Anti-TGF-beta antibody preparations and their uses

Abstract

The present disclosure provides pharmaceutical compositions comprising anti-TGF-β antibodies and methods of their use.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional 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 has been filed electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on Jun. 17, 2022, is named 022548_WO064_SL.txt and is 19,437 bytes in size. [Background technology]

[0003] Transforming growth factor beta (TGF-β) is a cytokine that controls many important 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. Deregulation of TGF-β leads to pathological conditions, such as birth defects, cancer, chronic inflammation, and autoimmune and fibrotic diseases.

[0004] TGF-β has three known isoforms-TGF-β1, 2, and 3. All three isoforms are initially transcribed as precursor peptides. After cleavage, the mature C-terminus remains associated with the N-terminus (called latency-associated peptide or LAP) to form a small latent complex (SLC) that is secreted from the cell. The inability of SLC to bind to TGF-β receptor II (TGFβRII) prevents receptor engagement. Activation by dissociation of the N- and C-termini occurs by one of several mechanisms, including proteolytic cleavage, acidic pH, or integrin conformational changes (Non-Patent Document 1).

[0005] TGF-β1, 2, and 3 are pleiotropic in their function and are expressed in different patterns across cell and tissue types. They are similar in activity in vitro, but individual knockouts in specific cell types suggest non-identical roles in vivo, even though they share the ability to bind to the same receptor (Non-Patent Document 2). Upon TGF-β binding to TGFβRII, the constitutive kinase activity of the receptor phosphorylates and activates TGF-β receptor I (TGFβRI), phosphorylating SMAD2 / 3, allowing binding to SMAD4, localization to the nucleus, and transcription of TGF-β responsive genes. Ibid. In addition to this classical signaling cascade, non-classical pathways signal through other factors, including p38 MAPK, PI3K, AKT, JUN, JNK, and NF-κB. TGF-β signaling is also regulated by other pathways, including WNT, Hedgehog, Notch, INF, TNF, and RAS. Thus, the end result of TGF-β signaling is the crosstalk of all of these signaling pathways integrating the state and environment of the cell. Id. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Connolly et al., Int J Biol Sci. (2012) 8(7):964-78 [Non-Patent Document 2] Akhurst et al., Nat Rev Drug Discov. (2012) 11(10):790-811 Summary of the Invention [Problem to be solved by the invention]

[0007] Because of the diverse functions of TGF-β, there is a need for effective pan-TGF-β specific antibody therapies. [Means for solving the problem]

[0008] The present disclosure provides a pharmaceutical composition comprising an anti-TGF-β antibody. In one aspect, the present disclosure provides a pharmaceutical composition, the composition comprising a heavy chain variable domain (V) corresponding to residues 1 to 120 of SEQ ID NO:1. H ) amino acid sequence and the light chain variable domain (V L The pharmaceutical composition is an aqueous solution comprising 20-200 mg / ml of an anti-TGFβ antibody comprising the amino acid sequence of 10-50 mM acetic acid, optionally 25 mM acetic acid, and 5-15% w / v sucrose, optionally 8% w / v sucrose, the aqueous solution having a pH of 5.0±0.2 or 5.0±0.3. In some embodiments, the composition is an aqueous solution having a pH of 4.7-5.3.

[0009] In some embodiments, the antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO:1 (with or without the C-terminal lysine) and the light chain amino acid sequence set forth in SEQ ID NO:2.

[0010] In some embodiments, the anti-TGFβ antibody is at a concentration of 40-180 mg / ml, optionally 50 mg / ml or 150 mg / ml.

[0011] In some embodiments, the composition comprises a surfactant, such as a polysorbate (e.g., polysorbate 80 (PS80)). In certain embodiments, the composition comprises PS80 at a concentration of 0.01-0.10% w / v, optionally 0.06% w / v.

[0012] In some embodiments, the composition optionally comprises a chelating agent selected from EDTA and DPTA, hi certain embodiments, the chelating agent is at a concentration of 0-20 μM, optionally 10 μM.

[0013] In certain embodiments, the composition comprises anti-TGFβ antibody at 50 mg / ml, 75 mg / ml, or 150 mg / ml, 25 mM acetic acid, 10 μM EDTA, 0.06% PS80, and 8% w / v sucrose, pH 5.0±0.3. In certain embodiments, the antibody comprises a heavy chain amino acid sequence set forth in SEQ ID NO:1 (with or without a C-terminal lysine) and a light chain amino acid sequence set forth in SEQ ID NO:2.

[0014] The disclosure also provides an article of manufacture comprising a vial and instructions for use, the vial containing about 16 ml of the composition.

[0015] Also provided herein is a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the composition. In some embodiments, the method further comprises administering an additional anti-cancer treatment. In certain embodiments, the composition is administered intravenously at a dose of 5 mg / kg or 15 mg / kg, optionally every other week. The present disclosure also provides the composition for use in treating a patient in need thereof in these methods, and the use of the composition for the manufacture of a medicament for treating a patient in need thereof in the treatment method of the present disclosure.

[0016] Other features, objects and advantages of the present invention will become apparent from the following detailed description. It will be understood, however, that the detailed description, while illustrating embodiments and aspects of the present invention, is given by way of illustration only and not by way of limitation. 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]

[0017] [Figure 1] Photographs showing the degree of protein turbidity in acetate or histidine buffer formulations of Ab1 80 mg / ml over a range of pH values. [Figure 2-1]Figures 2A-C are panels of bar graphs showing the evolution of 2 μm (Figure 2A), 10 μm (Figure 2B), and 25 μm (Figure 2C) subvisible particles in acetate and histidine formulations over 4 weeks of storage at 5 °C, 25 °C, or 40 °C. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 2-5] Continued from Figure 2-4. [Figure 2-6] Continued from Figure 2-5. [Diagram 3] 1 is a bar graph showing viscosity values ​​(in centipoise (cP)) of acetic acid and histidine formulations immediately after preparation (T0) and after 4 weeks of storage at 40° C. [Figure 4-1] 4A and 4B are panels of bar graphs showing the pH values ​​of acetate and histidine formulations upon storage at 40° C., 25° C., or 5° C. over a four week period. [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 4-4] Continued from Figure 4-3. [Figure 4-5] Continued from Figure 4-4. [Diagram 5] FIG. 1 is a scatter plot showing absorbance values ​​(340-360 nm) of acetic acid (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. [Figure 6-1] FIG. 13 is a panel of graphs showing HMWS evolution in formulations with different polysorbate (PS80) concentrations over 2 weeks of storage at 5° C. (top-left panel), 2 weeks of storage at 40° C. (top-right panel), 48 hours of rigorous agitation (bottom-left panel), and freeze / thaw (FT) cycles from −30° C. to room temperature (bottom-right panel). SR_# or Ch_#: # is the % concentration of PS80. SR and Ch represent two different vendors of PS80. [Figure 6-2] Continued from Figure 6-1. [Figure 7A-1] 1 is a pair of bar graphs showing Ab1 concentrations after dilution in saline or dextrose in polyolefin (PO) or polyvinyl chloride (PVC) IV bags. [Figure 7A-2] Continued from Figure 7A-1. [Figure 7B-1] A pair of bar graphs showing subvisible (≧10 μm) particles after dilution in saline (S) or dextrose (D) in PO or PVC IV bags. T0: zero time. T24: 24 hours. T48: 48 hours. [Figure 7B-2] Continued from Figure 7B-1. [Figure 8A-1] 8A-8B are graphs showing HMWS evolution in Ab1 formulations at various concentrations over 12 weeks of storage at 5° C., 25° C., and 40° C. (FIG. 8A) and over 6 months at −20° C. (FIG. 8B). [Figure 8A-2] Continued from Figure 8A-1. [Figure 8A-3] Continued from Figure 8A-2. [Figure 8B] 8A-8B are graphs showing HMWS evolution in Ab1 formulations at various concentrations over 12 weeks of storage at 5° C., 25° C., and 40° C. (FIG. 8A) and over 6 months at −20° C. (FIG. 8B). [Figure 9-1] FIG. 1 is a pair of graphs showing M252 oxidation (left panel) and HMWS% (right panel) evolution in metal-loaded Ab1 formulations. [Figure 9-2] Continued from Figure 9-1. [Figure 10-1] FIG. 1 is a pair of bar graphs showing HMWS and subspecies evolution in various formulations after 1 month of storage at 40° C. (left panel) or 3 months of storage at 25° C. [Figure 10-2] Continued from Figure 10-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure provides a stable pharmaceutical composition comprising a pan-TGF-β specific monoclonal antibody in an aqueous solution. One such antibody is Ab1. Ab1 is an IgG4 monoclonal antibody that targets all three isoforms of human TGF-β (TGF-β1, TGF-β2, and TGF-β3) and has a heavy chain amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence of SEQ ID NO:2.

[0019] The therapeutic success of monoclonal antibodies depends in part on the manufacturability, stability, and delivery properties of the antibody drug candidate. Unfavorable solution behavior, such as high solution viscosity or proteinaceous turbidity, greatly impacts the developability of antibody drugs. Formulation studies of Ab1 have shown that this antibody is surface active and has a high tendency to form sub-visible and visible particles upon solution agitation or under other interfacial stress conditions. The inventors have found that the present formulation, based on an acetate buffer with an acidic pH of approximately 5.0, significantly improves the stability of the formulation during storage and transportation, including reduced particle formation. The inventors have found that Ab1 exhibits undesirable solution behavior, such as proteinaceous turbidity and unfavorable colloidal stability at high pH (e.g., pH 6.0). The inventors have also found that particle formation in solution is further mitigated by the addition of a surfactant, and the inclusion of a chelating agent also helps improve the formulation.

[0020] I. Pan-specific anti-TGFβ monoclonal antibodies The monoclonal antibodies formulated herein comprise complementarity determining regions (CDRs) in Ab1. Such antibodies are collectively referred to herein as "Ab1-related antibodies" and include Ab1 itself. In some embodiments, the antibody is a fully human antibody comprising a human IgG4 constant region and a human kappa light chain constant region. In further embodiments (e.g., Ab1), the human IgG4 constant region has a mutation at position 228 (EU numbering). In some embodiments (e.g., Ab1), the mutation is a serine to proline mutation (S228P).

[0021] The heavy and light chain amino acid sequences of Ab1 are shown below as SEQ ID NO: 1 and 2, respectively. Position S228P is boxed and in bold in the sequence of SEQ ID NO: 1. The variable domains are in italics. The CDRs are boxed. The glycosylation site in the constant domain of the heavy chain is in bold (N297).

[0022] [ka]

[0023] In some embodiments, the antibodies herein are antibodies (e.g., human antibodies) with the above CDRs, i.e., the antibodies have the following heavy and light chain CDR amino acid sequences: 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 can include SEQ ID NOs: 3, 4, 5, 6, 7, and 8.

[0024] In further 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 described above. In certain embodiments, the antibodies formulated herein do not have a C-terminal lysine in the heavy chain.

[0025] In certain embodiments, the antibody formulated herein is Ab1. Ab1 has a predicted molecular weight of 144 KDa when non-glycosylated. Ab1 has a molecular weight of 147.011 kDa, a theoretical experimental isoelectric point (pI) of 6.78, and an experimental pI of about 5.9 to 7.1, as determined by mass spectrometry.

[0026] II. Methods for Producing Antibodies Ab1-related antibodies are produced by methods well established in the art. The DNA sequences encoding the antibody heavy and light chains are inserted into an expression vector such that the genes are operably linked to the necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The antibody light chain coding sequence and the antibody heavy chain coding sequence are inserted into separate vectors and 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 operably linked to the same expression control sequence (e.g., a common promoter), to separate same expression control sequences (e.g., promoters), or to different expression control sequences (e.g., promoters). The antibody coding sequence is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0027] In addition to the antibody chain genes, the recombinant expression vector may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as 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 promoters such as the native immunoglobulin and actin promoters.

[0028] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may have additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. For example, the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes may include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification), the neo gene (for G418 selection), and glutamate synthesis genes.

[0029] An expression vector encoding an antibody of the present disclosure is introduced into a host cell for expression. The host cell is cultured under conditions suitable for expression of the antibody, then harvested and isolated. The host cell includes mammalian, plant, bacterial or yeast host cells. Mammalian cell lines available as hosts for expression are well known in the art and include many 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), NIH3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. Cell lines are selected based on their expression levels. Other cell lines that may be used are insect cell lines such as Sf9 or Sf21 cells.

[0030] Furthermore, expression of the antibody can be enhanced using a number of known techniques, for example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions.

[0031] The tissue culture medium for the host cells may or may not contain animal derived components (ADCs) such as bovine serum albumin. In some embodiments, ADC-free culture medium is preferred for human safety. Tissue culture is performed using fed-batch, continuous perfusion, or any other method appropriate for the host cells and the desired yield.

[0032] III. Antibody Preparations The formulation confers superior stability to Ab1-related anti-TGF-β antibodies, including Ab1. "Stable" or "stability" refers to the ability of the antibody in the composition to retain its physical stability, chemical stability, and / or biological activity during storage and / or when subjected to physical or chemical stress. Stability is in the context of a selected temperature, e.g., under frozen conditions (e.g., -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, e.g., 16 weeks, 24 weeks, 36 weeks, 4 months, 6 months, 1 year, 2 years, 3 years, or more. Protein stability is measured in assays performed within shorter time periods, but whose results are indicative of stability in a clinical context. Such assays include freeze / thaw cycling assays, in which the protein composition is subjected to one or more freeze-thaw cycles; or agitation assays, in which the protein composition is subjected to a mechanical agitation procedure for a pre-determined period of time. Protein stability is determined by storing a protein composition at a specified storage temperature (e.g., 2-8°C) for a selected period of time and analyzing its structural and functional properties, such as the extent of dimerization or aggregation (e.g., measured by size-exclusion HPLC or protein gels), proteolysis (e.g., measured by size-exclusion HPLC or protein gels), color change of the composition, clarity of the liquid composition, enzyme activity, glycan content and composition, receptor binding affinity, methionine residue oxidation, and biological activity of the composition. See also the Examples below for a more detailed description of methods for examining the stability of antibody formulations.

[0033] An antibody described herein "retains its chemical stability" in a pharmaceutical composition if the chemical stability at a given time is such that the antibody is believed to maintain its biological activity, as defined below. To assess chemical stability, chemically modified forms of the antibody are detected and quantified. Chemical modifications include size modifications, which are 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 modifications include charge modifications, which may result, for example, from deamidation or oxidation, and are assessed by ion exchange chromatography, mass spectrometry, or size exclusion chromatography. In some embodiments, the type of chemical modification that occurs during accelerated storage of a composition comprising an antibody described herein includes oxidation of the antibody. In some embodiments, residues M252 and M428 of SEQ ID NO: 1 are oxidized in metal-loaded samples at accelerated storage.

[0034] The compositions of the present disclosure contain one or more pharma- ceutically 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 used as a diluent, vehicle, carrier, preservative, binder, or stabilizer for the active ingredient(s) of a drug. For example, the compositions may contain a buffer, an isotonicity agent, and / or a stabilizer such as an antioxidant. In some cases, one agent may serve more than one of these purposes. In some embodiments, the compositions of the present invention contain an anti-TGF-β antibody as described herein, a buffer such as acetic acid, a stabilizer such as sucrose, and a surfactant such as polysorbate 80 (PS80). The anti-TGF-β antibodies described herein have improved stability due to the combination of certain ingredients in the composition. The compositions of the present invention can be an aqueous solution or a lyophilized preparation. In a preferred embodiment, the compositions of the present invention are aqueous solutions.

[0035] In some embodiments, the composition includes a stabilizing agent such as L-methionine, hi certain embodiments, the composition is an aqueous solution composition that includes 5 to 20 mM (e.g., 10 mM) L-methionine.

[0036] In some embodiments, the composition comprises a bulking agent such as mannitol, hi certain embodiments, the composition is an aqueous solution composition comprising 1-10% (e.g., 3.5%) mannitol (w / v).

[0037] In some embodiments, the composition is an aqueous solution composition comprising a buffer, such as an L-histidine buffer, hi certain embodiments, the aqueous solution composition comprises 5-20 mM (e.g., 10 mM) L-histidine.

[0038] In some embodiments, the pH of the buffer ranges from about 4.0 to about 6.0. In some embodiments, the pH of the buffer is 6.0. In preferred embodiments, the pH of the buffer is 5.0±0.3. In some embodiments, the pH of the buffer is adjusted with sodium hydroxide.

[0039] In some embodiments, the composition is an aqueous solution composition comprising 40-180 mg / ml (e.g., 50-150 mg / ml) of Ab1-related antibody (e.g., Ab1); 10-50 mM (e.g., 10-30 mM) acetic acid; and 1-10% (e.g., 6-8%) w / v sucrose. In some other embodiments, the composition is an aqueous solution composition comprising 15-40 mg / mL of an anti-TGF-β monoclonal antibody, 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 solution composition may be 4.0-6.0 (e.g., 4.7-5.5).

[0040] In some embodiments, the aqueous solution composition comprises 0.01-0.07% w / v surfactant(s). Exemplary surfactants include non-ionic detergents such as polysorbates (e.g., polysorbate 20 and 80) and poloxamers (e.g., poloxamer 188). In some embodiments, the aqueous solution 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) may help reduce turbidity / protein cloudiness of the liquid composition.

[0041] In some embodiments, the aqueous solution composition comprises 0-50 μM (eg, 10 μM) chelating agent(s), such as EDTA or DPTA.

[0042] In a preferred embodiment, the composition is an aqueous solution composition comprising 50 or 150 mg / ml of anti-TGF-β monoclonal antibody, 25 mM acetic acid, 10 μM EDTA or DPTA, 0.06% PS80, and 8% w / v sucrose. In a particular embodiment, the aqueous solution composition has a pH of 5±0.3.

[0043] In some embodiments, the composition is an aqueous solution composition comprising 25 mg / mL of anti-TGF-β monoclonal antibody, 10 mM L-histidine, 2% (w / v) sucrose, 3.5% (w / v) mannitol, 10 mM L-methionine, 0.01% (w / v) polysorbate 80, and a pH of 6.0. In certain embodiments, the composition is an aqueous solution composition comprising 25 mg / mL of anti-TGF-β monoclonal antibody, 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.

[0044] [Table 1]

[0045] The aqueous solution composition is prepared by mixing Ab1, which has been produced by recombinant techniques and subsequently purified from host cells, with an excipient as described herein in water and adjusting the resulting mixture to the desired pH. For example, the anti-TGF-β monoclonal antibody and the desired excipients are added to, or buffer exchanged into, an acetate buffer at the desired pH.

[0046] In some embodiments, the aqueous solution compositions are prepared by reconstituting the lyophilized compositions of the invention with a pharma- ceutically acceptable liquid, such as sterile water, saline (e.g., 0.9% sodium chloride), or acetate buffered saline.

[0047] IV.Product The compositions of the invention are supplied in an article of manufacture (e.g., a kit) that includes instructions for use and, optionally, other therapeutic agents for treating the disorder. The active pharmaceutical ingredient (API) (e.g., Ab1) in the article of manufacture is provided in an amount that is easily administered according to the dosing regimen described herein.

[0048] For example, the product may include a vial containing 800 mg of Ab1 in 16 mL of aqueous solution containing 25 mM acetic acid, 8% sucrose, 10 μM EDTA or DTPA, 0.06% PS80 at pH 5.0±0.3. In some embodiments, the vial contains 800 mg of Ab1, 15 mg of acetic acid, 800 mg of sucrose, and 6 mg of PS80. In some embodiments, the vial contains 800 mg of Ab1, 24 mg of acetic acid, 1280 mg of sucrose, and 9.6 mg of PS80. In certain embodiments, the vial is a pretreated glass vial with a standard closure. For example, the vial may be an ISO 20R Type 1 tube glass vial with a West 20 mm stopper as the closure.

[0049] The composition is stored at -3°C to 5°C for 2 years or longer.

[0050] V. Uses of Ab1 and Related Antibodies TGF-β receptors are widely expressed on immune cells, resulting in the broad effects of TGF-β in both the innate and adaptive immune systems. TGF-β is associated with many disease states, such as birth defects, cancer, chronic inflammation, autoimmunity, and fibrotic diseases. Therapeutic amounts of Ab1 or related antibodies are used to treat these conditions. A "therapeutically effective" amount refers to an amount of Ab1, related antibodies, or another therapeutic agent referred to herein that alleviates one or more symptoms of the treated condition. This amount will vary based on the condition or patient being treated and will be determined by a medical professional using well-established principles.

[0051] The appropriate dosage level of the pharmaceutical compositions described herein is determined based on a variety of factors, including the patient's age, weight, disease state, general health, and medical history, as well as the route and frequency of drug administration, the pharmacodynamics and pharmacokinetics of the Ab1 active ingredient in the drug, and any other drugs taken by the patient concomitantly. In some embodiments, Ab1 or related antibodies are administered at 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 is administered at 5 mg / kg and 15 mg / kg. The administration frequency can be, for example, daily, every 2, 3, 4, or 5 days, weekly, biweekly, or every 3 weeks, monthly, or bimonthly. In some embodiments, the administration frequency is biweekly. The interval between successive administrations is 2 weeks, or shorter or longer than 2 weeks as determined by the physician as appropriate.

[0052] The antibody is administered intravenously (eg, by intravenous infusion over a period of 0.5 to 8 hours), subcutaneously, topically, or by any other route of administration appropriate to the condition and formulation.

[0053] Although Ab1 and related antibodies are derived from human antibody genes and therefore have low immunogenicity in humans, patients are monitored for adverse events when treated with Ab1 or related antibodies.

[0054] In some embodiments, efficacy of the antibodies of the invention is demonstrated in a patient (e.g., in a diseased tissue, such as a tumor tissue, of a patient) by one or more of the following: (1) a decrease in the level or activity of TGF-β, (2) an increase in MIP2 and / or KC / GRO levels, (3) an increase in INF-γ-positive CD8 + CD8 like T cells + T cell activation or infiltration into tumor tissue, and (4) increased clustering of natural killer (NK) cells.

[0055] The patient can be an adult (e.g., a patient 18 years of age or older, including geriatric patients who are 65 years of age or older). The patient can be a pediatric patient (a patient younger than 18 years of age, e.g., a patient from newborn to 6 years of age, 6 to 12 years of age, or 12 to 18 years of age).

[0056] In certain embodiments, a pharmaceutical Ab1 composition containing Ab1 at 50 mg / ml, 25 mM acetic acid, 8% sucrose, 10 μM EDTA or DPTA, and 0.06% PS80 (pH 5.0±0.3) (e.g., supplied in a 10 mL vial) is administered intravenously to a patient at 5 mg / kg or 15 mg / kg every other week until the desired therapeutic endpoint is achieved. For IV administration, the Ab1 composition is diluted in saline or IV dextrose solution (typically containing 5% dextrose in water). For example, a PO or PVC IV bag is used. In some embodiments, the Ab1 formulation is diluted in saline in a PVC bag prior to use. In some embodiments, the Ab1 formulation is diluted in IV dextrose solution in a PVC bag prior to use. In some embodiments, the Ab1 formulation is diluted in saline in a PO bag prior to use. In some embodiments, the Ab1 formulation is diluted in IV dextrose solution in a PO bag prior to use.

[0057] A. Non-oncological disease conditions Conditions treated by Ab1 and related antibodies include, but are not limited to, bone defects (e.g., osteogenesis imperfecta), glomerulonephritis, nerve or skin injuries, lung or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation-induced fibrosis, liver fibrosis, myelofibrosis, scleroderma, immune-mediated diseases (rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Berger's disease, and transplant rejection), and Dupuytren's contracture.

[0058] They are also useful for treating, preventing and reducing the risk of developing 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 (e.g., polycystic kidney disease, 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 when combined with antagonists of the renin-angiotensin-aldosterone system, including, but not limited to: renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, AngII receptor antagonists (also known as "AngII receptor blockers"), and aldosterone antagonists. See, for example, WO 2004 / 098637, the disclosure of which is incorporated herein by reference in its entirety.

[0059] Ab1 and related antibodies are useful in treating diseases and conditions associated with ECM deposition, such as systemic sclerosis, post-surgical adhesions, keloids and hypertrophic scars, proliferative vitreoretinopathy, glaucoma surgery, corneal injuries, cataracts, Peyronie's disease, adult tachycardia syndrome, liver cirrhosis, post-myocardial infarction scarring, restenosis after angioplasty, scarring after subarachnoid hemorrhage, post-laminectomy fibrosis, fibrosis after tendon and other repairs, biliary cirrhosis (including sclerosing cholangitis), pericarditis, pleurisy, tracheotomy, penetrating CNS injury, eosinophilic myalgia syndrome, vascular restenosis, venous occlusive disease, pancreatitis, and psoriatic arthropathy.

[0060] Ab1 and related antibodies are further useful in conditions where promotion of re-epithelialization is beneficial, including, but not limited to, skin conditions such as venous ulcers, ischemic ulcers (pressure ulcers), diabetic ulcers, transplant sites, transplant donor sites, abrasions and burns, conditions of the bronchial epithelium such as asthma, ARDS, mucositis associated with cytotoxic treatments, conditions of the intestinal epithelium such as esophageal ulcers (reflex disease), gastroesophageal reflux disease, gastric ulcers, small and large intestinal lesions (inflammatory bowel disease).

[0061] Further uses of Ab1 and related antibodies are in conditions where endothelial cell proliferation is desirable, for example, in stabilizing atherosclerosis, promoting healing of vascular anastomoses, or in conditions where inhibition of smooth muscle cell proliferation is desirable, such as in arterial disease, restenosis, and asthma.

[0062] Ab1 and related antibodies are also useful in enhancing immune responses to macrophage-mediated infections such as those caused by Leishmania spp., Trypanosoma cruzi, Mycobacterium tuberculosis and Mycobacterium leprae, as well as Toxoplasma gondii, Histoplasma capsulatum, Candida albicans, Candida parapsilosis, and Cryptococcus neoformans. They are also useful in reducing immunosuppression caused, for example, by tumors, AIDS, or granulomatous diseases.

[0063] Ab1 and related antibodies are also useful in the prevention and / or treatment of ophthalmic conditions such as glaucoma and post-fibrous zonectomy scarring.

[0064] B. Oncological Disease Conditions TGF-β regulates several biological processes, including cell proliferation, epithelial-mesenchymal transition (EMT), matrix remodeling, angiogenesis, and immune function. Each of these processes contributes to tumor progression. A broad detrimental role for TGF-β in cancer patients across indications has also been suggested by its elevation 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 malignant conditions, TGF-β can induce EMT, and the resulting mesenchymal phenotype leads to increased cell migration and invasion.

[0065] Compositions comprising Ab1 and related antibodies are useful for treating hyperproliferative diseases such as cancers including, but not limited to, skin cancer (e.g., melanoma, including unresectable or metastatic melanoma, 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 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., head and neck squamous cell carcinoma), brain cancer, glioblastoma, glioma, mesothelioma, leukemia and lymphoma.

[0066] In some embodiments, composition Ab1 and related antibodies are useful for treating cancer in patients who have failed or are expected to fail a previous treatment based on anti-PD-1, anti-PD-L1, or anti-PD-L2 therapeutic agents, i.e., patients who are non-responders or are expected to be non-responders to anti-PD-1, anti-PD-L1, or anti-PD-L2 treatment. In some embodiments, Ab1 and related antibodies are useful for treating cancer in patients who have relapsed from a previous anti-PD-1, anti-PD-L1, or anti-PD-L2 treatment. As used herein, the term "predicted" means that a person skilled in the medical arts can predict, based on his / her general medical knowledge and the particular condition of the patient, without administering the treatment, whether the patient will be a responder or non-responder, and whether the treatment will fail or be ineffective.

[0067] In some embodiments, the cancer is a mesenchymal subtype 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) promotes cell migration and invasion properties by downregulating epithelial cell genes and enhancing mesenchymal gene expression. EMT is a hallmark of tumor progression and invasion. Up to a quarter of colorectal and ovarian cancers are mesenchymal. Thus, by inhibiting TGF-β and its induction of EMT, Ab1 or related antibodies are used to treat mesenchymal solid tumors. Several genetic markers and pathological tests identify mesenchymal subtypes of solid tumors. Markers include ACAT2, VIM, MGP, ZEB2, and ZWINT, which are detected by qRT-PCR or immunohistochemistry. Such markers are used to select patients for anti-TGF-β monotherapy or combination therapy of the present invention.

[0068] In some embodiments, Ab1 and related antibodies are useful for treating patients with advanced solid tumors.

[0069] Compositions containing Ab1 and related antibodies are also used in the treatment of hematopoietic disorders or malignancies such as multiple myeloma, myelodysplastic syndromes (MDS), Hodgkin's lymphoma, non-Hodgkin's lymphoma, and leukemia, as well as various sarcomas such as Kaposi's sarcoma.

[0070] Compositions comprising Ab1 and related antibodies are also useful for inhibiting cyclosporine-mediated malignancy or cancer progression (eg, metastasis).

[0071] In the context of cancer therapy, it will be understood that "treatment" includes any medical procedure that results in partial remission of cancer, slowing the growth of cancer, delaying the progression or recurrence of cancer, or reducing cancer metastasis, as well as extending the patient's life expectancy.

[0072] C. Combination Therapies in Oncology It has been observed that the level of cytotoxic T cell infiltration in cancer is associated with favorable clinical outcome (Fridman et al., Nat Rev Cancer (2012) 12(4):298-306; and Galon et al., Immunity (2013) 39(1):11-26). Furthermore, the level of cytotoxic T cells (CD4 + T H1 Helper T cells, which help immune cells (such as T cells) and the cytokines they produce (e.g., IFN-γ) are also often associated with positive patient outcomes. In contrast, the presence of Treg cells has been shown to be associated with poor patient prognosis (Fridman, supra).

[0073] TGF-β suppresses nearly all aspects of the antitumor immune response. The cytokine promotes iTreg differentiation and inhibits cytotoxicity (CD8 + ) reduces cell proliferation and invasion. Inhibition of TGF-β by Ab1 or related antibodies, as described above, alleviates the immunosuppressive tumor microenvironment and provides positive outcomes for cancer patients.

[0074] Furthermore, the inventors have discovered that by alleviating the immunosuppressive tumor microenvironment, Ab1 and related antibodies can enable checkpoint modifiers, such as anti-PD-1 antibodies, to better induce an immune response, and as a result, more patients will benefit from immunotherapies, such as anti-PD-1, anti-PD-L1, or anti-PD-L2 treatments.

[0075] Ab1 and related antibodies, with or without therapeutic agents targeting immune checkpoint molecules, are also used in conjunction with other cancer therapies, such as chemotherapy (e.g., platinum or taxoid-based therapies), radiation therapy, and therapies targeting cancer antigens or oncogenic drivers.

[0076] Cancers that may be treated with a combination comprising Ab1 or a related antibody and an immune checkpoint inhibitor, such as an anti-PD-1 antibody, include those cancers listed in the subsections above.

[0077] In some embodiments, the cancer is refractory to prior anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy, such as advanced or metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, and Hodgkin's lymphoma. Refractory patients are those who experience disease progression without any evidence of response, e.g., radiologically confirmed, within 12 weeks of initiating treatment.

[0078] In some embodiments, Ab1 or a related antibody is used in conjunction with another cancer therapy, such as anti-PD-1 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 squamous cell carcinoma. See also discussion above.

[0079] Examples of anti-PD-1 antibodies are nivolumab, pembrolizumab, pidilizumab, MEDI0608 (formerly AMP-514; see, e.g., WO 2012 / 145493 and U.S. Pat. No. 9,205,148), 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 (H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, H2M7798N, H4H9019P, H4xH9034P2, H4xH9035P2, H4xH9037P2 in Table 1 of the PCT publication). , H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P and H4xH9008P, and those 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.

[0080] For example, CDRs, V H and V L Antibodies and related antibodies disclosed in WO 2015 / 112800, including antibodies and antigen-binding fragments having the same PD-1 epitope as the antibodies disclosed in the PCT publication, or heavy and light chain sequences, are used in conjunction with the Ab1 or related antibodies of the present disclosure to treat cancer. In related embodiments, useful anti-PD-1 antibodies have the heavy and light chain amino acid sequences shown below as SEQ ID NOs:9 and 10, respectively; the V and V of SEQ ID NOs:9 and 10, respectively; H and V Lor one or more (eg, all six) CDRs (shown in boxes) of SEQ ID NOs: 9 and 10.

[0081] [ka]

[0082] In other related embodiments, useful anti-PD-1 antibodies may comprise the heavy and light chain amino acid sequences set forth below as 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 (shown in boxes) in SEQ ID NOs: 11 and 12. In related embodiments, useful anti-PD-1 antibodies may comprise the heavy and light chain amino acid sequences set forth below as 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 (shown in boxes) in SEQ ID NOs: 9 and 10.

[0083] [ka]

[0084] In some embodiments, antibodies of the disclosure, such as anti-PD-1 antibodies, do not have a C-terminal lysine in their heavy chains. The C-terminal lysine is removed during manufacturing or by recombinant techniques (i.e., the coding sequence for the heavy chain does not contain a codon for the C-terminal lysine). Thus, antibodies comprising the heavy chain amino acid sequence of SEQ ID NO:3 without the C-terminal lysine are also contemplated within the invention.

[0085] D. Biomarkers of Treatment Efficacy The efficacy of Ab1 and related antibodies is determined by biomarkers or target occupancy. For example, in tumor tissue, target occupancy is assayed by assessing the levels of active TGF-β in biopsies using the Meso Scale Discovery (MSD) assay. In blood, target engagement is assayed by assessing the effect of reduction of circulating TGF-β on peripheral blood mononuclear cells such as lymphocytes (T cells, B cells, NK cells) and monocytes. For example, circulating CD8 + Increased T cell proliferation was observed by expressing CD45 as a marker in flow cytometry + RO + CCR7 + CD28 + Ki67 + Activation of circulating NK cells is assessed using CD3 as a marker for flow cytometry. - CD56 high / dim CD16 + or CD137 + In addition, Ki-67, PD-1, and ICOS are used as PD markers associated with T cell activation.

[0086] Immune modulation upon treatment with Ab1 or related antibodies is assayed, for example, by assessing changes in infiltrating immune cells and immune markers by multiplex immunohistochemistry (IHC) assays using the NeoGenomics platform. In particular, NeoGenomic's MultiOmyx TIL Panel staining for a panel of immune markers allows for quantitative determination of density and localization of various immune cells. Immune markers include iTreg differentiation; CD8 + T cell infiltration and proliferation; and CD8 + Ab1 can indicate the production of IFNγ by CD4 T cells. + Inhibits differentiation of T cells into iTregs (see, e.g., Example 3 of U.S. Patent Publication No. 2018 / 0244763), and inhibits CD8 +It was shown to increase T cell proliferation and their production of IFNγ (as shown in a mixed lymphocyte reaction assay; data not shown). Thus, the efficacy of treatment with Ab1 or related antibodies was confirmed by inhibition of iTregs, CD8 + Induction of T cell proliferation and infiltration into tumors or other diseased tissues, increased IFNγ production, and / or CD8 + Immunomodulation upon treatment with Ab1 or related antibodies also increased the ratio of CD8 T cells to Treg cells. + T cells, Treg cells, NK cells, and other immune cells are assayed in peripheral blood by methylation PCR-based quantitative immune cell counting. The efficacy of treatment can be demonstrated clinically as a delay or amelioration in disease progression, such as tumor progression.

[0087] Unless otherwise stated herein, scientific and technical terms used in connection with this disclosure have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in practicing or testing this disclosure. In case of conflict, the present specification, including definitions, controls. In general, the nomenclature and techniques used in connection with neurology, medicine, medical and pharmaceutical chemistry, and cell biology described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques were performed according to manufacturer's instructions as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular includes plural and plural terms also include singular. Throughout this specification and embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises" or "comprising" are understood to mean the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. All literature and other references mentioned herein are incorporated herein by reference in their entirety. Although many texts are cited herein, this citation does not acknowledge that any of these texts form part of the common general knowledge in the art. As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the reference value mentioned. In certain embodiments, the term refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (more or less) of the reference value mentioned, unless otherwise stated or clear from the context.

[0088] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. EXAMPLES

[0089] The following example describes a study evaluating various formulations of Ab1 to arrive at the formulation with the best biological activity and long-term stability. We evaluated the effect of buffer properties and pH on the physical and chemical stability of Ab1 liquid formulations during refrigerated, accelerated, and stressed storage conditions. Acetate and histidine buffer systems were selected for this study with and without the addition of sodium chloride.

[0090] We evaluated the optimal concentration of polysorbate 80 (PS80) required for stabilization of Ab1 liquid formulations at various storage temperatures, freeze-thaw cycles, and agitation-induced stress.

[0091] We also evaluated the physical stability of Ab1 drug product (DP) after dilution and incubation at room temperature in intravenous (IV) infusion bags for up to 48 hours. 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 PS80 concentration in the liquid DP was also examined for its ability to protect Ab1 after dilution.

[0092] We further investigated the effect of transition metals on the chemical and physical stability of proteins. Transition metals can leach into drug substances (DS) during manufacturing. EDTA and DTPA were evaluated for their ability to chelate and protect proteins during a worst-case set of experiments.

[0093] To ensure that the proposed targeted formulation matrix is ​​sufficient to stabilize high concentration DS and low concentration DP solutions, we evaluated the stability of the solutions during freeze-thaw cycles and under frozen and liquid storage conditions, and over a range of concentrations of all excipients, including the API.

[0094] The experimental materials and methods are as follows.

[0095] drug substance High concentration Ab1 drug substance (DS) was prepared using ultrafiltration / diafiltration (UFDF). DS concentrations were generally prepared at up to 180 mg / ml. Results from the UFDF simulator were used for concentration correction of buffer salts that were accumulated or depleted during the UF process steps due to the Donnan effect. After formulation of drug product (DP) samples into target protein and excipient concentrates, the formulations were then purified through 0.22 μm filters under laminar flow before aseptic fill packaging.

[0096] Inspection of particles visible to the naked eye Macroscopic particles were analyzed under a visual inspection unit. DP vials were cleaned with lens paper prior to inspection to remove dust and fingerprints from the outer surface.

[0097] pH The pH of buffers and formulated mAb solutions was measured using Thermo-Scientific™ pH probes and meters. Results were considered comparable if the differences between replicate measurements were within 0.1 pH units.

[0098] Osmolarity Osmolarity measurements were performed on 20 μL of sample (n=2 or 3) using a freezing point depression osmometer (Advanced Instrument, OsmoPRO). Osmolarity standards were run before and after sample analysis to ensure measurement accuracy.

[0099] Total protein concentration Total protein concentration was determined by measuring ultraviolet (UV) absorbance at 280 nm using Variable Pathlength Technology with a 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 with a microfluidic chip from Unchained Labs' Big Lunatic system. Measurements were performed in duplicate with 2–5 μL of sample.

[0100] DSC of structure and thermal stability Differential scanning calorimetry (DSC) was performed on a Malvern Microcal calorimeter with a thermal gradient from 15°C to 105°C at a heating rate of 0.5°C / min. Protein solutions were measured at a protein concentration of 1mg / ml. OriginPro software was used for analysis and thermal unfolding temperature (Tm) determination.

[0101] Solution turbidity and optical density Sample turbidity was quantified by measuring the optical density (OD) from 340 nm to 360 nm on a Molecular Devices SpectraMax® i3 Microplate Reader. For each sample, 200 μL was loaded into a UV-Vis transparent 96-well plate. OD was determined as the average of the absorbance values ​​at 340 nm, 345 nm, 350 nm, 355 nm, and 360 nm.

[0102] Size-exclusion HPLC for high molecular weight species Analysis of protein aggregates (high molecular weight species or HMWS) was performed by size exclusion chromatography (SEC). Samples were run on a 1260 series HPLC (Agilent, Santa Clara, CA) equipped with a TSK-GEL® G3000SWXL (Tosoh Bioscience, Tokyo, Japan) analytical column and a matching 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 min. Triplicate injections were performed for each sample. Detection was by UV absorbance at 280 nm, and chromatographic peaks were integrated to determine the relative percentage of each eluting species.

[0103] Microflow Imaging (MFI) of particles invisible to the naked eye Particles invisible to the naked eye were analyzed using a Protein Simple MEI™ Model DPA-4200. The system was flushed extensively with 0.22 μm filtered, degassed MilliQ® water before measuring the 2, 10, and 25 μm standards. Samples (n=1 or 2) were run using the 1 mL method with a flow rate of 0.17 mL / min. As the sample flowed through the flow cell, it was illuminated by a light source and a camera quickly captured an image as the sample passed through the flow cell. Particles were identified by MFI™ software, and then the size, clarity, and morphology of each individual particle were calculated.

[0104] High-precision liquid particle counter for particles invisible to the naked eye Particles invisible to the naked eye were also measured by light obscuration on a Hach® High Accuracy (HIAC) Liquid Particle Counter Model 9703+. The system was flushed with 0.22 μm filtered, degassed MilliQ® water until particle counts were below 20 particles / mL. Accurate particle counts were confirmed by measuring 2 μm, 10 μm, and 25 μm standards and extensive washing to remove any background. Using the 1 mL protocol, samples were measured by five separate injections of 0.2 mL. The first sample measurement was ignored and the following four were averaged.

[0105] Capillary isoelectric focusing of charge variants Protein charge heterogeneity was measured by UV absorbance at 280 nm and by capillary isoelectric focusing (cIEF) using a Protein Simple iCE3 instrument. Samples (1 mL) and standard solutions were diluted to 2.5 mg / ml in water. On-board mixing was used to mix samples and MasterMix before analysis. Isoelectric focusing of samples included a prefocusing time at 1500 V for 1 min, followed by focusing at 3000 V for 10 min. Detection covered 5 exposures, followed by sample loading for 55 s for each formulation. Results were considered comparable if the difference was equal or less than 10%.

[0106] PTM quantification by LC-MS Protein samples were diluted to 2 mg / ml, vortexed, and autodigested using 40 μg protein. Digestion buffer was 25 mM Tris, pH 8.5. For each sample, 15 μL of digest (containing approximately 5 μg protein) was injected onto a C18 column for LC-MS analysis. Samples were analyzed on a Q Exactive™ using the DDA top8 LC-MS / MS method.

[0107] LC-MS / MS data acquired on the Q Exactive™ were processed by BioPharma Finder™ 3.0 on server WLSD58 for identification and relative quantification of modifications including Met / Trp oxidation, deamidation, Asp isomerization and HC C-terminal modifications. Due to low levels of modifications not able to produce good MS / MS spectra for identification by BioPharma Finder™, MS-only peptide mapping was used for peptide assignment. All data were then processed using Progenesis to provide retention time alignment and peptide amounts after peak picking. Manual adjustment of peak picking was required for some deamidated and isomerized peptides where Progenesis could not perform this task accurately.

[0108] efficacy 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 the binding of TGF-β to the TGF-β cell surface receptor, thus allowing cell proliferation. In the Ab1 potency assay, various levels of Ab1 were incubated with TGF-β2 and then added to mink lung cells.

[0109] Cells were incubated with Ab1 and TGF-β2 for 3 days, then PrestoBlue™ reagent was added. PrestoBlue™ contains a cell-permeable, non-fluorescent compound, resazurin, which is metabolized and reduced by viable cells, resulting in the production of a fluorescent product, resorufin. Thus, cell proliferation is directly related to the intensity of the fluorescent signal. Five hours after PrestoBlue™ addition, fluorescence was measured using a plate reader. The bioassay software was run on a Log 10 After the reference and sample curves were determined to be suitable, the curves were confined and the final result of the assay was the EC 50 Reference EC divided by 50 and reported as percent relative potency (%RP). EXAMPLES

[0110] Buffers and pH Screening This example describes an experiment in which various buffer and pH conditions were screened to identify a suitable formulation of Ab1. Because liquid drug products are more convenient to prepare and administer in both hospitals and at home (compared to lyophilized drug products), various aqueous buffers were tested. Table 1 represents the formulation conditions and sample codes used in this study.

[0111] [Table 2]

[0112] Protein turbidity is a macroscopic manifestation of attractive protein-protein interactions. It was observed with the naked eye that the Ab1 formulation exhibited significant pH-dependent protein turbidity (Figure 1). The solution was almost clear and transparent below pH 5.3, but at pH >5.3, protein turbidity increased with increasing pH. Acetic acid formulations generally exhibited lower protein turbidity than histidine formulations (Figure 1). These macroscopic appearance images indicated that the optimal formulation pH was in the range of 4.7-5.0 using acetic acid as the buffer species.

[0113] After storage at 5°C and 25°C for up to 4 weeks, the solutions in Table 1 showed no significant change in high molecular weight species (HMWS)%. However, after 4 weeks of storage at 40°C, all formulations showed an increase in HMWS% of about 0.5%. This change was not particularly significant at the given stress conditions. Furthermore, the addition of sodium chloride to the formulations showed no significant effect on the degree of protein aggregation and HMWS%.

[0114] With respect to subvisible particles, HIAC particle counts demonstrated that subvisible particle growth was sensitive to the nature of the buffer type and solution pH (Figures 2A-2C). Acetate formulations buffered at pH 4.7 and 5.0 resulted in the generation of the fewest particles over time compared to any other pH of acetate formulations, and all histidine formulations studied. However, there was no discernible or clear effect of temperature on particle growth in any condition.

[0115] The acetate and histidine formulations, with or without the addition of sodium chloride, showed comparable viscosities at T0 at any given pH and after 4 weeks at 40°C (Figure 3). The measured viscosity values ​​were all within 2-3 cP, well below any limits that may present challenges during DP administration to patients or during the manufacturing process. There was also no significant change in the viscosity values, indicating that the formulations tested did not undergo significant chemical degradation. There was no significant change in the pH value of any of the formulations after storage at the three temperatures over a period of 4 weeks (Figures 4A and 4B). These results indicate that acetate and histidine did not undergo any significant chemical degradation and maintained their buffering ability even after up to 4 weeks of stress storage at 40°C.

[0116] Plate UV measurements were performed to follow possible turbidity and protein turbidity changes over time (Figure 5). The same pH-dependent protein turbidity observed during prior visual inspection at T0 was detected by spectroscopic measurements. The measured OD values ​​increased with pH, ​​with the histidine formulations being more turbid than the acetate formulations. After 4 weeks of storage at 25°C and 40°C, a slight increase in the OD of most formulations was observed. The increase in OD over time was due to the formation of particles that were not visible to the naked eye. The results confirmed that a pH around 4.7-5.0 resulted in the least amount of sub-visible particles.

[0117] Osmolality values ​​of buffer only and buffered protein solutions were compared (Table 2). The acetate formulation was slightly more osmolal than the histidine. The addition of protein increased the osmolality of all formulations. Osmolality values ​​were all reasonable considering that the DP was only administered after dilution into an IV infusion bag.

[0118] [Table 3]

[0119] The chemical stability of Ab1 was similar in the acetate and histidine formulations, as seen when comparing the amount and monomer percentage of the acidic isoforms: after 4 weeks at 40°C, there was a slight increase in the relative amount of the acidic isoform produced when the pH of the histidine formulation reached solutions of 6.0 and 6.5.

[0120] All formulations had the same relative potency of approximately 1.0, indicating that they were all acceptable. These potency results are in line with previous aggregation and HMWS% results, as all HMWS% values ​​were low (<2%) and therefore potency was not expected to change significantly. EXAMPLES

[0121] Surfactant Screening This example describes experiments evaluating the addition of the surfactant polysorbate 80 (PS80) to Ab1 formulations. The formulation conditions and sample codes used in these experiments are shown in Table 3 below.

[0122] [Table 4]

[0123] Plate turbidity and optical density (OD; 340-360 nm) values ​​of the formulations were evaluated for formulations stored at 5°C or 40°C for 1 week or after 48 hours of strong 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. No differences were observed with various concentrations of PS80, 0.025%, 0.05% and 0.1%, using two different commercial sources of PS80 (A and B). However, OD values ​​slightly decreased in formulations without added surfactant over time at both 5°C and 40°C storage. Slightly higher turbidity was also observed in formulations without surfactant. These observations indicated that PS80 has a solubilizing effect on Ab1 protein.

[0124] The levels of small soluble aggregates were monitored by SEC under different storage and interface stress conditions (Figure 6). Storage temperature only had a slight effect, resulting in a 0.5% increase in aggregation after up to 2 weeks of storage; PS80 concentration had no noticeable effect on aggregation levels. Up to 10 worst-case freeze-thaw cycles between -30°C and room temperature did not show any negative impact on protein stability (Figure 6, bottom-right panel).

[0125] On the other hand, strong stirring or shaking shows a large effect on aggregation (Figure 6, bottom-left panel). Formulations without added surfactant produced up to 8% HMWS after 48 hours of stirring. These aggregation levels were highly sensitive to the presence and concentration of surfactant. PS80 concentrations as low as 0.025% were sufficient to reduce aggregation levels to below 2% of the aggregation levels of formulations without PS80. Increasing the PS80 concentration even higher than 0.05 or 0.1% slightly reduced the HMWS%. These data indicated that 0.05% should be used as the lower limit of PS80 concentration targeted to stabilize Ab1 formulations.

[0126] The SEC data above indicates that Ab1 did not exhibit a high risk of aggregation in most of the conditions studied in this section. Therefore, HIAC is a critical assay required to track the large soluble and insoluble aggregates filtered by SEC, which are in the sub-visible particle range in size. The HIAC data show that under any given storage or interface stress condition, the formulations that did not contain any PS80 produced substantially more sub-visible particles. The data further show that PS80 had a concentration-dependent effect on reducing the number of particles in Ab1 formulations. PS80 concentrations as low as 0.025% were sufficient to initiate a reduction in particle counts. Increasing the PS80 concentration to 0.05% and higher resulted in a continued reduction in particle counts.

[0127] The pH values ​​of the formulations were measured at T0 and after 2 weeks of storage. There was no significant change in the pH of the formulations at any time point or temperature. There was also no concentration-specific effect of PS80 on the formulation pH. The concentration of PS80 did not induce any change in the ability of the acetate buffer to maintain the pH of the 150 mg / ml Ab1 formulation.

[0128] All studies in this example showed no effect of PS80 source on formulation stability for any of the conditions tested. EXAMPLES

[0129] IV Bag Dilution Study This example describes experiments testing the stability of Ab1 formulations in different IV bags. The formulation conditions used in these experiments are shown in Table 4 below.

[0130] [Table 5]

[0131] Prior to IV infusion, the DP was diluted in an IV bag. Without a sufficient concentration of surfactant in the DP, protein molecules could be absorbed into the IV bag depending on the type of material the bag was made from. As a result, the DP diluted in the IV bag had a lower API concentration than the intended dose administered to the patient.

[0132] We followed the absorption behavior and physical stability of Ab1 diluted in bags spiked with different concentrations of PS80 and coated with PS80. PS80 was diluted by spiked to concentrations of 0.001%, 0.0005%, 0.0003%, and 0.0002% to determine the optimal concentration required to start the DP formulation. The aforementioned PS80 concentrations correspond to PS80 concentrations of 50-300 times dilutions of the starting DP. DP 150 mg / ml containing no PS80 was then diluted to 0.5 or 1.0 mg / ml. The effect of diluting the formulation and dilution into four different combinations of IV bag materials was evaluated: saline in a PVC bag, saline in a PO bag, dextrose in a PVC bag, and dextrose in a PO bag.

[0133] The diluted formulations were then incubated and measured after 24 and 48 hours. The data show that saline in PO and PVC bags did not significantly affect protein absorption at any of the added surfactant concentrations studied (Figure 7A). However, the use of dextrose as a diluent slightly affected absorption depending on the bag material. In particular, the PVC dextrose bag combination caused noticeable protein absorption as the PS80 concentration decreased (Figure 7A). The results had direct implications for recommendations made for DP administration.

[0134] Subvisible (>10 μm) particle generation after DP dilution was highly sensitive to the type of diluent used. Saline-based solutions produced higher levels of particles than dextrose-based solutions (Figure 7B). This was independent of the concentration of PS80 used in the formulation. The difference in subvisible particle counts between 0.001% and 0.003% PS80 was not significant. The IV bag material did not appear to have a significant effect on particle formation, with particle counts being comparable.

[0135] Aggregation of diluted DP samples was also evaluated after incubation in dextrose and saline in combination with PO and PVC bags. Protein diluted into saline bags produced slightly more HMWS% than those in dextrose bags. PO bags also produced more aggregates than PVC bags. Protein stability was also protein concentration dependent, with the lower 0.5 mg / ml dilution generally showing higher aggregation levels than the 1.0 mg / ml dilution. However, PS80 concentration did not show a significant effect on protein stability. EXAMPLES

[0136] DS and DP stability This example describes a study to evaluate the long-term stability of drug substance (DS) and drug product (DP). Table 5 shows the formulations tested, in which sucrose was used as the cryoprotectant, PS80 was used as the surfactant, and DTPA was used as the chelating agent.

[0137] [Table 6]

[0138] These data in 20 mM acetate buffer containing 50 μM DTPA determined that a formulation of UF / DF purified Ab1 containing 25 mM acetate, providing the same pH range, and 10 μM EDTA would exhibit the same solution behavior. Therefore, we tested this preliminary proposed target formulation of DS and DP: 25 mM acetate, 8% sucrose, 0.06% PS80, 10 μM chelator (DTPA was tested, but EDTA is equivalent), pH 5.0 (Table 5). The ability of this formulation matrix to stabilize Ab1 at concentrations spanning the full range of DS and DP concentrations was evaluated for storage stability. After 3 months of storage at 5° C. or 25° C., there was no significant change in HMWS% for either formulation (FIG. 8A). At 40° C., there was a maximum increase of about 0.5%, but this increase is considered to be minor under these types of accelerated stress storage conditions, reflecting the overall ability of the proposed formulation matrix to optimize stability and shelf life.

[0139] Ab1 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 HMWS% (Figure 8B). Low molecular weight species (LMWS)% was also tracked by SEC, with no significant change in fragmentation observed for any of the formulations or storage conditions. Additionally, all formulations met USP 1000 for both 10 and 25 μm subvisible particles. <787> It was within the specifications.

[0140] There was no dramatic change in the chemical stability of Ab1 under either condition. In summary, the proposed formulation matrix has the capacity to stabilize a range of lowest possible DP concentrations of 50 mg / ml and highest possible DS concentrations of 165 mg / ml. EXAMPLES

[0141] Metal loading studies and chelating agent compatibility During the manufacturing of biopharmaceuticals, there is a small risk of transition metal contamination of the DS and DP. If contaminated, the transition metals can lead to chemical instability and aggregation in liquid solutions. This example describes a study that evaluated the effect of metals and chelators on Ab1 formulations. Table 6 shows the formulations used to test metal loading:

[0142] [Table 7]

[0143] As shown in the table, Ab1 samples were spiked with iron and copper, with and without the addition of the chelator DTPA. In the absence of added chelator, iron-spiked samples showed significant M252 oxidation and aggregation, while copper-spiked samples also showed increased oxidation and aggregation, but to a lesser extent (Figure 9). However, in the presence of DTPA, there was a dramatic decrease in proteolysis.

[0144] We also tested another chelating agent, EDTA. The storage stability of Ab1 formulations was compared with the addition of 10 μM DTPA or 10 μM EDTA. The data showed that these two chelating agents provided similar levels of protection to protein molecules in solution. These chelating agents selectively reduced metal-induced particle formation or aggregation. EXAMPLES

[0145] Formulation robustness with concentration ranges for API and excipients This example describes a study evaluating the stability of Ab1 formulations over a range of antibody and excipient concentrations. The formulations used for the study are shown in Table 7 below.

[0146] [Table 8]

[0147] We assessed aggregation by analyzing HMWS as dimer, trimer, and tetramer subspecies. This allowed the ability to track the effect of formulation component variations on the details of direct aggregation. pH 4.7 and pH 5.3 were the most stable and did not produce significant amounts of HMWS, and furthermore, the predominant HMW species was dimer (Figure 10). The pH 5.0 formulation produced approximately 0.6% more total aggregates, with dimer levels comparable to pH 4.7 and 5.3, which showed 0.6% primarily trimer species. Nevertheless, the total HMWS% levels were acceptable.

[0148] There was no dramatic change in the number of subvisible particles (10 and 25 μm) across the different formulations with either time or storage temperature (data not shown). There was also no dramatic change in the number of subvisible particles (10 and 25 μm) after aggregation or freeze-thaw cycle stress (data not shown).

[0149] In conclusion, acetate buffered formulations offered optimal physical and chemical stability compared to histidine. Subvisible particle counts were highly sensitive to the nature of the buffer species and pH, with acetate formulations at low pH (4.7 and 5.0) producing the lowest particle counts. Turbidity and proteinaceous haze were also pH dependent, with formulations in low pH acetate conditions producing much less proteinaceous haze, indicating that low pH is less likely to induce protein-protein interactions. Solutions with less proteinaceous haze were also shown to require shorter processing times during the UFDF procedure, an important manufacturing consideration required to produce high concentrations of drug substance.

[0150] The addition of PS80 to the final formulation is necessary to reduce the risk of aggregation, especially particle formation, which has been shown to be accelerated by interfacial stresses such as freeze / thaw cycles. PS80 was also effective in reducing or decreasing protein absorption into IV infusion components (e.g., IV bags). PS80 concentrations ≥ 0.05% provided optimal stability.

[0151] Significant oxidation and moderate levels of aggregation of metal-loaded samples were observed under accelerated storage conditions. Metal chelators were added to the formulation to provide protection from possible metal-induced protein and excipient degradation. 10 μM EDTA and 10 μM DTPA provided comparable levels of protection, resulting in similar changes in HMWS, oxidation, and PS80 concentrations.

[0152] 8% sucrose, which equates to approximately 80 mg / ml, was found to be a suitable ratio for protection of both DP and DS at concentrations of 50 mg / ml and 150 mg / ml, respectively.

[0153] Results from these stability studies demonstrated the robustness of the targeted formulation to stabilize frozen DS as well as liquid DP under a variety of storage and other stress conditions. EXAMPLES

[0154] Alternative formulations of Ab1 [powder for solution for injection] A further exemplary Ab1 formulation is a sterile lyophilized product. The drug product was filled into USP Type 1 borosilicate glass vials at 10.3 mL / vial with 0.3 mL excess. The vials were capped with siliconized gray butyl rubber stoppers and sealed with aluminum seals and flip-off caps. Table 8 provides exemplary composition information for Ab1 drug product.

[0155] For administration, each vial was reconstituted with 9.7 mL of water for injection to yield 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).

[0156] Excipient screening showed that 2% sucrose reduced the formation of aggregates during freezing and thawing. The bulking agent, mannitol, did not significantly affect protein stability but aided in producing superior lyophilized cakes.

[0157]

Table 9

Claims

1. A pharmaceutical composition, wherein the composition comprises A heavy chain variable domain (V H ) amino acid sequence corresponding to residues 1 to 120 of SEQ ID NO: 1 and a light chain variable domain (V L ) anti-TGFβ antibody containing an amino acid sequence of 20 to 200 mg / ml, 10 - 50 mM acetic acid, optionally 25 mM acetic acid, and 5 - 15% w / v sucrose, optionally 8% w / v sucrose in an aqueous solution, and the solution has a pH of 5.0 ± 0.2 or 5.0 ± 0.

3.

2. The composition according to claim 1, wherein 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.

3. The composition according to claim 1, further comprising a surfactant.

4. The composition according to claim 3, wherein the surfactant is polysorbate, optionally polysorbate 80 (PS80).

5. The composition according to claim 4, wherein PS80 has a concentration of 0.01 - 0.10% w / v, optionally 0.06% w / v.

6. The composition according to claim 1, wherein the anti-TGFβ antibody has a concentration of 40 - 180 mg / ml, optionally 50 mg / ml or 150 mg / ml.

7. The composition according to claim 1, further comprising a chelating agent selected from EDTA and DPTA, optionally.

8. The composition according to claim 7, wherein the chelating agent has a concentration of 0 - 20 μM, optionally 10 μM.

9. The composition according to claim 1, wherein the aqueous solution has a pH of 4.7 - 5.

3.

10. 50 mg / ml, 75 mg / ml, or 150 mg / ml of anti-TGFβ antibody, 25 mM acetic acid, 10 μM EDTA, 0.06% PS80, and 8% w / v sucrose in a composition according to claim 1, and having a pH of 5.0 ± 0.

3.

11. The composition according to claim 10, wherein the antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 1 and the light chain amino acid sequence set forth in SEQ ID NO:

2.

12. A product comprising a vial and instructions for use, wherein the vial contains about 16 ml of the composition according to claim 11.

13. Use of the composition according to any one of claims 1 - 11 in the manufacture of a medicament for use in treating cancer in a patient in need thereof.

14. The use according to claim 13, wherein further anti-cancer treatment is to be administered.

15. The use according to claim 13, wherein the composition is to be administered intravenously at a dose of 5 mg / kg or 15 mg / kg, optionally every other week.

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