during experiments under the most stringent conditions.

[0098] To ensure that the proposed target formulation base is suitable for stabilizing high-concentration DS and lower-concentration DP solutions, the present inventors evaluated the stability of the solutions during freeze-thaw cycles, as well as under frozen and liquid storage conditions, and over the entire concentration range for all excipients, including the API.

[0099] The materials and methods of the experiments are as follows. Medicinal substance

[0100] Drug substance (DS) with high Ab1 concentration was prepared using ultrafiltration / diafiltration (UFDF) methods. DS concentration was typically prepared at a level of no more than 180 mg / mL. Results from the UFDF simulator were used to adjust the concentration of buffer salts, which may accumulate or be depleted during UF process steps due to the Donnan effect. After formulating drug substance (DP) samples at the target protein and excipient concentrations, the formulations were then clarified using 0.22 μm filters under laminar flow before filling under aseptic conditions. Visible particulate matter control

[0101] Visible particles were analyzed under a visual inspection device. Before inspection, DP vials were wiped with optical glass cleaning paper to remove dust and fingerprints from the outer surface.

[0102] The pH of the buffers and formulated mAb solutions was measured using a Thermo-Scientific™ pH probe and pH meter. Results were considered comparable when the difference between duplicate measurements was within 0.1 pH units. Osmolality

[0103] Osmolality measurements were performed on 20 µL samples (n=2 or 3) using a freezing point depression osmometer (Advanced Instruments, OsmoPRO). Testing with osmolality standards was performed before and after sample analysis to ensure measurement accuracy.Total protein concentration

[0104] Total protein concentration was determined by measuring ultraviolet (UV) absorbance at 280 nm using variable beam path technology using the SoloVPE system from C technologies. Measurements were performed on 20 μL of sample (n = 2 or 3). Total protein concentration was also determined by measuring UV absorbance at 280 nm on microfluidic chips using the Big Lunatic system from Unchained Labs. Measurements were performed in duplicate on 2-5 μL of sample. DSC for conformational and thermal stability analysis

[0105] Differential scanning calorimetry (DSC) was performed on a Malvern Microcal calorimeter with a linear temperature increase from 15°C to 105°C at a heating rate of 0.5°C / min. Protein solutions were measured at a protein concentration of 1 mg / mL. To analyze and determine the thermal unfolding temperature of the protein (T m ) OriginPro software was used. Turbidity and optical density of the solution

[0106] Sample turbidity was quantified by measuring the optical density (OD) from 340 nm to 360 nm using a SpectraMax® i3 microplate reader from Molecular Devices. 200 µL of each sample was loaded into a transparent 96-well UV-Vis plate. OD was determined as the average of the absorbance values ​​at 340 nm, 345 nm, 350 nm, 355 nm, and 360 nm. Size-exclusion HPLC for high molecular weight compound analysis

[0107] Protein aggregates (high-molecular-weight species or HMWS) were analyzed using size-exclusion chromatography (SEC). Samples were analyzed on a 1260 Series HPLC System (Agilent, Santa Clara, CA, USA) equipped with a TSK-GEL® G3000SWXL analytical column (Tosoh Bioscience, Tokyo, Japan) and a corresponding guard column. The mobile phase used was 40 mM phosphate and 150 mM sodium chloride, pH 7.2, at a flow rate of 0.5 mL / min for 30 minutes. Three injections were performed for each sample. Detection was performed using UV absorption at 280 nm and chromatographic peaks were integrated to determine the relative percentage of each eluted species. Microflow imaging (MFI) for analysis of particles not visible to the naked eye

[0108] Invisible particles were analyzed using the Protein Simple MFI™ model DPA-4200. The system was thoroughly rinsed with 0.22 µm-filtered and degassed MilliQ® water before measuring 2, 10, and 25 µm standards. Samples (n=1 or 2) were analyzed using the 1 mL method at a flow rate of 0.17 mL / min. A light source was illuminated as the sample flowed through the flow cell, and a camera rapidly captured images as the sample passed through the flow cell. Particles were identified using the MFI™ software, which then calculated the size, opacity, and morphology of each individual particle. High-precision liquid particle counter for analyzing invisible particles

[0109] Invisible particles were also measured using the photometric counting method on a Hach® Model 9703+ High Accuracy Liquid Particle Counting (HIAC) instrument. The system was rinsed with 0.22-μm-filtered, degassed MilliQ® water until particle counts were less than 20 particles / mL. Standards of 2 μm, 10 μm, and 25 μm were measured to ensure accurate particle counts, followed by thorough rinsing to remove any background. Using the 1-mL protocol, samples were measured using five separate injections of 0.2 mL. The first sample measurement was discarded, and the values ​​for the next four measurements were averaged. Capillary isoelectric focusing for analysis of charge-differing variants

[0110] Protein charge heterogeneity was measured by capillary isoelectric focusing (cIEF) using a ProteinSimple iCE3 instrument using UV absorbance at 280 nm. Samples (1 mL) and standards were diluted to 2.5 mg / mL in water. The built-in mixing function was used to mix the samples and master mix before analysis. Isoelectric focusing of the samples involved a 1-minute pre-focusing period at 1500 V, followed by 10 minutes of focusing at 3000 V. Detection spanned 5 exposures, and sample loading lasted 55 seconds for each formulation. Results were considered comparable if the difference was 10% or less. LC-MS quantification of P™

[0111] Protein samples were diluted to 2 mg / mL, vortexed, and 40 μg of protein were used for automated digestion. The digestion buffer was 25 mM Tris, pH 8.5. For each sample, 15 μL of the digested sample (containing approximately 5 μg of protein) was injected onto a C18 column for LC-MS analysis. Samples were analyzed using the LC-MS / MS method with DDA of the 8 most abundant ions using Q Exactive™.

[0112] LC-MS / MS data collected with Q Exactive™ were processed with BioPharma Finder™ 3.0 on the WLSD58 server to identify and quantify modifications, including Met / Trp oxidation, deamidation, Asp isomerization, and C-terminal HC modification. For low-level modifications that were unable to generate MS / MS spectra suitable for identification by BioPharma Finder™, peptide mapping using MS alone was used to distribute peptides. All data were then processed using Progenesis to obtain relative peptide abundances after retention time alignment and peak sorting. Some deamidated and isomerized peptides required manual adjustment of peak sorting when Progenesis was unable to perform this task correctly. Activity

[0113] When TGF-β is incubated with mink lung cells, it inhibits cell proliferation. Ab1 is an anti-TGFβ antibody that, when bound to TGF-β, inhibits TGF-β binding to TGF-β receptors on the cell surface, thereby promoting cell proliferation. In the Ab1 activity assay, varying levels of Ab1 were incubated with TGF-β2 and then added to mink lung cells.

[0114] Cells were incubated with Ab1 and TGF-β2 for three days, and then PrestoBlue™ reagent was added. PrestoBlue™ contains a cell-permeable, non-fluorescent compound, resazurin, which is metabolized and reduced by living cells, resulting in the formation of a fluorescent product, resorufin. Therefore, cell proliferation directly correlates with the intensity of the fluorescent signal. Five hours after the addition of PrestoBlue™, fluorescence was measured using a plate reader. Log transformation was performed in biological analysis software. 10 and fitted to a four-parameter model. Once the curves for the standard and sample were determined to be suitable, the curves were constrained, and the final analytical result was determined as the EC ratio 50 standard divided by EC 50of the test sample and recorded as a percentage of relative activity (%RP). Example 1. Screening for the evaluation of buffers and pH

[0115] This example describes experiments in which various buffers and pH conditions were screened to identify suitable formulations for Ab1. Because liquid drug products are more convenient for preparation and administration in both clinical and home settings (compared to lyophilized drug products), various liquid aqueous buffers were tested. Table 1 presents the formulation parameters and sample codes used in this study.Table 1. Buffer Parameters Link Parameter Ab1 (mg / ml) Ace_4.7 pH 4.7, 20 mM acetate 80 Ace_5.0 pH 5.0, 20 mM acetate 80 Ace_5.3 pH 5.3, 20 mM acetate 80 Ace_5.5 pH 5.5, 20 mM acetate 80 Ace-NaCl_5.5 pH 5.5, 20 mM acetate, 25 mM NaCl 80 Hist-NaCl_5.5 pH 5.5, 10 mM histidine, 25 mM NaCl 80 Hist_5.5 pH 5.5, 10 mM histidine 80 Hist_6.0 pH 6.0, 10 mM histidine 80 Hist_6.5 pH 6.5, 10 mM histidine 80

[0116] Opalescence is a visual manifestation of protein-protein interactions involving attraction. Ab1-based formulations were visually observed to exhibit significant pH-dependent opalescence (Fig. 1). At pH less than 5.3, solutions were almost always clear and translucent, but at pH >5.3, opalescence increased with increasing pH. Acetate formulations were generally characterized by less opalescence than histidine formulations (Fig. 1). These visual images indicate that the optimal pH of the formulation is in the range of 4.7-5.0 using acetate as a type of buffer.

[0117] After no more than 4 weeks of storage at 5°C and 25°C, no significant change in % high molecular weight species (HMWS) was observed for the solutions in Table 1. However, an approximately 0.5% increase in % HMWS content was observed for all formulations after 4 weeks of storage at 40°C. This change was not particularly significant given the stress conditions. Furthermore, the addition of sodium chloride to the formulations did not significantly affect the degree of protein aggregation or the % HMWS content.

[0118] Regarding the invisible particles, HIAC particle counting showed that the growth of invisible particles responded to the type of buffer and the pH of the solution (Figs. 2A-2C). Acetate formulations buffered at pH 4.7 and 5.0 resulted in the lowest particle numbers over time compared to any other pH acetate formulation and all histidine formulations studied. However, no clear or distinct effect of temperature on particle growth was observed for any parameter.

[0119] The acetate and histidine formulations exhibited comparable viscosity values ​​at T0 and after 4 weeks at 40°C for any given pH with or without the addition of sodium chloride (Fig. 3). All measured viscosity values ​​were within 2-3 cP, which is well below any limits that may pose problems during DP administration to patients or during manufacturing processes. There were also no significant changes in viscosity values, indicating that the tested formulations did not undergo significant chemical degradation. There was also no significant change in pH for any formulation over the 4-week period after storage at the three temperatures (Figs. 4A and 4B). These results indicate that acetate and histidine did not undergo any significant chemical degradation and retained their ability to perform buffering function even after nearly 4 weeks of storage under stress conditions at 40°C.

[0120] UV plate measurements were performed to monitor any changes in turbidity and opalescence that may have occurred over time (Fig. 5). The same pH-dependent opalescence observed during visual inspection earlier at T0 was detected using spectrophotometry. The measured OD values ​​increased with pH, ​​with the histidine formulations being more turbid than the acetate formulations. For most formulations, a slight increase in OD was observed after 4 weeks of storage at 25°C and 40°C. The increase in OD over time was due to the formation of non-visible particles. The results confirm that a pH of approximately 4.7–5.0 results in the formation of the fewest non-visible particles.

[0121] Osmolality values ​​of the buffer alone and buffered protein solutions were compared (Table 2). Osmolality was slightly higher in the acetate formulations than in the histidine formulations. The addition of protein increased osmolality for all formulations. All osmolality values ​​were acceptable, considering that DP is added to the IV infusion bag only after dilution. Table 2. Osmolality values ​​of the buffer alone and buffered Ab1 solutions Sample Osmolality (mOsm / kg) Buffer separately (two repeats.) 80 mg / ml Ab1 Ace_4.7 24,5 92 Ace_5.0 28 40 Ace_5.5 32 46 Ace-NaCl_5.5 79,5 101 His-NaCl_5.5 71,5 74 Hist_5.5 13,5 26 Hist_6.0 11,5 21 Hist_6.5 9 15

[0122] The chemical stability of Ab1 was similar for the acetate and histidine formulations, as demonstrated by comparing the amounts of acidic isoforms and the percentages of monomers. A slight increase in the relative amounts of acidic isoforms formed was observed as the solution pH approached 6.0 and 6.5 for the histidine formulations after 4 weeks at 40°C.

[0123] All formulations had similar relative activity of approximately 1.0, indicating that they are all acceptable. These activity evaluation results are consistent with the previous aggregation and HMWS % content evaluation results, as all HMWS % content values ​​were low (<2%), and therefore the activity is not expected to change significantly. Example 2. Screening for Surfactant Evaluation

[0124] This example describes experiments evaluating the addition of the surfactant polysorbate 80 (PS80) to Ab1-based formulations. The formulation parameters and sample codes used in these experiments are shown in Table 3 below.Table 3. Formulations with Polysorbate 80 Compound Source PS80 Link PS80 Ab1 Buffer pH 1 A PS80_CSR_0% 0% 150 mg / ml 20 mM acetate + 8% sucrose 5,0 2 PS80_CSR_0.025% 0,025% 3 PS80_CSR_0.05% 0,05% 4 PS80_CSR_0.1% 0,1% 5 B PS80_ChP_0.05% 0,05% 150 mg / ml 20 mM acetate + 8% sucrose 5,0 6 PS80_ChP_0.1% 0,1%

[0125] Turbidity and optical density (OD; at 340-360 nm) values ​​of the plate formulations were assessed for formulations stored for one week at 5°C or 40°C, or after 48 hours of vigorous agitation. The data show that for formulations containing PS80, OD values ​​did not change after 1 week of storage at either 5°C or 40°C, or after 48 hours of agitation. There were also no observable differences across varying concentrations of 0.025%, 0.05%, and 0.1% PS80 using two different commercial PS80 sources (A and B). However, OD values ​​did decrease slightly over time for formulations without added surfactant when stored at both 5°C and 40°C. Slightly higher turbidity was also observed for formulations without surfactant. These observations indicate that PS80 had a solubilizing effect on Ab1 protein.

[0126] Levels of small soluble aggregates were monitored by SEC under different storage conditions and interfacial stress (Fig. 6). Storage temperature had only a minor effect, resulting in a 0.5% increase in aggregation after almost 2 weeks of storage; there was no detectable effect of PS80 concentration on aggregation levels. The most severe conditions, performing freeze-thaw cycles from -30°C to room temperature no more than 10 times, also had no negative effect on protein stability (Fig. 6, lower right panel).

[0127] On the other hand, vigorous stirring or shaking had a dramatic effect on aggregation (Figure 6, lower left panel). Formulations without added surfactant formed up to 8% HMWS after 48 hours of stirring. These aggregation levels were highly responsive to the presence and concentration of surfactant. PS80 concentrations as low as 0.025% were sufficient to reduce aggregation levels to less than 2% of those in PS80-free formulations. Increasing the PS80 concentration even above 0.05 or 0.1% only slightly further reduced the HMWS content. These data indicate that 0.05% can be used as a lower limit for the PS80 concentration intended to stabilize Ab1-based formulations.

[0128] The above SEC data indicate that Ab1 is not at high risk of aggregation under most of the conditions studied in this section. Therefore, HIAC was a key assay to monitor larger soluble and insoluble aggregates filtered by SEC, which may be in the range of particles not visible to the naked eye. HIAC data show that under any given storage or interfacial stress conditions, formulations that do not contain PS80 formed significantly more particles not visible to the naked eye. Furthermore, the data show that PS80 had a concentration-dependent effect on reducing particle counts in Ab1-based formulations. PS80 concentrations as low as 0.025% were sufficient to initiate particle count reduction. Increasing PS80 concentrations to 0.05% and above continued to reduce particle counts.

[0129] The pH values ​​of the formulations were measured at T0 and after 2 weeks of storage. There were no significant changes in pH for any formulation at any time or temperature. There was also no effect on the pH of the formulation related to the PS80 concentration. PS80 concentrations did not induce any changes in the ability of the acetate buffer to maintain the pH of Ab1-based formulations at a concentration of 150 mg / mL.

[0130] Across all studies in this example, there appeared to be no effect of PS80 source on the stability of the formulation for any of the test conditions.Example 3. Dilution Study in IV Bags

[0131] This example describes experiments that tested the stability of Ab1-based formulations in different IV bags. The formulation parameters used in these experiments are shown below in Table 4. Table 4. Formulations and Sample Codes for Dilution Studies in IV Bags PO Diluted PS80 (%) PO_dextrose PO_salt solution 0.5 mg / ml Ab1 1.0 mg / ml Ab1 0.5 mg / ml Ab1 1.0 mg / ml Ab1 0,0010% PO_D_0,5_a PO_D_1,0_a PO_S_0,5_a PO_S_1,0_a 0,0005% PO_D_0,5_b PO_D_1,0_b PO_S_0,5_b PO_S_1,0_b 0,0003% PO_D_0,5_c PO_D_1,0_c PO_S_0,5_c PO_S_1,0_c 0,0002% PO_D_0.5_d PO_D_1,0_d PO_S_0,5_d PO_S_1,0_d PVC Diluted PS80 (%) PVC_dextrose PVC_saline solution 0.5 mg / ml Ab1 1.0 mg / ml Ab1 0.5 mg / ml Ab1 1.0 mg / ml Ab1 0,0010% PVC_D_0,5_a PVC_D_1,0_a PVC_S_0,5_a PVC_S_1,0_a 0,0005% PVC_D_0,5_b PVC_D_1,0_b PVC_S_0,5_b PVC_S_1,0_b 0,0003% PVC_D_0,5_c PVC_D_1,0_c PVC_S_0,5_c PVC_S_1,0_c 0,0002% PVC_D_0,5_d PVC_D_1,0_d PVC_S_0,5_d PVC_S_1,0_d

[0132] Before IV infusions, DP is diluted in IV bags. Without sufficient surfactant concentrations in the DP, protein molecules may be adsorbed by the bag, depending on the type of material the IV bag is made of. As a result, the diluted DP in the IV bag may have a lower API concentration than intended for the dose administered to the patient.

[0133] The present inventors monitored the adsorption properties and physical stability of Ab1 diluted in bags added and coated with different concentrations of PS80. PS80 was diluted by adding to concentrations of 0.001%, 0.0005%, 0.0003%, and 0.0002% to determine the optimal concentration required in the original DP formulation. The above PS80 concentrations correspond to 50- to 300-fold dilutions of the PS80 concentrations in the original DP. Next, 150 mg / mL of PS80-free DP was diluted to 0.5 or 1.0 mg / mL. The effects of dilution of formulations in four different combinations of IV bag materials and dilution solutions were assessed: saline in PVC bags, saline in PO bags, dextrose in PVC bags, and dextrose in PO bags.

[0134] The diluted formulations were then incubated and measured after 24 and 48 hours. The data show that the saline solution in the PO and PVC bags did not significantly affect protein adsorption at any of the added surfactant concentrations studied (Figure 7A). However, when dextrose was used as a diluent, the effect on adsorption was slightly dependent on the bag material. In particular, the combination of dextrose and the PVC bag caused appreciable protein adsorption as the PS80 concentration decreased (Figure 7A). The results directly impact the recommendations that will be provided for the administration of DP.

[0135] The formation of subvisible particles (>10 μm) after DP dilution was highly dependent on the type of diluent used. Saline-based solutions produced higher levels of particles than dextrose-based solutions (Fig. 7B). This was independent of the PS80 concentration used in the formulation. There was no significant difference in the number of subvisible particles between the 0.001% and 0.003% PS80 formulations. The IV bag material did not appear to have a significant effect on particle formation, and particle numbers were comparable.

[0136] Aggregation was also assessed in diluted DP samples after incubation in dextrose / saline combinations and PO and PVC pouches. Protein diluted in saline pouches formed slightly higher HMWS % than in dextrose pouches. PO pouches also formed more aggregates than PVC pouches. A concentration-dependent dependence of protein stability was also observed, with dilutions with concentrations less than 0.5 mg / mL generally exhibiting higher levels of aggregation than dilutions with concentrations of 1.0 mg / mL. However, PS80 concentration did not significantly affect protein stability. Example 4. DS and DP Stability

[0137] This example describes studies to evaluate the long-term stability of a drug substance (DS) and a drug product (DP). Table 5 shows the test formulations, where sucrose was used as a cryoprotectant, PS80 was used as a surfactant, and DTPA was used as a chelating agent. Table 5. Formulations for the Long-Term Stability Test Serial number Ab1 (mg / ml) Buffer (pH 5.0) % sucrose %PS80 DTPA (μM) D_1 50 20 mM acetate 8 0,06 50 D_2 100 20 mM acetate 8 0,06 50 D_3 135 20 mM acetate 8 0,06 50 D_4 150 20 mM acetate 8 0,06 50 D_5 165 20 mM acetate 8 0,06 50

[0138] Given these data for 20 mM acetate buffers containing 50 μM DTPA, it was determined that formulations of UF / DF-purified Ab1 containing 25 mM acetate, which would provide the same pH range, and 10 μM EDTA would exhibit similar solution properties. Based on this, we tested this preliminary proposed target formulation for DS and DP: 25 mM acetate, 8% sucrose, 0.06% PS80, 10 μM chelating agent (DTPA was tested, but EDTA is equivalent), pH 5.0 (Table 5). The ability of this formulation base to stabilize Ab1 at concentrations that cover the concentration range of DS and DP was assessed for storage stability. After 3 months of storage at 5°C or 25°C, there was no significant change in % HMWS content in any of the formulations (Fig. 8A).In the 40°C group, no more than approximately 0.5% increase was observed; however, this increase was considered insignificant under these types of accelerated storage conditions and reflects the overall ability of the proposed formulation base to optimize stability and shelf life.

[0139] Ab1-based formulations were also frozen at -80°C and then stored at -20°C. After 6 months of storage under these conditions, there was no significant change in the % HMWS content (Fig. 8B). The % low molecular weight compounds (LMWS) content was also monitored by SEC, and there were no significant changes in fragmentation observed in any formulation or under any storage condition. Furthermore, each formulation was within USP specifications. <787> for particles invisible to the naked eye, both 10 and 25 microns in size.

[0140] There were also no significant changes in the chemical stability of Ab1 under any condition. In conclusion, the proposed formulation base has the ability to stabilize the range from the lowest effective concentration of DP, which is 50 mg / mL, to the highest effective concentration of DS, which is 165 mg / mL. Example 5. Study of metal addition and chelating agent compatibility

[0141] During the manufacture of biological products, there is a small risk of contamination of DS and DP with transition metals. If contamination is present, transition metals can lead to chemical instability and aggregation in liquid solution. This example describes studies that assessed the effect of metal and chelating agents on Ab1-based formulations. Table 6 shows the formulations used for metal addition testing. Table 6. Formulations for Metal Addition Testing Serial number Ab1 (mg / ml) Buffer (pH 5.0) % sucrose %PS80 DTPA (μM) Fe (ppb) Cu (ppb) M_1 150 20 mM acetate 8 0,06 - - - M_2 150 20 mM acetate 8 0,06 - - 500 M_3 150 20 mM acetate 8 0,06 50 - 500 M_4 150 20 mM acetate 8 0,06 - 500 - M_5 150 20 mM acetate 8 0,06 50 500 -

[0142] As shown in the table, iron and copper were added to Ab1 samples with and without the addition of the chelating agent DTPA. Without the addition of the chelating agent, significant oxidation and aggregation of M252 was observed in the iron-added samples; however, the copper-added samples also showed increased oxidation and aggregation, but to a lesser extent (Fig. 9). However, in the presence of DTPA, a significant decrease in protein degradation was observed.

[0143] The present inventors also tested another chelating agent, EDTA. To evaluate storage stability, 10 μM DTPA or 10 μM EDTA were added to Ab1-based formulations and compared. The data show that these two chelating agents provided similar levels of protection for protein molecules in solution. These chelating agents selectively reduced the extent of metal-induced particle formation or aggregation. Example 6. Stability of Formulations to Changes in API and Excipient Concentration Ranges

[0144] This example describes studies evaluating the stability of Ab1-based formulations across ranges of antibody and excipient concentrations. The formulations used for the studies are shown in Table 7 below.Table 7. Formulations for API and Excipient Concentration Range Studies pH according to Ref pH Ab1 (mg / ml) Acetate (mM) % sucrose %PS80 DTPA (μM) 4,7_F1 4,7 40 16 9,6 0,045 50 4,7_F2 4,7 40 24 6,4 0,075 50 4,7_F3 4,7 40 24 9,6 0,060 40 4,7_F4 4,7 57,5 16 6,4 0,045 40 4,7_F5 4,7 75 16 8,0 0,075 50 4,7_F6 4,7 75 20 9,6 0,075 40 4,7_F7 4,7 75 24 6,4 0,045 45 5,0_F1 5,0 40 16 6,4 0,075 40 5,0_F2 5,0 57,5 20 8,0 0,060 45 5,0_F3 5,0 75 24 9,6 0,045 50 5,3_F1 5,3 40 16 9,6 0,075 45 5,3_F2 5,3 40 20 6,4 0,045 50 5,3_F3 5,3 40 24 8,0 0,045 40 5,3_F4 5,3 57,5 24 9,6 0,075 50 5,3_F5 5,3 75 16 6,4 0,060 50 5,3_F6 5,3 75 16 9,6 0,045 40 5,3_F7 5,3 75 24 6,4 0,075 40

[0145] The present inventors assessed aggregation by analyzing HMWS into dimer, trimer, and tetramer subspecies. This made it possible to directly monitor the effects of variations in the formulation components on the aggregation characteristics. The formulations with pH 4.7 and pH 5.3 were the most stable and did not form large amounts of HMWS; moreover, the predominant HMW species was the dimer (Fig. 10). The formulations with pH 5.0 formed approximately 0.6% more aggregates; the dimer levels were comparable to those at pH 4.7 and 5.3, and the 0.6% was mainly the trimer species. However, the overall HMWS level in % was acceptable.

[0146] There was no significant change in the number of non-visible particles (10 and 25 μm) among the different formulations, either with time or with storage temperature (data not shown). There was also no significant change in the number of non-visible particles (10 and 25 μm) after agitation or freeze-thaw stress (data not shown).

[0147] In conclusion, acetate-buffered formulations provided optimal physical and chemical stability compared to histidine. The number of particles not visible to the naked eye responded strongly to the type of buffer compound and pH, with acetate formulations characterized by lower pH (4.7 and 5.0) producing the lowest particle numbers. Turbidity and opalescence were also pH-dependent; formulations were significantly less opalescent under lower pH conditions in acetate, indicating fewer protein-protein interactions involving attraction at lower pH. Less opalescent solutions were also shown to have shorter processing times during UFDF operations, an important manufacturing consideration necessary for obtaining highly concentrated drug substances.

[0148] The addition of PS80 to the final formulation was necessary to reduce the degree of aggregation and, in particular, the risk of particle formation, which has been shown to be accelerated by interfacial stress, such as agitation or freeze / thaw cycles. PS80 was also effective in reducing or attenuating the degree of protein adsorption on IV infusion components (e.g., IV bags). PS80 concentrations > 0.05% provided optimal stability.

[0149] Under accelerated storage conditions, significant levels of oxidation and moderate levels of aggregation were observed in samples with added metals. Metal-based chelating agents were added to the formulation to provide protection against potential metal-induced protein and excipient degradation. 10 μM EDTA and 10 μM DTPA provided comparable levels of protection and resulted in similar changes in HMWS, oxidation, and PS80 concentrations.

[0150] It was found that 8% sucrose, equivalent to about 80 mg / ml, constitutes a ratio suitable for protecting the concentrations of both DP and DS, which are 50 mg / ml and 150 mg / ml, respectively.

[0151] The results of these stability studies demonstrate the stability of the target formulation required to stabilize frozen DS as well as liquid DP under a variety of storage conditions and other stress conditions.Example 7. Alternative formulation based on Ab1 [powder for preparation of infusion solution]

[0152] The additional illustrative formulation based on Ab1 is a sterile, lyophilized product. The drug is filled into a USP Type 1 borosilicate glass vial at 10.3 mL per vial with a 0.3 mL excess. The vial is closed with a silicone-coated gray butyl rubber stopper and hermetically sealed with an aluminum seal and a cap with a tear-off tab. Table 8 provides information on the illustrative composition of the Ab1-based drug.

[0153] For administration, the contents of each vial are diluted with 9.7 ml of water for injection to obtain a protein concentration of 25 mg / ml in an aqueous solution containing 10 mM L-histidine, 2% (w / v) sucrose, 3.5% (w / v) mannitol, 10 mM L-methionine, 0.01% (w / v) polysorbate 80, pH 6.0 at 22°C (Table 8).

[0154] ​​Screening for excipient evaluation revealed that 2% sucrose reduced the degree of aggregate formation during freezing and thawing. The bulking agent, mannitol, did not significantly affect protein stability but contributed to the production of an excellent lyophilized mass. Table 8. Alternative illustrative formulation for Ab1 Medicinal product, 25 mg / ml 10 mM L-histidine-HCL 2% (w / v) sucrose 3.5% (w / v) mannitol 10 mM L-methionine 0.01% PS80 pH 0.6 --->SEQUENCE LIST <110> GENZIM CORPORATION <120> TGF-beta antibody-based formulations and their use <130> 022548.WO064 <140> <141> <150> 63 / 212,473 <151> 2021-06-18 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 447 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic polypeptide <400> 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Asn20 25 30Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45Gly Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Thr Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Ser Thr Leu Gly Leu Val Leu Asp Ala Met Asp Tyr Trp Gly Gln100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr AlaAla130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu260 265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro340 345 350Pro Ser GlnGlu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys435 440 445 <210> 2 <211> 215 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic polypeptide <400> 2Glu Thr Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Ser20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Asp SerPro85 90 95Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val180 185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys195 200 205Ser Phe Asn Arg Gly Glu Cys210 215 <210> 3 <211> 5 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic peptide <400> 3Ser Asn Val Ile Ser1 5 <210> 4 <211> 17 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic peptide <400> 4Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe Lys1 5 10 15Gly <210> 5 <211> 11 <212> PROTEIN <213> Artificialsubsequence <220> <223> Description of the artificial sequence: synthetic peptide <400> 5Thr Leu Gly Leu Val Leu Asp Ala Met Asp Tyr1 5 10 <210> 6 <211> 12 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic peptide <400> 6Arg Ala Ser Gln Ser Leu Gly Ser Ser Tyr Leu Ala1 5 10 <210> 7 <211> 7 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic peptide <400> 7Gly Ala Ser Ser Arg Ala Pro1 5 <210> 8 <211> 9 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic peptide <400> 8Gln Gln Tyr Ala Asp Ser Pro Ile Thr1 5 <210> 9 <211> 444 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic polypeptide <400> 9Glu Val Gln Leu Leu Glu Ser Gly Gly Val Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe20 25 30Gly MetThr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ser Gly Ile Ser Gly Gly Gly Arg Asp Thr Tyr Phe Ala Asp Ser Val50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Gly Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Lys Trp Gly Asn Ile Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser180 185 190Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn195 200 205Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro210 215 220Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val245 250 250 255Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro275 280 285 285Arg Glu Glu Glu P He Sern Sern Val Ser Val Leu Thr290 295 300Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Pro Lys Thr Ile Ser Lys Ala325 330 Gly Glyn Pro Glu3 Gln Val Tyr Thr Leu Pro Pro Ser Gln340 345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Glyn Pro37 370 Thr Asn Tyr Asn 08 Lyn Thr Pro Pro Val Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 4040 234 Thr Lyr Lys Ser Leu Ser Leu Ser Leu Gly Lys435 440 <210> 10 <211> 214 <212> PROTEIN <213> Artificial sequence <220> <223> Descriptionartificial sequence: syntheticpolypeptide <400> 10Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ala Ser Val Gly1 5 10 15Asp Ser Ile Thr Ile Thr Cys Arg Ala Ser Leu Ser Ile Asn Thr Phe20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Leu Pro 40 Leu I 45 I Ala Ser Ser Ser Leu His Gly Gly Val Pro Ser Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Thr Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Pro Tyr Cys Gln Gln Ser Ser Asn Thr Phe 85 Gly Gly 95 Gly Phe 95 Thr Val Val Asp Phe Arg Arg Thr Val Ala Ala100 100 105 110Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly115 120 125Thr Ala Ser Val Cys Val Leu Asn Asn Phe Pro Arg Glu Ala130 130 130 130 130 130 135 130 130 130 130 135 Gl Val Gl Val Glyp Alan Val Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 18 018 Cys Val Thr His Gln Gly Leu Ser Ser Pro Val ThrLys Ser195 200 205Phe Asn Arg Gly Glu Cys210 <210> 11 <211> 448 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic polypeptide <400> 11Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln165 170175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu340 345 350Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser370 375 380Asn Gly Gln Pro Glu Asn AsnTyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys435 440 445 <210> 12 <211> 214 <212> PROTEIN <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic polypeptide <400> 12Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly115 120125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205Phe Asn Arg Gly Glu Cys210<---

Claims

1. A pharmaceutical composition for the treatment of cancer in a patient in need thereof, wherein the composition is an aqueous liquid solution containing: 20-200 mg / ml of an antibody to TGF-β, wherein the antibody comprises an amino acid sequence of a heavy chain variable domain (VH) corresponding to residues 1-120 of SEQ ID NO: 1, and an amino acid sequence of a light chain variable domain (VL) corresponding to residues 1-108 of SEQ ID NO: 2, wherein the antibody comprises a constant region of human IgG4 and a constant region of a human κ light chain, surfactant, chelating agent, 10-50 mM acetate, optionally 25 mM acetate, 5-15% w / v sucrose, optionally 8% w / v sucrose, and water, where the solution is characterized by a pH of 5.0 ± 0.2 or 5.0 ± 0.

3.

2. The composition of claim 1, wherein the antibody comprises the heavy chain amino acid sequence set forth under SEQ ID NO: 1 (with or without a C-terminal lysine) and the light chain amino acid sequence set forth under SEQ ID NO:

2.

3. The composition of claim 1, wherein the surfactant is a polysorbate, optionally polysorbate 80 (PS80).

4. The composition of claim 3, wherein PS80 is present in a concentration of 0.01-0.10% w / v, optionally 0.06% w / v.

5. A composition according to any of the preceding claims, wherein the antibody to TGF-β is present at a concentration of 40-180 mg / ml, optionally 50 mg / ml or 150 mg / ml.

6. A composition according to any one of the preceding claims, wherein the chelating agent is selected from EDTA and DPTA.

7. The composition of claim 6, wherein the chelating agent is present at a concentration of 0-20 μM, optionally 10 μM.

8. The composition according to any one of the preceding claims, wherein the aqueous liquid solution is characterized by a pH of 4.7-5.

3.

9. The composition according to claim 1, containing: 50 mg / ml, 75 mg / ml or 150 mg / ml anti-TGF-β antibody, 25 mM acetate, 10 μM EDTA, 0.06% PS80 and 8% w / v sucrose, at a pH of 5.0 + 0.

3.

10. The composition of claim 9, wherein the antibody comprises the heavy chain amino acid sequence set forth under SEQ ID NO: 1 and the light chain amino acid sequence set forth under SEQ ID NO:

2.

11. A finished product for the treatment of cancer in a patient in need thereof, comprising a vial and instructions for use, wherein the vial contains 16 ml of the composition according to claim 10.

12. Use of a composition according to any one of claims 1-10 for the manufacture of a medicinal product for use in the treatment of cancer in a patient in need thereof.

13. The use according to claim 12, which includes the administration of an additional anticancer therapeutic agent.

14. Use according to claim 12 or 13, wherein the composition is intended for intravenous administration at a dose of 5 mg / kg or 15 mg / kg, optionally once every two weeks.

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