preparation

Abstract

The present invention relates to a pharmaceutical formulation comprising: (i) a therapeutically effective amount of a bispecific protein comprising a soluble T cell receptor (TCR) and a scFV; and (ii) a surfactant. The w / w ratio of the surfactant to the protein is in the range of 0.75:1 to 1.5:1. The formulation may also include a filler and / or a stabilizer. Another pharmaceutical formulation comprises: (i) a therapeutically effective amount of a bispecific protein comprising a soluble T cell receptor (TCR) and a scFV; (ii) a filler; and (iii) a stabilizer. The w / w ratio of the stabilizer to the filler may be greater than 1:1.

Description

Technical Field

[0001] The present invention relates to formulations. In particular, the present invention relates to pharmaceutical formulations of bispecific proteins and scFVs, wherein the bispecific proteins comprise soluble T cell receptors (TCR). Background Art

[0002] Advances in biotechnology have made it possible to manufacture proteins for pharmaceutical applications. Since proteins are larger and more complex than traditional organic and inorganic drugs (e.g., in addition to complex three-dimensional structures, they also have multiple functional groups), there are special problems in the preparation of such proteins. In order for proteins to maintain biological activity, preparations must fully preserve the conformational integrity of at least one core sequence of amino acids of the protein while protecting multiple functional groups of the protein from degradation. The degradation pathways of proteins may involve chemical instability (e.g., any process involving the formation or cleavage of bonds to obtain new chemical entities to modify proteins) or physical instability (e.g., changes in the higher-order structure of proteins). Chemical instability can be caused by deamidation, racemization, hydrolysis, oxidation, β elimination, or disulfide exchange. For example, physical instability can be caused by denaturation, aggregation, precipitation, or adsorption. The three most common protein degradation pathways are protein aggregation, deamidation, and oxidation (Cleland et al Critical Reviews in Therapeutic Drug Carrier Systems 10(4):307-377(1993)).

[0003] Antibodies, including fragments and variants, are a class of therapeutic proteins for which successful pharmaceutical preparations have been described (Wang et al. J Pharm Sci. 2007 Jan; 96 (1): 1-26.). However, the technology of stable pharmaceutical preparations involving bispecific proteins comprising soluble T cell receptors (TCRs) and scFvs is extremely limited. Due to the complexity of known protein preparations and the numerous differences between TCR bispecific proteins and antibodies, it is not expected that the technology developed for preparing antibodies will be applied to these bispecific TCR proteins. For example, in antibody preparations, high protein concentrations (i.e., greater than 1 mg / ml) are required to minimize the volume of the drug product administered to the patient while maintaining the desired therapeutic effect. TCR-scFv bispecific proteins are designed to be particularly effective at low protein concentrations, so preparations with low protein concentrations (i.e., less than 1 mg / ml) are required. Low protein concentrations are particularly problematic because even low levels of aggregation or transient contact with the surface of the vial (i.e., adsorption loss) can result in significant loss of protein activity.

[0004] Furthermore, unlike antibodies, TCRs are known to be inherently unstable in solution, which means that maintaining protein stability at high temperatures (e.g., above -30°C or -20°C, e.g., 2°C to 8°C) and / or during long-term storage is not straightforward. Stability of therapeutic proteins at high temperatures and during long-term storage is a desirable feature, allowing for convenient and low-cost transportation to and storage at clinical centers. Finally, glycosylation is an important consideration in antibody formulations because changes in glycosylation can affect the rate of protein degradation. TCR-scFv bispecific proteins are typically produced in Escherichia coli (E. coli) and are therefore not glycosylated. Summary of the invention

[0005] It is desirable to provide pharmaceutical formulations for TCR-scFv bispecific proteins. It is particularly desirable that such formulations maintain protein stability at low protein concentrations and during long-term storage at elevated temperatures.

[0006] In a first aspect, the present invention provides a pharmaceutical preparation comprising:

[0007] a therapeutically effective amount of a bispecific protein comprising a soluble T cell receptor (TCR) and a scFv; and

[0008] Surfactants;

[0009] The w / w ratio of surfactant to protein is in the range of 0.75:1 to 1.5:1.

[0010] The inventors have found that the w / w ratio of surfactant to protein provides a stable formulation in the range of 0.75: 1 to 1.5: 1. Surfactants are commonly used excipients in protein formulations to prevent protein aggregation and adsorption to the surface. It has been shown in the art that a wide range of surfactant concentrations (0.02 mg / mL to 2.0 mg / mL or 0.002% to 0.2%) are suitable for preventing aggregation, and higher concentrations are more effective in preventing precipitation (Wang, Eur J Pharm Biopharm. 2017 May; 114: 263-277). In addition, in known protein formulations, the weight of the protein usually significantly exceeds the weight of the surfactant. For example, in the commercially available therapeutic antibody trastuzumab, the weight of the protein is 0.1: 1.5: 1. and bevacizumab In the formulation of , the antibody is more than 50 times higher than the surfactant. The inventors have found that in TCR-scFv bispecific protein formulations, increasing or decreasing the w / w ratio of surfactant to protein outside the narrow range of 0.75:1 to 1.5:1 unexpectedly leads to a significant loss of protein stability. Without being bound by theory, the inventors hypothesize that the low concentration of protein in the formulation is a result of the high potency of the drug, which means that the protein is more sensitive to small changes in surfactant concentration.

[0011] The w / w ratio of surfactant to protein may be in the range of 0.8: 1 to 1.2: 1, 0.9: 1 to 1.1 : 1 or 0.95: 1 to 1.05: 1. The ratio may be 0.75: 1, 0.8: 1, 0.85: 1, 0.9: 1, 0.95: 1, 1: 1, 1.05: 1, 1.1: 1, 1.15: 1, 1.2: 1. The preferred ratio is 1: 1.

[0012] The preparation of the present invention may contain one or more pharmaceutically acceptable surfactants. Examples of pharmaceutically acceptable surfactants include: polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80); poloxamers (e.g., poloxamer 188); Triton; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauroyl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearoyl sarcosine; linoleyl betaine, myristyl sarcosine, linoleyl sarcosine, or stearoyl sarcosine; Betaine or cetyl betaine; lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine or isostearamidopropyl betaine (e.g. lauramidopropyl); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl cocoyl taurate; polyethylene glycol, polypropylene glycol and copolymers of ethylene glycol and propylene glycol. Preferably, in the formulation of the present invention, the surfactant is a polysorbate, such as polysorbate 20 (trade name 20) or polysorbate 80 (trade name 80). More preferably, the surfactant is polysorbate 20 ( 20). The surfactant may be present at a w / v concentration of 0.1% or less. For example, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%. The concentration of the surfactant may be 0.02%. The concentration of the surfactant may be 0.05%. Preferably, the surfactant is polysorbate 20, present at a concentration of 0.02%.

[0013] The formulation of the first aspect may further comprise a filler and / or a stabilizer. Preferably, the ratio of the stabilizer to the filler is greater than 1:1.

[0014] In a second aspect, the present invention provides a pharmaceutical preparation comprising:

[0015] A therapeutically effective amount of a bispecific protein comprising a soluble T cell receptor (TCR) and a scFV;

[0016] Fillers; and

[0017] Stabilizers;

[0018] The w / w ratio of the stabilizer to the filler is greater than 1:1.

[0019] The present inventors have found that the use of fillers and stabilizers provides a stable formulation that is easy to freeze-dry, wherein the w / w ratio of stabilizer to filler is greater than 1:1. It is known that a mixture comprising a disaccharide stabilizer (e.g., trehalose or sucrose) and a filler (e.g., mannitol) is a suitable excipient for freeze-dried protein formulations (Johnson, J Pharm Sci. 2002 Apr; 91 (4): 914-22). Mannitol is easy to form a crystalline structure and promotes the formation of a stable cake that does not collapse during the drying process. However, in the crystalline state, mannitol cannot interact with protein, which may cause aggregation. Adding sucrose that forms an amorphous phase can prevent aggregation. It has been proposed in the art that a weight ratio of at least 3:1 of mannitol to sucrose is required to provide a sufficient balance between cake stability and prevention of aggregation; lower ratios have been shown to inhibit mannitol crystallization (Jena et al, Pharm Res. 2016 Jun; 33(6): 1413-25; Johnson et al, J Pharm Sci. 2002 Apr; 91(4): 914-22). Contrary to the teachings of the art, the inventors have found that a ratio of filler (e.g., mannitol) to stabilizer (e.g., sucrose / trehalose) of less than 1:1 (preferably 1:5) will produce a stable cake that minimizes aggregation. The inventors hypothesize that when crystalline mannitol is excessive, it may cause excessive strain on the structure of the TCR-scFv bispecific protein in the lyophilized state, subsequently leading to unfolding, denaturation, and aggregation.

[0020] The w / w ratio of stabilizer to filler may preferably be in the range of 3:1 to 7:1, more preferably in the range of 4:1 to 6:1. For example, the ratio may be 2:1 or 3:1 or 4:1 or 5:1 or 6:1. The ratio of stabilizer to filler may preferably be about 5:1, for example 5.5:0.9 to 4.5:1.1. The ratio of stabilizer to filler may more preferably be 5:1. The concentration of stabilizer may be in the range of 4.5% (w / v) to 5.5% (w / v), for example 5%. The concentration of filler used in the formulation may be 0.9% (w / v) to 1.1% (w / v), for example 1%.

[0021] Fillers are pharmaceutically acceptable compounds that act to increase the volume of the freeze-dried (lyo) cake. Compounds used as fillers are known in the art and include polyols (e.g., mannitol or sorbitol), carbohydrates, monosaccharides, polymers, or proteins. Preferably, the filler is a polyol. More preferably, the filler is mannitol. Preferably, mannitol is present at a concentration of 1%.

[0022] Stabilizers are pharmaceutically acceptable compounds that act as lyoprotectants / cytoprotectants when frozen or lyophilized (freeze drying), thereby preventing aggregation and / or other chemical or physical instability. Compounds used as stabilizers are known in the art. Examples of stabilizers include sugars, sugar alcohols and amino acids. Preferably, the stabilizer is a disaccharide. More preferably, the stabilizer is trehalose or sucrose. The stabilizer can be a mixture of trehalose and sucrose. Preferably, the stabilizer is trehalose present at a concentration of 5%. In a preferred formulation of the present invention, the stabilizer is trehalose and the filler is mannitol. The w / w ratio of trehalose to mannitol is preferably 5:1.

[0023] The formulations of the present invention may also include a buffer. Pharmaceutically acceptable buffers are known in the art. Examples include phosphate, citrate, lysine, histidine, acetate, succinate, and tris (tris) buffers. Any of these buffers may be used in the present invention. A preferred buffer is phosphate-citrate. The concentration of the buffer may be from about 20 mM to about 50 mM, such as 20 mM, 30 mM, 40 mM, or 50 mM. Preferably, the buffer concentration is 50 mM. Preferably, the formulation comprises a 50 mM phosphate-citrate buffer.

[0024] The optimal pH of the pharmaceutical formulation depends on the primary sequence of the protein and its concentration, as well as the properties and concentrations of other excipients. In preferred formulations of the present invention, the pH is in the range of about pH 6 to about pH 7, preferably 6.3 to 6.7. For example, the pH may be pH 6.0, or pH 6.1, or pH 6.2, or pH 6.3, or pH 6.4, or pH 6.5, or pH 6.6, or pH 6.7, or pH 6.8, or pH 6.9, or pH 7.0, with pH 6.5 being preferred and pH 6.0 also being preferred.

[0025] The preparation can be an aqueous preparation. Preferably, the aqueous carrier is a pharmaceutically acceptable (safe and non-toxic to human administration) aqueous carrier. The aqueous carrier can be distilled water or sterile water. In some embodiments, the aqueous preparation can be frozen. Alternatively, the aqueous preparation can be lyophilized to produce a lyophilized preparation. Suitable protocols and methods for preparing lyophilized pharmaceutical preparations from liquid preparations are known in the art. Methods for reconstituting lyophilized preparations before administration are known in the art. In short, freeze drying is accomplished by freezing the preparation and then sublimating ice from the frozen material at a temperature suitable for primary drying. Typically, the primary drying range is about -30°C to 25°C (assuming that the product remains frozen during the primary drying period) at a suitable pressure ranging from about 0.05mBar to about 0.5mBar. The size and type of the preparation, the container (e.g., a glass vial) containing the sample, and the volume of the liquid will primarily determine the time required for drying, which may vary from a few hours to a few days. The secondary drying stage can be performed at about 0°C to 40°C, depending primarily on the type and size of the container and the type of protein used. The time and pressure required for secondary drying will be the time and pressure to produce a suitable lyophilized cake (depending on, for example, temperature and other parameters). The secondary drying time depends on the residual moisture content required in the product, and generally requires at least about 5 hours (e.g., 15 hours to 24 hours). The pressure can be the same as the pressure used in the primary drying step. Freeze drying conditions can vary according to formulation and vial size.

[0026] The pharmaceutical formulations of the present invention are preferably stable. As used herein, the term "stability" or "stable" refers to a formulation in which one or more biophysical and / or chemical properties of the bispecific protein remain substantially unchanged when stored for a long time at 2°C to 8°C. A long time refers to up to at least 2 years, more preferably up to at least 5 years or longer. Additional or alternative conditions can be used as indicators of the potential long-term stability of the formulation at 2°C to 8°C. For example, stable can mean that the protein remains substantially unchanged after a defined number of freeze-thaw cycles, such as at least 5 or at least 10 freeze-thaw cycles; or stable can mean that the protein remains substantially unchanged after short-term storage at high temperature, such as storage at a temperature of about 30°C for at least one month.

[0027] The stability of the preparation can be evaluated by checking one or more biophysical or chemical properties, including but not limited to appearance (color and turbidity), protein concentration, pH, the presence of (subvisible) particles invisible to the naked eye, aggregation, denaturation and protein activity. Those skilled in the art know various analytical techniques that can be used to evaluate the stability of protein preparations. Examples include but are not limited to visual inspection, turbidimetry, light transmission method, absorbance at 280nm, capillary gel electrophoresis (Capillary gel electrophoresis, CGE), anion exchange chromatography (Anion exchange chromatography, AIEX), size exclusion chromatography (Size exclusion chromatography, SEC), protein activity assay. For freeze-dried samples, other analytical techniques may include assessing the physical stability of the cake and determining the moisture content (e.g., by Karl Fischer assessment). Specific techniques are further provided in the example section. Preferably, the stability of the preparation is determined by assessing one or more of the following characteristics, and preferably all characteristics: pH, visual appearance, protein concentration, the presence of particles invisible to the naked eye, CGE, SEC, AIEX and protein activity.

[0028] Bispecific protein comprises soluble TCR and scFv antibody fragment, preferably merged by joint.Joint sequence is usually flexible, because they are made up of amino acid (such as glycine, alanine and serine that do not have the bulky side chain that may limit flexibility).Available or optimal length joint sequence is easy to determine.Usually joint sequence length is less than about 12, for example, less than 10, or is 5 to 10 amino acids.TCR is made up of two disulfide bonded chains.It is generally believed that each chain (α and β) has two domains, i.e. variable domain and constant domain (referred to as TRAV / TRBV and TRAC / TRBC respectively).The variable domain of each chain is located at N-terminal, and comprises three complementary determining regions (ComplementarityDetermining Region, CDR) embedded in framework sequence.CDR comprises recognition site for peptide-MHC combination.Soluble TCR can comprise TRAV and TRBV and can be preferably human. Soluble TCR may comprise a mutation relative to a natural or wild-type (preferably human) TCR, so that it has a superphysiological affinity for antigens (examples of antigens include NY-ESO, MAGEA4 or PRAME). Soluble TCR may be a heterodimeric αβTCR polypeptide pair, in which the α polypeptide and the β polypeptide each have a TCR variable region and a constant region, but lack a TCR transmembrane region and a cytoplasmic region. Non-natural disulfide bonds may exist between the residues of the constant regions of the TCR α and β polypeptides. Soluble TCR can recognize antigens present on diseased cells, such as cancer cells or cells infected by viruses or bacteria. The two chains of soluble TCR can be produced as single chains (WO2004 / 033685) or as disulfide-linked dimers (WO03 / 020763). In a specific embodiment, the soluble TCR comprises an αβ heterodimer having a TRAV region and a TRBV region and an extracellular α chain TRAC constant domain sequence and an extracellular β chain TRBC1 or TRBC2 constant domain sequence. The TRAC or TRBV sequence can be modified by truncating or replacing the natural disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The TRAC or TRBV sequence can be modified by replacing the Thr48 position of TRAC and the Ser57 position of TRBC1 or TRBC2 with a cysteine ​​residue, which forms a disulfide bond between the α and β constant domains of the TCR. The constant regions of the α and β polypeptides can be connected by a natural disulfide bond between Cys4 of exon 2 of TRAC*01 and Cys2 of exon 2 of TRBC1 or TRBC2.The soluble TCR can be a single-chain αβTCR polypeptide of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ or Vα-L-Vβ-Cβ type, wherein Vα and Vβ are TCRα and β variable regions, respectively, Cα and Cβ are TCRα and β constant regions, respectively, and L is a linker sequence.

[0029] The term "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the structure required for antigen binding. The scFv may be derived from a human antibody, or it may have been humanized. The scFv may be derived from an antibody that recognizes CD3. In a specific embodiment, the anti-CD3 scFv may be derived from an UCHT1 antibody (Shalaby et al. J Exp Med. 1992 Jan 1; 175(1): 217-25, US5821337). The scFv may be fused to the N-terminus or C-terminus of a soluble TCR. It is preferred if the scFv is fused to the N-terminus of a soluble TCR (WO2010 / 133828).

[0030] The TCR-scFv bispecific protein can be present at a concentration of less than 1 mg / ml, less than 0.5 mg / ml, and preferably less than 0.3 mg / l. The concentration of the TCR-scFv bispecific protein can be in the range of 0.05 mg / ml to 0.5 mg / ml or 0.1 mg / ml to 0.3 mg / ml, or can be 0.5 mg / ml, 0.4 mg / ml, 0.3 mg / ml, 0.2 mg / ml, or 0.1 mg / ml. The preferred concentration is 0.2 mg / ml.

[0031] Specific TCR-scFv bispecific proteins that can be included in the formulation of the present invention include those disclosed in WO2011001152, WO2017 / 109496, WO2017 / 175006, and WO2018234319. The specific TCR-scFv bispecific protein of WO2011 / 001152 has: an alpha chain of SEQ ID No: 45, wherein amino acids 1 to 109 are replaced with SEQ ID No. 8, and the amino acid at position 1 is A, and the C-terminus of the alpha chain is truncated by 8 amino acids from F196 to S203 (including the end value) based on the numbering of SEQ ID No: 45; and a beta chain of SEQ ID No: 36, wherein residues 259 to 370 correspond to SEQ ID No. 27, and the amino acids at positions 1 and 2 are A and I, respectively. The pH of the formulation containing such a TCR-scFv bispecific protein may preferably be 6.5.

[0032] Specific formulations according to the present invention include:

[0033] A TCR-scFv bispecific protein present at a concentration of 0.2 mg / ml and having: an alpha chain of SEQ ID No: 45 of WO2011001152, wherein amino acids 1 to 109 are replaced with SEQ ID No. 8 of WO2011001152, and the amino acid at position 1 is A, and the C-terminus of the alpha chain is truncated by 8 amino acids from F196 to S203 (inclusive) based on the numbering of SEQ ID No: 45; and a beta chain of SEQ ID No: 36 of WO2011001152, wherein residues 259 to 370 correspond to SEQ ID No. 27 of WO2011001152, and the amino acids at positions 1 and 2 are A and I, respectively;

[0034] 50 mM phosphate-citrate buffer;

[0035] 5% trehalose;

[0036] 1% mannitol; and

[0037] 0.02% w / v Polysorbate 20,

[0038] And the pH of the specific formulation of the present invention is 6.5.

[0039] In an alternative preferred embodiment, the formulation comprises:

[0040] 0.5 mg / ml of a TCR-scFv bispecific protein, such as described in WO2017 / 109496;

[0041] 50 mM phosphate-citrate buffer;

[0042] 5% trehalose;

[0043] 1% mannitol; and

[0044] 0.05% w / v Polysorbate 20,

[0045] And the pH of the preparation was 6.0.

[0046] In an alternative preferred embodiment, the formulation comprises:

[0047] 0.2 mg / ml of a TCR-scFv bispecific protein, such as described in WO2017 / 175006;

[0048] 50 mM phosphate-citrate buffer;

[0049] 5% trehalose;

[0050] 1% mannitol; and

[0051] 0.02% w / v Polysorbate 20,

[0052] And the pH of the preparation was 6.0.

[0053] Preferably, the bispecific protein in the formulation of the invention is non-glycosylated.

[0054] In another aspect, the present invention also provides a method for preparing a pharmaceutical preparation, the method comprising:

[0055] Formulated with: (i) a therapeutically effective amount of a bispecific protein comprising a soluble T cell receptor (TCR) and a scFV; and (ii) a surfactant,

[0056] The w / w ratio of surfactant to protein is in the range of 0.75:1 to 1.5:1.

[0057] In another aspect, the present invention also provides a method for preparing a pharmaceutical preparation, the method comprising:

[0058] Formulated with: (i) a therapeutically effective amount of a bispecific protein comprising a soluble T cell receptor (TCR) and a scFV; (ii) a filler; and (iii) a stabilizer.

[0059] The ratio of stabilizer to filler is greater than 1:1.

[0060] The present invention also provides the use of the preparation of the present invention in medicine. The preparation can be used in the method for treating a disease including cancer or an infectious disease. The cancer can be a melanoma (including but not limited to skin melanoma or uveal melanoma (also known as ocular melanoma), malignant lentigo melanoma, superficial spreading melanoma, acral lentigo melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue melanoma, small cell melanoma with small nevus-like cells, and spitzoid melanoma. Alternatively, the cancer can be breast cancer (including triple-negative), ovarian cancer, endometrial cancer, esophageal cancer, lung cancer (NSCLC and SCLC), bladder cancer, colon cancer, gastric cancer, liver cancer, pancreatic cancer, prostate cancer, connective tissue cancer (i.e., sarcoma) or head and neck cancer; or the cancer may be a leukemia or lymphoma.

[0061] In a preferred embodiment, the formulation can be administered by injection, such as intravenous injection. The formulation can be administered by continuous infusion (e.g., several hours, days or weeks) or by intermittent infusion (e.g., once, twice or three times a week, or less frequently).

[0062] Alternatively, the formulation may be suitable for administration by any other suitable route, including intratumoral subcutaneous, intramuscular, intrathecal, enteral (including oral or rectal), inhalation, or intranasal.

[0063] In another aspect, the present invention provides a method of treating a human subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation disclosed herein.

[0064] Preferred features of any one aspect of the invention apply mutatis mutandis to each of the other aspects of the invention.The prior art documents mentioned herein are incorporated herein to the maximum extent permitted by law. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The present invention is further described in the following non-limiting examples. With reference to the accompanying drawings, in which:

[0066] Figure 1 SE-UPLC, AE-HPLC, and CGE analysis results of a formulation containing 0.2 mg / ml TCR-scFv bispecific protein, 50 mM phosphate-citrate, pH 6.5, 5% trehalose, 1% mannitol, and 0.02% Tween 20 are provided;

[0067] Figure 2 SE-UPLC, AE-HPLC and CGE analysis results of a formulation containing 0.2 mg / ml of a selective TCR-scFv bispecific protein, 50 mM phosphate-citrate at pH 6.5, 5% trehalose, 1% mannitol and 0.02% Tween 20 are provided;

[0068] Figure 3 Provided are the results of light transillumination analysis of formulations comprising 0.2 mg / ml TCR-scFv bispecific protein, 50 mM phosphate-citrate, pH 6.5, 5% trehalose, 1% mannitol, and 0.06%, 0.04%, 0.02%, 0.01%, or 0.005% Tween 20;

[0069] Figure 4Provides results of Design of Experiments (DoE) analyses of formulations comprising 0.2 mg / ml TCR-scFv bispecific protein, 50 mM phosphate-citrate, pH 6.5, 5% trehalose, 1% mannitol, and 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, or 0.08% Tween 20;

[0070] Figure 5 SE-UPLC, AE-HPLC, and CGE analysis results of a formulation containing 0.5 mg / ml TCR-scFv bispecific protein, 50 mM phosphate-citrate, pH 7.5, 120 mM NaCl, and 0.05% Tween 20 are provided;

[0071] Figure 6 The results of turbidity analyses of formulations containing 0.2 mg / ml TCR-scFv bispecific protein, 50 mM phosphate-citrate, pH 6.5, 5% mannitol, and 0.02% Tween in the presence or absence of 1% sucrose are presented. DETAILED DESCRIPTION

[0072] Example

[0073] Example 1 - Stable Aqueous Formulation of Soluble TCR-scFv Bispecific Protein

[0074] A)

[0075] A bispecific protein comprising a soluble TCR fused to an anti-CD3 scFv was prepared using known methods (the alpha chain of SEQ ID No: 45 of WO2011 / 001152, wherein amino acids 1 to 109 are substituted with SEQ ID No. 8 of WO2011001152, and the amino acid at position 1 is A, and the C-terminus of the alpha chain is truncated by 8 amino acids from F196 to S203 (inclusive) based on the numbering of SEQ ID No: 45; and the beta chain of SEQ ID No: 36 of WO2011 / 001152, wherein residues 259 to 370 correspond to SEQ ID No. 27 of WO2011 / 001152, and the amino acids at positions 1 and 2 are A and I, respectively), and the buffer was exchanged into a formulation buffer using size exclusion chromatography (SEC). The protein was then diluted to a final concentration of 0.20 ± 0.02 mg / mL using formulation buffer and filled into bottles in a laminar airflow (LAF) bench.

[0076] The formulation buffer contained 50 mM phosphate-citrate, 5% trehalose, 1% mannitol, and 0.02% Tween 20. The pH was 6.5.

[0077] Protein stability was monitored for 24 months at <-60°C or +5°C. Stability at each temperature was assessed using a variety of analytical methods, including capillary gel electrophoresis (CGE), anion exchange high-performance liquid chromatography (AE-HPLC), and size exclusion ultra-performance liquid chromatography (SE-UPLC). Other assessments included visual inspection, measurement of protein content and pH, detection of particles invisible to the naked eye by light transillumination, and activity assays to confirm functionality. Ten freeze-thaw cycles were performed to demonstrate protein stability.

[0078] SE-UPLC was used to detect the presence of multimers and other higher molecular weight species and was performed on a UPLC system using a 1.7 μm, 4.6 mm × 300 mm UPLC SE column, a 0.2 M phosphate mobile phase at pH 7.5, and an isocratic phosphate mobile phase flow rate of 0.2 ml / min. The method involves separating the protein components in the test sample on the SE column using fluorescence detection on the UPLC system after injecting the appropriate amount onto the column. Figure 1 A shows the purity assessment measured by SE-UPLC at eight time points over the course of 24 months at +5°C. Purity at 0 months was 99.2% and at 24 months was 99.3%. Minimum acceptance criteria = > 95%

[0079] AE-HPLC was used to detect charge-based degradation and was performed on an HPLC system using a 2 mm x 250 mm weak AE column with a mobile phase of phosphate at pH 8.2 flowing at 0.2 ml / min. The protein was eluted with a NaCl gradient in phosphate buffer at pH 8.2. The method involved separating the protein components in the test sample on an AE column using fluorescence detection on the HPLC system after injecting the appropriate amount onto the column. Figure 1 B shows the charge state assessment measured by AE-HPLC at eight time points during 24 months at +5°C. The % main peak at 0 months was +3.4% and the reading at 24 months was -1.9% compared to the reference, indicating that the product was stable in this formulation (acceptance criteria within ±15% of the reference).

[0080] CGE is used to determine the presence of low molecular weight impurities and degradation products (e.g., fragments). The method is performed using a chip and standard CGE. In the chip method, the sample is prepared to a final concentration of 0.2 mg / mL and heated at 70°C for 5 minutes. After cooling, the denatured sample is loaded onto the chip and separated on a bioanalyzer (Agilent Technologies). Standard CGE is run on a PA 800plus instrument with a diode array detector (Beckman Coulter) using reagents and separation capillaries (57 cm×50 μm ID, bare fused quartz) from the Proteolab SDS Mw analysis kit. The sample is prepared at 0.2 mg / mL, then diluted in SDS sample buffer and heated at 70°C for 5 minutes. The analysis is read at 220 nm and the migration time and corrected peak area% are recorded. Minimum acceptance criteria=>95%

[0081] Figure 1 C shows the purity assessment measured by CGE at seven time points over the course of 24 months at +5°C. The purity at 0 months was 99.6% and at T = 24 months was 99.7%, indicating that the product was stable in this formulation.

[0082] Stability assessment after multiple freeze-thaw cycles was used as a further indicator of long-term stability. Freeze-thaw cycles were performed by rapidly freezing the sample to <-60°C and then incubating at room temperature (about 22°C). Stability was assessed by SE-UPLC as described above. The results showed that the purity was 99.6% before exposure to 10 freeze-thaw cycles and 99.5% after that, indicating that the product was stable in this formulation.

[0083] Visual analysis was performed according to the principles of the pharmacopoeial method (Ph.Eur.2.2.1, 2.2.2 and 2.9.20). Clear, colorless solutions essentially free of particles were observed after 0 and 24 months at +5°C. In addition, no visual changes were detected after 10 freeze-thaw cycles.

[0084] After 24 months, protein concentration and pH were within + / - 0.02 mg / ml and 0.2 pH units of the specified values.

[0085] Evaluation of particles invisible to the naked eye by laser light transmission showed that after 24 months, particles ≥25 μm were present at ≤600 / bottle and particles ≥10 μm were present at ≤6000 / bottle (i.e. within the acceptance criteria). Turbidity measurements were within the acceptable range (<3 NTU (opalescence standard I)).

[0086] These data indicate that the formulation is stable during long term storage at temperatures between 2°C and 8°C.

[0087] The freeze-dried formulation was prepared as follows: 0.6 ml of the sample was frozen and then kept in a 2R glass vial at -45°C for 2 hours, followed by primary drying at -15°C / 0.07 mbar for 22 hours and secondary drying at +40°C / 0.07 mbar for 23 hours. The sample was then sealed in a vial with a rubber stopper under vacuum. The freeze-dried sample was stored at +5°C for a long term. The sample was reconstituted by adding 0.6 ml of sterile water for injection to the vial and mixing.

[0088] B)

[0089] A second bispecific protein comprising an alternative soluble TCR fused to an anti-CD3 scFv (as described in WO2017 / 175006) was prepared at a final concentration of 0.20 ± 0.02 mg / mL. The formulation buffer contained 20 mM phosphate-citrate, 5% trehalose, 1% mannitol and 0.02% Tween 20. The pH was 6.0.

[0090] Stability assessment was performed over a 12 month period using the same methodology as described in Part A.

[0091] SE-UPLC, AE-HPLC and CGE data are shown in Figure 2 A to Figure 2 C. The purity obtained by SE-UPLC was 98.9% at 0 months and 99.3% at 12 months. The charge state obtained by AE-HPLC was -12.2% of the main peak at 0 months and -19.5% at 12 months compared to the reference (acceptance criteria within ±20% of the reference). The purity obtained by CGE was 98.8% at 0 months and 98.3% at 12 months. In each case, extrapolation of the data indicated that stability ≥ 24 months can be expected.

[0092] SE-UPLC also demonstrated stability after 10 freeze-thaw cycles.

[0093] These data indicate that the formulation is stable during long term storage at temperatures between 2°C and 8°C.

[0094] Example 2 - Formulation Optimization

[0095] A-Detergent Protein Ratio

[0096] The weight ratio between detergent and protein was found to be a critical factor in producing a stable formulation.

[0097] Evidence for this is obtained from laser light obscuration (LO) measurements, which assess the presence of particles invisible to the naked eye of 2 μm and larger after 5 freeze-thaw cycles. The formulation buffer contains the TCR-scFv bispecific protein of Example 1A, 50 mM phosphate-citrate, 5% trehalose, 1% mannitol, and one of 5 different concentrations of Tween 20. The pH is 6.5. The protein concentration is kept constant at 0.2 mg / ml. The assay is performed on a PAMAS SVSS-C or equivalent particle size analyzer. The samples were analyzed in pure form with a pre-run volume of 0.4 ml and a measurement volume of 0.1 ml in triplicate. Particle counts of ≥2 μm, ≥10 μm, and ≥25 μm were determined.

[0098] Figure 3 It is shown that when the ratio of Tween 20 to protein is 1, the particle count of particles ≥ 10 μm is the lowest, and thus the stability is the highest.

[0099] Further evidence for the importance of the detergent-to-protein ratio was obtained from a design of experiments (DoE) study. In this experiment, the stability of the bispecific protein of Example 1A was examined under 32 different conditions at a fixed protein concentration of 0.2 mg / ml. The variable included detergent concentration. In each case, the formulation was stored at +30°C ± 3°C for 1 month and stability was monitored by 5 freeze-thaw cycles at elevated temperature. The effect of Tween 20 concentration (thus the w / w ratio of Tween 20 / protein ranged from 1 to 4) was evaluated by multiple analytical methods including SE-UPLC, AE-HPLC and CGE. In addition, the bispecific protein activity was assessed using ELISA. The results are summarized in Figure 4 middle.

[0100] B-Mannitol + Sucrose / Trehalose

[0101] The inclusion of mannitol and either sucrose or trehalose was found to be a critical factor in the formulation and was necessary to provide a formulation that was easily lyophilized.

[0102] The bispecific protein of Example 1A was prepared in a formulation buffer containing 50 mM phosphate-citrate, 120 mM NaCl and 0.05% Tween 20 at a final protein concentration of 0.5 mg / ml. The pH was 7.5. Stability was assessed using the same method as described in Example 1. Figure 5 A to Figure 5C shows SE-UPLC, AE-HPLC and CGE data during 36 months at +5°C. The data indicate that the formulation is unstable after 12 months. Using the DoE method, it was subsequently found that adding 5% mannitol or 5% sucrose improved stability. Omitting NaCl is also preferred. However, as determined by laser light transmission (LO) and turbidity (NTU) measurements, the formulation containing 5% mannitol resulted in high levels of aggregation after 10 freeze-thaw cycles. Turbidity is measured by turbidimetry using a turbidimeter and calibrated according to standards of known turbidity.

[0103] Figure 6 It was shown that the addition of 1% sucrose reduced freeze-thaw induced aggregation.

[0104] To further investigate the lyophilization preparation, a formulation containing 50 mM phosphate-citrate, 5% mannitol, 1% sucrose, 0.02% Tween 20 was prepared with a final protein concentration of 0.2 mg / ml. The pH was 6.5. The samples were lyophilized in glass DP lyophilization vials and stored at 50°C, 25°C, and 40°C for 1, 2, and 3 months. The samples were subsequently rehydrated by adding 0.5 ml of ultrapure water and incubated at room temperature for 1 hour before evaluation using a variety of techniques including turbidity, laser light transmission, SE-UPLC, AE-HLPC, CGE, and protein activity. Increased aggregation and significant loss of protein activity were observed at higher temperatures, indicating that the formulation is not suitable for long-term storage.

[0105] In order to improve long-term stability and maintain liquid stability, it was subsequently found that the ratio of mannitol to either sucrose was important for maintaining stability, and trehalose could be interchanged with sucrose. Lyophilization and storage at 25°C or 40°C for 4 weeks were followed by evaluation of the formulations of the bispecific protein of Example 1A containing two different mannitol / sucrose ratios. Stability was evaluated using a variety of techniques, including turbidity, laser light transmission, SE-UPLC, AE-HLPC, and protein activity. Turbidity data are shown in the table below, which shows that higher concentrations of mannitol relative to sucrose can lead to increased aggregation.

[0106]

Claims

1. A pharmaceutical preparation comprising: A therapeutically effective amount of a bispecific protein comprising a soluble T cell receptor (TCR) and a scFV; Surfactants; Fillers; and Stabilizers; wherein the w / w ratio of the surfactant to the protein is in the range of 0.75:1 to 1.5:1, the ratio of the stabilizer to the filler is in the range of 3:1 to 7:1, the surfactant is polysorbate 20, the filler is mannitol, the stabilizer is sucrose and / or trehalose, the bispecific protein is present at a concentration of less than 1 mg / ml and the pH is in the range of 6 to 7.

2. The preparation according to claim 1, wherein The w / w ratio of the surfactant to the protein was 1:

1.

3. The formulation according to claim 1, further comprising a buffer.

4. The preparation according to claim 3, wherein The buffer was phosphate / citrate.

5. The preparation according to claim 1, wherein (a) The TCR-scFv bispecific protein has: (i) an alpha chain having the following sequence: GDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRAGNY IATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT; (ii) a beta strand having the following sequence: AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGASPISFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD; or (b) The TCR-scFv bispecific protein has: (i) An α-chain having the following sequence: GDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT; and (ii) A β-chain having the following sequence: AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGASPIRFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD; or (c) The TCR-scFv bispecific protein has: (i) an α chain having the following sequence: GDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT; and (ii) a β chain having the following sequence: AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGG GSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGSAPIRFGPGTRLTVTEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

6. The formulation of claim 5, wherein the TCR-scFv bispecific protein has: (i) an alpha chain having the following sequence: GDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNY IATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT; (ii) a beta strand having the following sequence: AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGG GSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGASPIRFGPGTRLTVTEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

7. The formulation of claim 1, wherein the bispecific protein has an α chain and a β chain, the α chain having the following sequence: AQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDGSTPMQFGKGTRLSVIANIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT The beta strand has the following sequence: AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGG GSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVT VSSGGGGSDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSWAQGDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWGAPYEQYFPGGTRLTVTEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

8. The preparation according to claim 1, comprising 50 mM phosphate-citrate buffer, 5% Trehalose, 1% mannitol, and 0.02% w / v Polysorbate 20, wherein the pH of the formulation is 6.0, 6.5 or 6.8 and the bispecific protein is present at a concentration of 0.2 mg / ml.

9. The preparation according to claim 1, comprising 20 mM phosphate-citrate buffer, 5% Trehalose, 1% mannitol, and 0.02% w / v Polysorbate 20, wherein the pH of the formulation is 6.0, 6.5 or 6.8 and the bispecific protein is present at a concentration of 0.2 mg / ml.

10. The preparation according to claim 1, wherein The formulation is aqueous.

11. The preparation according to claim 1, wherein The formulation is lyophilized.

12. A method for preparing an aqueous pharmaceutical preparation, comprising: Formulating: (i) a therapeutically effective amount of a bispecific protein comprising a soluble T cell receptor (TCR) and a scFV; (ii) surfactant; (iii) filler; and (iv) a stabilizer, wherein the w / w ratio of the surfactant to the protein is in the range of 0.75:1 to 1.5:1, the ratio of the stabilizer to the bulking agent is in the range of 3:1 to 7:1, the surfactant is polysorbate 20, the bulking agent is mannitol, the stabilizer is sucrose and / or trehalose, the bispecific protein is present at a concentration of less than 1 mg / ml and the pH is in the range of 6 to 7.

13. The method according to claim 12, wherein the aqueous pharmaceutical formulation is a formulation as defined in any one of claims 2 to 11.

14. Use of the preparation according to claim 1 in the preparation of a medicament for treating cancer.

15. The use according to claim 14, wherein the cancer is melanoma.

16. The use according to claim 15, wherein the melanoma is uveal melanoma.

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