Pharmaceutical formulations of masked antibodies

Abstract

The present disclosure relates to low pH pharmaceutical compositions and / or formulations comprising masked antigen binding proteins. In particular, the disclosure relates to pharmaceutical formulations of masked antibodies. Provided is a pharmaceutical composition comprising: (a) a masked antigen-binding protein comprising: (i) an antigen-binding antibody or antigen-binding fragment thereof (AB1); and (ii) a masking domain (MD1) conjugated to AB1, wherein the masking domain comprises: a. A masking peptide or polypeptide (MP1) that inhibits or reduces the binding of AB1 to the antigen; and b. A protein recognition site (PR1), wherein binding of a protein to the PR1 or cleavage of the PR1 by the protease increases binding of AB1 to the antigen; (b) at least one buffering agent; and (c) at least one polyol, wherein the pH of the pharmaceutical composition is lower than the isoelectric point (pI) of the AB1 and MD1 regions of the masked antigen binding protein.

Description

[0001] This application is a divisional application of an application filed on July 30, 2019, with application number 201980050417.X, and invention name “Pharmaceutical formulations of masked antibodies”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 712,906, filed on July 31, 2018, which is hereby incorporated by reference. Technical Field

[0004] The present disclosure relates to low pH pharmaceutical compositions and / or formulations comprising masked antigen binding proteins.

[0005] Cross-references to materials incorporated by reference

[0006] The computer-readable nucleotide / amino acid sequence listing filed concurrently with this article is incorporated by reference in its entirety and is identified as follows: ASCII (text) file named "52811_Seqlisting.txt", created on July 30, 2018, and 12,007 bytes in size. Background Art

[0007] Protein-based drugs are one of the fastest growing therapeutic agents in clinical (pre) development and commercial products. Compared with small chemical drugs, protein drugs have high specificity and activity at relatively low concentrations, and typically provide treatment for high-impact diseases, such as various cancers, autoimmune diseases and metabolic disorders (Roberts, Trends Biotechnol. [Biological Technology Trends] July 2014; 32 (7): 372-80, Wang, Int J Pharm. [International Journal of Pharmacy] August 20, 1999; 185 (2): 129-88).

[0008] Due to the progress of commercial scale purification methods, protein-based drugs, such as recombinant proteins and antibodies, can now be obtained in high purity during manufacture. However, recombinant proteins (including antibodies) are only marginally stable and are highly susceptible to both chemical and physical degradation. Chemical degradation refers to modifications involving covalent bonds, such as deamidation, oxidation, cleavage or formation of new disulfide bridges, hydrolysis, isomerization or deglycosylation. Physical degradation includes protein unfolding, undesirable adsorption and aggregation on the surface. Dealing with these physical and chemical instabilities is one of the most challenging tasks in protein drug development (Chi et al., Pharm Res [Drug Research], Vol. 20, No. 9, September 2003, pp. 1325-1336, Roberts, Trends Biotechnol. [Biotechnology Trends] July 2014; 32 (7): 372-80).

[0009] Protein aggregation represents a major event in the physical instability of proteins and antibodies, and its occurrence is generally attributed to the inherent tendency to minimize thermodynamically unfavorable interactions between solvents and hydrophobic protein residues. This is particularly problematic because it is often encountered during production, refolding, purification, sterilization, transportation and storage. Aggregation can occur even under solution conditions where the native state of a protein or antibody is thermodynamically highly favorable (e.g., neutral pH and 37°C) and unstressed (Chi et al., Pharm Res, Vol. 20, No. 9, Sep 2003, pp. 1325-1336, Roberts, Trends Biotechnol. 2014 Jul;32(7):372-80, Wang, Int J Pharm. 1999 Aug 20;185(2):129-88, Mahler J Pharm Sci. 2009 Sep;98(9):2909-34).

[0010] Antibody aggregation is problematic because it impairs biological activity. In addition, antibody aggregation results in an undesirable drug product appearance and reduces product yield due to the complex purification steps required to remove aggregates from the final product. Recently, there has been increasing concern that aggregated antibodies (even humanized or fully human antibodies) can significantly increase the risk of patients developing an immune response to active antibody / protein monomers, leading to the formation of neutralizing antibodies and drug resistance, or other adverse side effects (Mahler J Pharm Sci. [Journal of Pharmaceutical Science] September 2009; 98(9): 2909-34).

[0011] In general, several efforts to minimize protein aggregation by multiple mechanisms have been reported.By modifying the primary structure of protein (including antibody), it is also possible to stabilize protein (including antibody) and therefore prevent aggregate formation and other chemical changes, thereby increasing internal hydrophobicity and reducing external hydrophobicity.However, the genetic engineering of protein or antibody may cause functional impairment and / or immunogenicity increase.Another method focuses on the dissociation of aggregate (called "depolymerization"), to recover functional natural monomers by using multiple mechanisms such as temperature, pressure, pH and salt.At present, protein and antibody aggregates are mainly removed as impurities in the refining step of downstream processing.However, in the case of high-level high molecular weight (HMW) antibody species, removing a large amount of HMW not only causes significant yield loss, and challenges (Chi et al., Pharm Res [drug research], volume 20, the 9th phase, September 2003, pp. 1325-1336) have been proposed to the robust downstream processing of design.

[0012] Maintaining protein stability and activity in biological and biotechnological applications faces serious challenges. There is a need in the art for optimized pharmaceutical compositions that provide enhanced stability of antibodies or therapeutic proteins and reduce aggregation and denaturation or degradation during formulation, filling, transportation, storage and administration, thereby preventing loss of function and adverse immunogenic reactions. Summary of the invention

[0013] The present disclosure provides pharmaceutical compositions and / or formulations for minimizing or reducing aggregation and / or high molecular weight species of masked antigen-binding proteins. In some embodiments, the present disclosure provides a pharmaceutical composition or formulation comprising: (A) a masked antigen-binding protein (including, for example, a Probody or ProTIA prodrug), the masked antigen-binding protein comprising: (i) an antibody or antigen-binding fragment thereof (AB1) that binds to an antigen; and (ii) a masking domain (MD1) coupled to AB1, wherein the masking domain comprises: (a) a masking peptide or masking polypeptide (MP1) that inhibits or reduces the binding of AB1 to the antigen; and (b) a protein recognition site (PR1), wherein the binding of a protein to the PR1 or the cleavage of the PR1 by a protease increases the binding of AB1 to the antigen; (B) at least one buffer; and (C) at least one polyol, wherein the pH of the pharmaceutical composition or formulation is lower than the isoelectric point (pI) of the AB1 and MD1 regions of the masked antigen-binding protein.

[0014] In some other embodiments, the present disclosure also provides a pharmaceutical composition or formulation, which comprises: (A) a masked antigen-binding protein (including, for example, a Probody or a ProTIA prodrug), the masked antigen-binding protein comprising: (i) a first antibody or antigen-binding fragment thereof (AB1) that binds to an antigen; and (ii) a first masking domain (MD1) coupled to AB1, wherein the masking domain comprises: (a) a masking peptide or masking polypeptide (MP1) that inhibits or reduces the binding of AB1 to the antigen; and (b) a protein recognition site (PR1), wherein the binding of a protein to the PR1 or the cleavage of the PR1 by a protease increases the binding of AB1 to the antigen. binding; (iii) a second antibody or antigen-binding fragment thereof (AB2) that binds to a second antigen; and (vi) a second masking domain (MD2) coupled to AB2, wherein the second masking domain comprises: (a) a masking peptide or masking polypeptide (MP2) that inhibits or reduces the binding of AB2 to the antigen; and (b) a second protein recognition site (PR2), wherein binding of a protein to the PR2 or cleavage of the PR2 by a protease increases the binding of AB2 to the second antigen; (B) at least one buffer; and (C) at least one polyol, wherein the pH of the pharmaceutical composition or formulation is lower than the isoelectric point (pI) of the AB1, AB2, MD1 and MD2 regions of the masked antigen-binding protein.

[0015] In some aspects, the AB1 and / or AB2 region of the masked antigen binding protein (including, for example, a Probody or ProTIA prodrug) is a scFv or Fab, and / or binds to an antigen expressed on the surface of a cell (including, for example, CD3).

[0016] The pharmaceutical composition or preparation of the present disclosure include at least one buffer. In certain embodiments, the buffer is selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate and 2-(N-morpholino) ethanesulfonate or a combination thereof. In certain embodiments, the at least one buffer is present in a composition or preparation in a concentration range of from about 5mM to about 200mM (e.g., 5mM to 200mM) or about 10mM to about 50mM (e.g., 10mM to 50mM).

[0017] The pharmaceutical composition or formulation of the present disclosure also includes at least one polyol. In certain embodiments, the polyol is sugar. In certain embodiments, the sugar is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched dextran and linear non-branched dextran or a combination thereof. In certain embodiments, the polyol is a sugar alcohol (e.g., sorbitol). In certain embodiments, the polyol is sucrose, trehalose, mannitol, sorbitol, xylitol, erythritol, isomalt, glycerol, cyclodextrin or sulfobutyl cyclodextrin (captisol). In certain embodiments, the at least one polyol is present in a composition or formulation in a concentration range of from about 1% (w / V) to about 20% (w / V) (or about 9% (w / V) to about 12% (w / V)).

[0018] In some cases, the pharmaceutical composition or formulation of the present disclosure may optionally further include at least one surfactant. In certain embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, pluronic F68, triton X-100, polyoxyethylene 3 and PEG 3350, PEG 4000, PEG 8000 or a combination thereof. In certain embodiments, the at least one surfactant is present in the composition in a concentration range of from 0.004% (w / V) to about 0.5% (w / V) (or about 0.001% (w / V) to about 0.01% (w / V)).

[0019] In some embodiments, the pH of the composition or formulation is greater than 3.5, and / or the pH is about 2 pH units lower than the pI of the AB1, AB2, MD1 and / or MD2 regions (described further below) of the masked antigen binding protein (including, e.g., Probody or ProTIA prodrug). In some embodiments, the pH of the composition or formulation is between 7.0 and 3.6. In some embodiments, the pH range of the composition or formulation is from about 3.5 to about 5.5 or ranges from 4.0 to 5.0. In some embodiments, the pH of the composition or formulation is about 4.2 or about 3.6. In some embodiments, the pH of the composition or formulation is 4.2 or 3.6.

[0020] The pharmaceutical compositions or formulations of the present disclosure may optionally further comprise an amino acid excipient. In some embodiments, the amino acid excipient is present in a concentration range from about 0.1% (mM) to about 20% (mM).

[0021] In some embodiments, the pharmaceutical composition or formulation of the present disclosure comprises 10 mM glutamate, 9% (w / V) sucrose and 0.01% (w / V) polysorbate 80, and wherein the pH of the liquid pharmaceutical composition is 4.2 or 3.6. In some embodiments, the masked antigen-binding protein is present in the composition at a concentration ranging from about 0.1 mg / mL to about 17 mg / mL, or from about 0.1 mg / mL to about 30 mg / mL. In some embodiments, the masked antigen-binding protein is present in the composition in an amount ranging from about 50 μg to about 200 mg.

[0022] The pharmaceutical composition of the present disclosure can be a lyophilized composition or a liquid composition. In some embodiments, the pharmaceutical composition is a reconstituted lyophilized composition.

[0023] In some embodiments, the pharmaceutical composition is a liquid composition that is stable after storage for 0, 1, 2, and / or 4 weeks at 2° C.-8° C., 4° C., and / or 25° C. In some embodiments, the pharmaceutical composition or formulation has about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold less antibody aggregation and / or high molecular weight antibody species as measured by ultra high performance liquid chromatography (SE-UHPLC) compared to a reference pharmaceutical composition consisting of 1 mg / ml masked antigen binding protein, 100 mM glycine, pH 4.9.

[0024] In another aspect, described herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition or formulation of the disclosure.

[0025] Specifically, this article provides the following:

[0026] 1. A pharmaceutical composition comprising:

[0027] (a) A masked antigen-binding protein comprising:

[0028] (i) an antibody or an antigen-binding fragment thereof (AB1) that binds to an antigen; and

[0029] (ii) a masking domain (MD1) coupled to AB1, wherein the masking domain comprises:

[0030] a. a masking peptide or masking polypeptide (MP1) that inhibits or reduces the binding of AB1 to the antigen; and

[0031] b. a protein recognition site (PR1), wherein binding of a protein to the PR1 or cleavage of the PR1 by a protease increases binding of AB1 to the antigen;

[0032] (b) at least one buffer; and

[0033] (c) at least one polyol,

[0034] The pH of the pharmaceutical composition is below the isoelectric point (pi) of the AB1 and MD1 regions of the masked antigen binding protein.

[0035] 2. The pharmaceutical composition of item 1, wherein the masked antigen-binding protein further comprises:

[0036] (iii) a second antibody or antigen-binding fragment thereof (AB2) that binds to a second antigen; and

[0037] (iv) a second masking domain (MD2) coupled to AB2, wherein the second masking domain comprises:

[0038] a. a masking peptide or masking polypeptide (MP2) that inhibits or reduces the binding of AB2 to the antigen; and

[0039] b. a protein recognition site (PR2), wherein binding of a protein to the PR2 or cleavage of the PR2 by a protease increases binding of AB2 to the antigen;

[0040] wherein the pH of the pharmaceutical composition is below the isoelectric point (pi) of the AB1, AB2, MD1 and MD2 regions of the masked antigen binding protein.

[0041] 3. The pharmaceutical composition of item 1 or 2, wherein the masked antigen binding protein is a Probody or ProTIA prodrug.

[0042] 4. The pharmaceutical composition according to any one of items 1 to 3, wherein AB1 and / or AB2 is scFv or Fab.

[0043] 5. The pharmaceutical composition according to any one of items 1 to 4, wherein AB1 and / or AB2 binds to an antigen expressed on the surface of a cell.

[0044] 6. A pharmaceutical composition as described in any of items 1-5, wherein the at least one buffer is selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate and 2-(N-morpholino)ethanesulfonate or a combination thereof.

[0045] 7. The pharmaceutical composition of claim 6, wherein the at least one buffer is present in a concentration range of about 5 mM to about 200 mM.

[0046] 8. The pharmaceutical composition of claim 7, wherein the at least one buffer is present in a concentration range of about 10 mM to about 50 mM.

[0047] 9. The pharmaceutical composition of any one of items 1-8, wherein the at least one polyol is selected from the group consisting of sugars, cyclic polysaccharides, sugar alcohols, linear branched glucans and linear unbranched glucans or a combination thereof.

[0048] 10. The pharmaceutical composition according to item 9, wherein the sugar is a monosaccharide or a disaccharide.

[0049] 11. The pharmaceutical composition of any one of items 1-10, wherein the polyol is selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, xylitol, erythritol, isomalt, glycerol, cyclodextrin, sulfobutyl cyclodextrin or a combination thereof.

[0050] 12. The pharmaceutical composition of any one of items 1-11, wherein the at least one polyol is present in a concentration range of about 1% (w / V) to about 20% (w / V).

[0051] 13. The pharmaceutical composition of claim 12, wherein the at least one polyol is present in a concentration range of about 9% (w / V) to about 12% (w / V).

[0052] 14. The pharmaceutical composition according to any one of items 1 to 13, further comprising at least one surfactant.

[0053] 15. A pharmaceutical composition as described in item 14, wherein the at least one surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, Pluronic F68, Triton X-100, polyoxyethylene 3 and PEG 3350, PEG 4000 and PEG 8000 or a combination thereof.

[0054] 16. The pharmaceutical composition of item 14 or 15, wherein the at least one surfactant is present in a concentration range of about 0.001% (w / V) to about 0.5% (w / V).

[0055] 17. The pharmaceutical composition of item 16, wherein the at least one surfactant is present in a concentration range of about 0.001% (w / V) to about 0.01% (w / V).

[0056] 18. The pharmaceutical composition of any one of items 1-17, wherein the pH of the composition is about 2 pH units lower than the pI of AB1, AB2, MD1 and / or MD2.

[0057] 19. The pharmaceutical composition of any one of items 1-17, wherein the pH of the composition is greater than about 3.5.

[0058] 20. The pharmaceutical composition of any one of items 1-19, wherein the pH of the composition is between 7.0 and 3.6.

[0059] 21. A pharmaceutical composition as described in any of items 1-20, wherein the pH of the composition is between 4.0 and 5.0.

[0060] 22. A pharmaceutical composition as described in any of items 1-21, wherein the pH of the composition is between 3.5 and 5.5.

[0061] 23. The pharmaceutical composition of any one of items 1-22, wherein the pH of the composition is about 4.2 or 3.6.

[0062] 24. The pharmaceutical composition of any one of items 1-23, which has an osmotic pressure range of 150 mOsm to 500 mOsm.

[0063] 25. The pharmaceutical composition according to any one of items 1 to 24, further comprising an amino acid excipient.

[0064] 26. The pharmaceutical composition of claim 25, wherein the amino acid excipient is present in a concentration range of about 0.1 mM to about 20 mM.

[0065] 27. A pharmaceutical composition as described in any of items 1-26, wherein the composition comprises 10 mM glutamate, 9% (w / V) sucrose and 0.01% (w / V) polysorbate 80, and wherein the pH of the pharmaceutical composition is 4.2 or 3.6.

[0066] 28. The pharmaceutical composition of any one of items 1-27, wherein the masked antigen binding protein is present in a concentration range of about 0.1 mg / ml to about 30 mg / ml.

[0067] 29. The pharmaceutical composition of any one of items 1-28, wherein the masked antigen binding protein is present in an amount ranging from about 50 μg to about 200 mg.

[0068] 30. The pharmaceutical composition of any one of items 1-29, wherein the pharmaceutical composition is a lyophilized composition.

[0069] 31. The pharmaceutical composition of any one of items 1-30, wherein the pharmaceutical composition is a liquid composition.

[0070] 32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is a reconstituted lyophilized composition.

[0071] 33. A pharmaceutical composition as described in any of items 1-32, wherein the composition is a liquid composition and is stable after storage at 2°C-8°C, 4°C and / or 25°C for 0 weeks, 1 week, 2 weeks, 4 weeks, 2 months, 6 months, 1 year and / or 2 years; or wherein the composition is a lyophilized composition and is stable after storage at 2°C-8°C, 4°C and / or 25°C for 0 weeks, 1 week, 2 weeks, 4 weeks, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years and / or 5 years.

[0072] 34. The pharmaceutical composition of any one of items 1-33, wherein the amount of antibody aggregation is reduced.

[0073] 35. The pharmaceutical composition of any one of items 1-34, wherein the amount of high molecular weight antibody species is reduced as measured by ultra high performance liquid chromatography (SE-UHPLC).

[0074] 36. A pharmaceutical composition as described in any of items 1-35, wherein the amount of antibody aggregation and / or high molecular weight antibody species is reduced by about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times or about 10 times compared to a reference pharmaceutical composition at a pH higher than that of the pharmaceutical composition.

[0075] 37. The pharmaceutical composition of any one of items 1-36, wherein the pI is measured or determined using the Bjellqvist algorithm.

[0076] 38. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition as described in any one of items 1-37.

[0077] Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to", and allowing for the presence of one or more features or components). It should be understood that although multiple embodiments in the specification are presented using "comprising" language, in many cases, "consisting of" or "consisting essentially of" language can also be used to describe related embodiments. Unless the context indicates otherwise, it should be noted that the term "a or an" refers to one or more, for example, "an immunoglobulin molecule" should be understood to represent one or more immunoglobulin molecules. Similarly, the terms "a or an", "one or more", and "at least one" can be used interchangeably. In addition, "and / or" used herein should be regarded as a specific disclosure of each of the two specified features or components with or without another specified feature or component. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0078] It should also be understood that when describing a range of values, the described feature can be a single value found within the range. For example, "a pH from about pH 4 to about pH 6" can be, but is not limited to, pH 4, 4.2, 4.6, 5.1, 5.5, etc., and any value between these values. In addition, "a pH from about pH 4 to about pH 6" should not be interpreted as meaning that the pH of the formulation under consideration changes by 2 pH units within the range of pH 4 to pH 6 during storage, but rather that the pH of the solution can be selected within the range, and the pH is kept buffered at about this pH. In some embodiments, when the term "about" is used, it means the listed number plus or minus 10% of the listed number.

[0079] In any range described herein, the endpoints of the range are included in the range. However, the description also contemplates the same range excluding the lower and / or higher endpoints. From the entirety of this application, including the drawings and specific embodiments, additional features and variations of the present invention will be apparent to those skilled in the art, and all of these features are intended as aspects of the present invention. Similarly, the features of the present invention described herein can be reorganized into additional embodiments, which are also intended as aspects of the present invention, regardless of whether the combination of these features is specifically mentioned above as an aspect or embodiment of the present invention. Moreover, only those limitations described herein as being essential to the present invention should be regarded as such limitations; variations of the present invention that are not described herein as being essential and lacking limitations are intended as aspects of the present invention.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide a comprehensive dictionary of many of the terms used in the present disclosure to the skilled person.

[0081] Units, prefixes and symbols are all expressed in their SI accepted form. Numerical ranges include numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino to carboxyl direction. The subheadings provided herein are not limitations of the various aspects or aspects of the disclosure, which can be obtained by reference to this specification as a whole.

[0082] All references cited herein are incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figures 1A-1Dare bar graphs providing the results of size exclusion ultra-high performance liquid chromatography (SE-UHPLC) analysis showing the % main peak (left graph) and % high molecular weight species (right graph) of the bispecific masked antigen binding protein in Formulation B at weeks 0, 2, and 4 when stored at 2°C-8°C or 25°C.

[0084] Figures 2A-2D are bar graphs providing the results of SE-UHPLC analysis showing the % main peak (left graph) and % high molecular weight species (right graph) of the bispecific masked antigen binding protein in Formulation C at weeks 0, 1, 2, and 4 when stored at 2°C-8°C or 25°C.

[0085] Figures 3A-3D are bar graphs providing the results of SE-UHPLC analysis showing the % main peak (left graph) and % high molecular weight species (right graph) of bispecific masked antigen binding proteins in Formulation D at weeks 0, 2, and 4 when stored at 2°C-8°C or 25°C.

[0086] Figures 4A-4D are bar graphs providing the results of SE-UHPLC analysis showing the % main peak (left graph) and % high molecular weight species (right graph) of the bispecific masked antigen binding protein in Formulation E at weeks 0, 2, and 4 when stored at 2°C-8°C or 25°C.

[0087] Figures 5A-5D are bar graphs providing the results of SE-UHPLC analysis showing the % main peak (left graph) and % high molecular weight species (right graph) of bispecific masked antigen binding proteins in Formulation F at weeks 0, 2, and 4 when stored at 2°C-8°C or 25°C.

[0088] Fig. 6A and 6B are bar graphs providing the results of SE-UHPLC analysis showing the % high molecular weight species of bispecific masked antigen binding proteins in Formulation A ("Starting Material"), Formulation F ("pH 7.0"), Formulation E ("pH 6.3"), Formulation D ("pH 5.2"), Formulation C ("pH 4.2"), or Formulation B ("pH 3.6") at Week 0 (lower graph) and Week 4 (upper graph) when stored at 2°C-8°C.

[0089] Fig. 7A and 7B are graphs showing reduced aggregation after dialysis into a low pH formulation (Formulation C) compared to the starting material (Formulation A).

[0090] Fig. 8A and8B are graphs showing the stability of bispecific masked antigen binding proteins (1 mg / mL) in a low pH formulation (Formulation C) as determined by size exclusion analysis when stored at 4°C or 25°C for 0, 1 or 2 weeks. Fig. 8A In the example above, the chromatogram is offset.

[0091] Fig. 9A and 9B are bar graphs showing the stability of bispecific masked antigen binding proteins in low pH formulations with high concentrations (e.g., 17 mg / mL) as determined by size exclusion (SEC) analysis. Formulation C with a protein concentration of 17 mg / mL had significantly lower aggregation / % high molecular weight species compared to Formulation A ("Starting Material") with a protein concentration of 11 mg / mL (left graph). Formulation C with a protein concentration of 17 mg / mL was also stable after storage at 4°C for 0, 2, or 4 weeks (right graph).

[0092] Figures 10A-10D are bar graphs providing the results of size exclusion ultra-high performance liquid chromatography (SE-UHPLC) analysis showing the % main peak (left graph) and % high molecular weight species (right graph) of the masked bispecific antibody in Formulation A at weeks 0, 2, and 4 when stored at 2°C-8°C or 25°C.

[0093] Fig.11 is a schematic representation of an exemplary masked antigen binding protein (also referred to as a "Probody").

[0094] Fig.12 is a diagram of an exemplary masked antigen binding protein (also referred to as a "ProTIA prodrug"). "ECBM" is an antigen binding domain (i.e., antigen binding domain (AB1)); "TABM" is a second antigen binding domain (i.e., second antigen binding domain (AB2)); "RS" is a peptide or polypeptide sequence that can be cleaved by a protease (i.e., protein recognition domain (PR)); "Bulking Moiety" is a peptide or polypeptide that inhibits or reduces the ability of ECMB and / or TABM to bind to an antigen (e.g., a masked domain (MD)). Once the precursor form of ProTIA is cleaved at the RS site, the binding of ECBM and / or TABM to the antigen is increased. DETAILED DESCRIPTION

[0095] Specific protein-based drugs (including antibodies) are generally unstable in liquid formulations for extended periods of time, and are particularly unstable at elevated temperatures (e.g., 25° C. and higher). The present disclosure describes low pH formulations comprising masked antigen-binding proteins that bind to one, two, or more antigens. In some embodiments, the bispecific or multispecific masked antigen-binding protein specifically binds to CD3 (including, e.g., the epsilon chain of CD3) and a cell surface antigen (including, e.g., EGFR) in such a way as to transiently bring malignant cells into proximity with activated T cells, thereby inducing T cell-mediated killing of antibody-bound malignant cells.

[0096] The general concept underlying the present invention is the discovery that the colloidal stability of liquid pharmaceutical compositions comprising masked antigen-binding proteins (including, for example, bispecific or multispecific masked antigen-binding proteins) is significantly improved at a pH that satisfies the following two points: (1) below the isoelectric point (pi) of the antibody or antigen-binding domain of the masked antigen-binding protein (e.g., VH / VL regions or antigen-binding fragments thereof or AB1 and / or AB2 regions as described below) and (2) below the pi of the masking domain of the masked antigen-binding protein (MD1 and MD2 regions described further below). The heavy and light chains of the masked antigen-binding protein typically have different isoelectric points (pi), with the intact heavy chain generally having a higher pi than the intact light chain. The pi of a masked antigen binding protein (including, e.g., a bispecific or multispecific masked antigen binding protein) and / or a region or domain within the masked antigen binding protein can be determined by any of a number of methods (including, e.g., using the Bjellqvist algorithm as described by Perez-Riverol, Y. et al., Isoelectric point optimization using peptide descriptors and support vector machines. Journal of proteomics 75, 2269-2274 (2012), which is incorporated herein by reference). See also, Kozlowski LP. IPC-Isoelectric Point Calculator. Biology Direct. 2016; 11:55 (incorporated herein by reference), which describes details of the isoelectric point calculator (IPC), available online at http: / / isoelectric.ovh.org.

[0097] The pi of the masked antigen binding protein region (such as the MD1 and / or MD2 region described further below) is typically different from the pi of the antigen binding portion of the masked antigen binding protein (including, for example, the heavy / light chain or antigen binding thereof, or the AB1 and / or AB2 region as described below).

[0098] The pIs of other representative theoretical AB and MD regions of theoretical masked antigen binding proteins are provided in Table 1. Also provided in Table 1 are recommended pHs to reduce aggregation and improve stability of these representative theoretical masked antigen binding proteins, bispecific masked antigen binding proteins, and multispecific masked antigen binding proteins.

[0099] Table 1

[0100]

[0101] Thus, herein we describe pharmaceutical compositions wherein the pH of the masked antigen binding protein composition is (1) below the isoelectric point (pi) of the antibody or antigen binding domain of the masked antigen binding protein (e.g., VH / VL region or antigen binding fragment thereof, or AB1 and / or AB2 region as further described below) and (2) below the pi of the masking domain of the masked antigen binding protein (MD1 and MD2 region as further described below). While not wishing to be bound by any particular theory, the low pH of the pharmaceutical composition ensures that the sub-regions of the masked antigen binding protein (including, e.g., antigen binding region and masking region or AB1 and / or AB2 region as further described below) carry a net positive charge, which results in inter-domain and intra-domain repulsion that reduces antibody aggregation and increases stability. In the case where a domain (e.g., a masking domain) has an acidic pI and another domain (e.g., an antibody or antigen binding domain) has a basic pI, if the pH of the pharmaceutical composition is between the pIs of each domain (e.g., at pH 7), dipoles can be generated, which lead to intramolecular and intermolecular electrostatic attractions that increase antibody aggregation and reduce stability. Although not wishing to be bound by any particular theory, electrostatic attraction can lead to aggregation and, therefore, to the formation of undesirable high molecular weight (HMW) species. HMW species critically affect the stability of solutions or the colloidal stability of dispersions. This is a particular problem for masked antigen binding proteins (including bispecific or multispecific masked antigen binding proteins), which may have multiple antigen binding domains and / or multiple masking polypeptides, each of which interacts locally with surrounding amino acids, leading to other complications. The low pH of the disclosed compositions allows protonation and electrostatic repulsion of various domains, resulting in an unexpectedly reduced incidence of HMW species in masked antigen binding proteins (including, e.g., bispecific or multispecific antigen binding proteins). In some embodiments, the pH range of the pharmaceutical composition is from 3.5 to 5.5. Preferably, the pH value is 4.0 to 5.5, for example, the pH value is 4.2 to 4.8. In some embodiments, the preferred pH value is a pH value of 3.6 to 4.2. In some embodiments, the pH of the composition is about 2 pH units lower than the pI of the AB1, AB2, MD1 and / or MD2 regions of the masked antigen-binding protein (described further below). In some embodiments, the pH of the composition is between about 7.0 and about 3.6. In some embodiments, the pH of the composition is greater than about 3.5.

[0102] Various aspects of the formulation are described below. The use of section headings is merely for ease of reading and is not intended to be limiting in itself. The entire document is intended to be viewed as a unified disclosure, and it should be understood that all combinations of features described herein may be contemplated.

[0103] Buffer

[0104] The pharmaceutical composition of the present disclosure comprises a buffer, which may optionally be selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate, potassium phosphate, potassium salt, divalent cation, calcium, magnesium, manganese, zinc, acetic acid / sodium acetate, citric acid / sodium citrate, succinic acid / sodium succinate, tartaric acid / sodium tartrate, histidine / histidine HCl, glycine, Tris, glutamate, amino acids, lysine, arginine, proline, and combinations thereof. In certain embodiments, the pharmaceutical composition comprises at least one buffer selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate, and combinations thereof.

[0105] Buffers are often used to control the pH in a formulation. In some embodiments, a buffer is added at a concentration to maintain the pH of the formulation at about 3.5 to about 5, or about 4.0 to about 5.5, or about 4.0 to about 5.0, or about 4.2 to about 4.8, or 4.2, or 3.6, or greater than 3.5. The effect of pH on a formulation can be characterized using any one or more of several methods, such as accelerated stability studies.

[0106] Organic acids, phosphates, and Tris are suitable buffers in protein formulations (Table 2). The buffering capacity of a buffer species is maximum at a pH equal to its pKa and decreases as the pH increases or decreases from this value. Ninety percent of the buffering capacity exists within one pH unit of its pKa. Buffering capacity also increases proportionally with increasing buffer concentration.

[0107] Several factors are typically considered when selecting a buffer. For example, the type of buffer and its concentration should be defined based on its pKa and the desired formulation pH. It is also important to ensure that the buffer is compatible with the protein drug, other formulation excipients, and does not catalyze any degradation reactions. Recently, polyanionic carboxylate buffers such as citrate and succinate have been shown to form covalent adducts with the side chain residues of proteins. The third aspect to consider is the stinging and irritation that the buffer may induce.

[0108] Table 2: Commonly used buffers and their pK a value

[0109]

[0110]

[0111] The buffer system present in the formulation is selected to be physiologically compatible and to maintain the desired pH.

[0112] Buffer can be suitable for maintaining the pH of preparation at any amount of predetermined level. Buffer can be in about 0.1mM and about 1000mM (1M) between or about 5mM and about 200mM between or about 5mM to about 100mM between or about 10mM and about 50mM between concentration exists. Suitable buffer concentration covers about 200mM or lower concentration. In certain embodiments, the buffer in preparation exists with about 190mM, about 180mM, about 170mM, about 160mM, about 150mM, about 140mM, about 130mM, about 120mM, about 110mM, about 100mM, about 80mM, about 70mM, about 60mM, about 50mM, about 40mM, about 30mM, about 20mM, about 10mM or about 5mM concentration. In some embodiments, the concentration of the buffer is at least about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, or 900 mM. In some embodiments, the concentration of the buffer is between about 1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 90 mM and 100 mM. In some embodiments, the concentration of the buffer is between about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30 or 40 mM and 50 mM. In some embodiments, the concentration of the buffer is about 10 mM.

[0113] Other exemplary pH buffers for buffering the formulations listed herein include, but are not limited to, glycine, glutamate, succinate, phosphate, acetate, and aspartic acid. Amino acids such as histidine, arginine, proline, and glutamic acid may also be used as buffers.

[0114] Polyols

[0115] The pharmaceutical compositions described herein also include at least one polyol. Polyols encompass a class of excipients including sugars (e.g., mannitol, sucrose, or sorbitol) and other polyols (e.g., glycerol and propylene glycol). Polymer polyethylene glycol (PEG) is included in this category. Polyols are commonly used as stabilizing excipients and / or isotonic agents in both liquid and lyophilized parenteral protein formulations. Polyols can protect proteins from physical and chemical degradation pathways.

[0116] Exemplary polyols suitable for use in the pharmaceutical compositions disclosed herein include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, polyols (including, for example, polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, sulfobutyl cyclodextrin, N-methylpyrrolidine, cellulose and hyaluronic acid), and sodium chloride (Carpenter et al., Develop. Biol. Standard 74:225, (1991)).

[0117] Other polyols suitable for pharmaceutical compositions disclosed herein include, but are not limited to, propylene glycol, glycerol (glycerol), threose, threitol, erythrose, erythritol, ribose, arabinose, arabitol, lyxose, maltitol, sorbitol, sorbose, glucose, mannose, mannitol, levulose, dextrose, maltose, trehalose, fructose, xylitol, inositol, galactose, xylose, fructose, sucrose, 1,2,6-hexanetriol, etc. Higher sugars include dextran, propylene glycol or polyethylene glycol. Compared with non-reducing sugars, reducing sugars, such as fructose, maltose or galactose are more easily oxidized. Other examples of sugar alcohols are glucitol, maltitol, lactitol or isomaltulose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyols. Monosaccharide glycosides include compounds obtained by reducing disaccharides such as lactose, maltose, lactulose, and maltulose.

[0118] In some embodiments, the at least one polyol is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched glucans and linear unbranched glucans or combinations thereof. In some embodiments, the at least one polyol is a disaccharide selected from the group consisting of sucrose, trehalose, mannitol and sorbitol or combinations thereof.

[0119] In some embodiments, the pharmaceutical composition comprises at least one polyol (e.g., sugar) at a concentration of about 0% w / v to about 40% w / v, or about 0% w / v to about 20% w / v, or about 1% w / v to about 15% w / v. In some embodiments, the pharmaceutical composition comprises at least one polyol (e.g., a sugar) at a concentration of at least 0.5% w / v, at least 1% w / v, at least 2% w / v, at least 3% w / v, at least 4% w / v, at least 5% w / v, at least 6% w / v, at least 7% w / v, at least 8% w / v, at least 9% w / v, at least 10% w / v, at least 11% w / v, at least 12% w / v, at least 13% w / v, at least 14% w / v, at least 15% w / v, at least 16% w / v, at least 17% w / v, at least 18% w / v, at least 19% w / v, at least 20% w / v, at least 30% w / v, or at least 40% w / v. In some embodiments, the pharmaceutical composition comprises at least one polyol (e.g., sugar) at a concentration of about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v to about 15% w / v. In some embodiments, the pharmaceutical composition comprises at least one polyol (e.g., sugar) at a concentration of about 1% w / v to about 15% w / v. In yet another embodiment, the pharmaceutical composition comprises at least one polyol (e.g., sugar) at a concentration of about 9% w / v, about 9.5% w / v, about 10% w / v, about 10.5% w / v, about 11% w / v, about 11.5% w / v, or about 12% w / v. In some embodiments, the pharmaceutical composition comprises at least one polyol (e.g., sugar) at a concentration of about 9% w / v to about 12% w / v. In some embodiments, the at least one polyol (e.g., sugar) is present in the composition at a concentration of about 9% w / v. In some embodiments, the at least one polyol is selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol or a combination thereof. In some embodiments, the polyol is sucrose and is present in the composition at a concentration range of from about 9% w / v to about 12% w / v.

[0120] If desired, the formulation may also include appropriate amounts of bulking and osmotic pressure regulating agents, such as sugars, suitable for forming a lyophilized "cake."

[0121] In preferred embodiments, the masked antigen binding protein pharmaceutical composition or formulation comprises 10 mM glutamate or 10 mM acetate, 9% (w / V) sucrose and 0.01% (w / V) polysorbate 80, wherein the pH of the pharmaceutical composition or formulation is 4.2 or 3.6.

[0122] Surfactants

[0123] The pharmaceutical compositions disclosed herein may further optionally include at least one surfactant. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to compete with proteins for interfacial positions. The hydrophobic portion of the surfactant molecule occupies the interfacial position (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (typically near the critical micelle concentration of the detergent), the surface layer of the surfactant molecule serves to prevent protein molecules from adsorbing at the interface. Surface-induced degradation is thereby minimized. Surfactants include, for example, fatty acid esters of sorbitol polyethoxylates, such as polysorbate 20 and polysorbate 80 (see, e.g., ). The two differ only in the length of the aliphatic chain, which gives the molecules C-12 and C-18 hydrophobic character, respectively. Therefore, polysorbate 80 is more surface active and has a lower critical micelle concentration than polysorbate 20. The surfactant poloxamer 188 has also been used in several commercially available liquid products, such as and

[0124] Detergents can also affect the thermodynamic conformational stability of proteins. The instability of detergents in proteins can be reasonably explained based on the hydrophobic tails of detergent molecules, which can specifically bind to partially or completely unfolded protein states. These types of interactions can cause conformational equilibrium to shift to a more extended protein state (i.e., increase the exposure of the hydrophobic part of the protein molecule to supplement the binding of polysorbate). Alternatively, if the native state of the protein exhibits some hydrophobic surfaces, the detergent combined with the native state can stabilize the conformation.

[0125] Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Typically, as raw materials, they contain sufficient amounts of peroxide to cause oxidation of protein residue side chains, especially methionine. The possibility of adding oxidative damage caused by stabilizers emphasizes that the excipient with the lowest effective concentration should be used in the formulation. For surfactants, the effective concentration of a given protein will depend on the stabilization mechanism. It has been assumed that if the mechanism of surfactant stabilization is related to preventing surface denaturation, the effective concentration will be near the critical micelle concentration of the detergent. On the contrary, if the stabilization mechanism is related to specific protein-detergent interactions, the effective surfactant concentration will be related to the stoichiometry of protein concentration and interaction (Randolph TW, et al., Pharm Biotechnol. [Drug Biotechnology], 13: 159-75 (2002)).

[0126] Surfactants can also be added in appropriate amounts to prevent surface-related aggregation during freezing and drying (Chang, B, J. Pharm. Sci. [Journal of Pharmaceutical Sciences] 85: 1325, (1996)). Exemplary surfactants include, but are not limited to, anionic surfactants, cationic surfactants, nonionic surfactants, zwitterionic surfactants and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium lauryl sulfate, 1-octane sulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate and glycodeoxycholic acid sodium salt. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate and cetyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to CHAPS, CHAPSO, SB3-10 and SB3-12. Nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, Tween 20, and Tween 80. In another embodiment, surfactants include lauromacrogol 400; polyoxyethylene 40 stearate; polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60; glycerol monostearate, polysorbate 20, 40, 60, 65, and 80; soy lecithin and other phospholipids such as DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid esters; methylcellulose and carboxymethylcellulose.

[0127] The pharmaceutical compositions described herein may further optionally comprise at least one surfactant, either alone or in mixtures of varying ratios. In some embodiments, the composition comprises a surfactant at a concentration of about 0.001% w / v to about 5% w / v. In some embodiments, the composition comprises a surfactant at a concentration of at least 0.001% w / v, at least 0.002% w / v, at least 0.003% w / v, at least 0.004% w / v, at least 0.005% w / v, at least 0.007% w / v, at least 0.01% w / v, at least 0.05% w / v, at least 0.1% w / v, at least 0.2% w / v, at least 0.3% w / v, at least 0.4% w / v, at least 0.5% w / v, at least 0.6% w / v, at least 0.7% w / v, at least 0.8% w / v, at least 0.9% w / v, at least 10% w / v, at least 11% w / v, at least 12% w / v, at least 13% w / v, at least 14% w / v, at least 15% w / v, at least 16% w / v, at least 17% w / v, at least 18% w / v, at least 19% w / v, at least 20% w / v, at least 21% w / v, at least 22% w / v, at least 23% w / v, at least 24% w / v In some embodiments, the composition comprises a surfactant at a concentration of about 0.004% w / v to about 0.5% w / v. In some embodiments, the composition comprises a surfactant at a concentration of about 0.004% w / v, about 0.005% w / v, about 0.007% w / v, about 0.01% w / v, about 0.05% w / v, about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, or about 0.4% w / v to about 0.5% w / v. In some embodiments, the composition comprises a surfactant incorporated at a concentration of about 0.001% w / v to about 0.01% w / v. In some embodiments, the surfactant is polysorbate 80, and the polysorbate 80 is present at a concentration of about 0.01% w / v.

[0128] Other considerations

[0129] As used herein, the term "pharmaceutical composition" relates to a composition suitable for administration to a subject in need thereof. The terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein and refer to any subject, particularly a mammalian subject, to whom the pharmaceutical composition is desired to be administered. Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc., preferably humans. The pharmaceutical compositions disclosed herein are stable and pharmaceutically acceptable, i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects in the subject to which the pharmaceutical composition is administered. The pharmaceutically acceptable compositions of the present invention may be particularly sterile and / or pharmaceutically inert. Specifically, the term "pharmaceutically acceptable" may mean approved for use in animals by a regulatory agency or other generally recognized pharmacopoeia, and more particularly for use in humans.

[0130] The pharmaceutical compositions provided by the present disclosure include masked antigen-binding proteins (e.g., bispecific or multispecific masked antigen-binding proteins). In some embodiments, a therapeutically effective amount of masked antigen-binding proteins is provided. "Therapeutically effective amount" means the amount of the masked antigen-binding protein (including, for example, bispecific or multispecific masked antigen-binding proteins) that induces the desired therapeutic effect. Therapeutic efficacy and toxicity, such as ED50 (dose effective in 50% of the population) and LD50 (dose lethal to 50% of the population) can be determined in cell culture or experimental animals by standard pharmaceutical methods. The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as an ED50 / LD50 ratio. Pharmaceutical compositions that exhibit a large therapeutic index are generally preferred.

[0131] Protein and antibody formulations, including pharmaceutical compositions disclosed herein, can be administered parenterally. When administered parenterally, they must be sterile. Sterile diluents include pharmaceutically acceptable (safe and nontoxic to human administration) and useful liquids for preparing liquid pharmaceutical compositions (e.g., pharmaceutical compositions reconstructed after lyophilization). Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution. Diluents may include aqueous solutions of salts and / or buffers.

[0132] Excipients are additives included in pharmaceutical compositions or formulations because they impart or enhance the stability, delivery, and manufacturability of the drug product. Regardless of the reason for including the excipient, the excipient must be safe and tolerated by the patient. For protein drugs and antibodies, the selection of excipients is particularly important because they can affect both the efficacy and immunogenicity of the drug. Therefore, it is necessary to develop protein formulations with the appropriate selection of excipients that provide suitable stability, safety, and marketability.

[0133] Excipients described herein are organized by their chemical type or their functional role in pharmaceutical compositions / preparations. In view of the teaching and guidance provided herein, those skilled in the art will be able to easily change the amount or scope of the excipient without increasing viscosity to an undesirable level. Excipients can be selected to achieve the desired osmotic pressure (i.e., isotonic, hypotonic or hypertonic) of the final solution, pH, desired stability, anti-aggregation or anti-degradation or anti-precipitation, protection under freezing, lyophilization or high temperature conditions, or other properties. Various types of excipients are known in the art. Exemplary excipients include salts, amino acids, other tonicity agents, surfactants, stabilizers, swelling agents, cryoprotectants, lyoprotectants, antioxidants, metal ions, chelating agents and / or preservatives.

[0134] Furthermore, when a particular excipient in a pharmaceutical composition or formulation is reported as, for example, a percentage (%) w / v, one skilled in the art will recognize that the equivalent molar concentration of that excipient is also taken into account.

[0135] Other stabilizers and bulking agents

[0136] The pharmaceutical composition described herein may further optionally include at least one stabilizer (including sugar, polymer and / or polyol). Stabilizers include a class of compounds that can be used as cryoprotectants, lyoprotectants and glass formers. Cryoprotectants are used to stabilize proteins at low temperatures during freezing or in a frozen state. By retaining the natural conformational properties of proteins during the dehydration phase of freeze drying, lyoprotectants stabilize proteins or antibodies in freeze-dried solid dosage forms. Glass properties are classified as "strong" or "fragile" according to their relaxation properties as a function of temperature. Importantly, cryoprotectants, lyoprotectants and glass formers maintain the same phase with proteins or antibodies to impart stability. Exemplary lyoprotectants include, but are not limited to, glycerol and gelatin, as well as melibiose, melezitose, raffinose, mannotriose and stachyose.

[0137] Amino Acids

[0138] In some embodiments, the pharmaceutical compositions described herein further comprise one or more amino acids. Amino acids have a variety of uses in protein and antibody formulations or pharmaceutical compositions as buffers, bulking agents, stabilizers, and antioxidants. Histidine and glutamic acid are used to buffer protein or antibody formulations in the pH range of 5.5–6.5 and 4.0–5.5, respectively. The pKa of the imidazole group of histidine is 6.0, and the pKa of the carboxyl group of the glutamic acid side chain is 4.3, which makes them suitable for buffering in their respective pH ranges. Glutamic acid is found in some formulations (e.g., Histidine is commonly found in commercially available protein / antibody formulations (e.g., ). It provides a good alternative to citrate, which is a buffer known to cause stinging when injected. Interestingly, histidine is also reported to have a stabilizing effect when used in high concentrations in both liquid and lyophilized presentations (Chen B, et al., Pharm Res. [Drug Research], 20 (12): 1952-60 (2003)). It has also been observed that histidine (up to 60mM) reduces the viscosity of high concentration antibody formulations. However, in the same study, the authors observed increased aggregation and discoloration in formulations containing histidine during freeze-thaw studies of antibodies in stainless steel containers. The authors attributed this to the effects of iron ions caused by corrosion of steel containers. Another note about histidine is that it undergoes photooxidation in the presence of metal ions (Tomita M, et al., Biochemistry [Biochemistry], 8 (12): 5149-60 (1969)). The use of methionine as an antioxidant in formulations appears promising; it has been observed to be effective against a variety of oxidative stresses (Lam XM, et al., J Pharm Sci., 86(11): 1250-5 (1997)).

[0139] The amino acids glycine, proline, serine, and alanine stabilize proteins or antibodies. Glycine is also a bulking agent in lyophilized formulations (e.g., Arginine has been shown to be an effective agent for inhibiting aggregation and has been used in both liquid and lyophilized formulations (e.g., Liquid). The pharmaceutical compositions described herein may further optionally comprise any one or more of the above-mentioned amino acids.

[0140] Antioxidants

[0141] In some embodiments, the pharmaceutical compositions described herein further comprise one or more antioxidants. Oxidation of protein residues comes from many different sources. In addition to the addition of specific antioxidants, preventing oxidative protein damage involves careful control of many factors throughout the manufacturing process and product storage, such as atmospheric oxygen, temperature, light exposure, and chemical contamination. The most commonly used pharmaceutical antioxidants are reducing agents, oxygen / free radical scavengers, or chelating agents. Antioxidants in therapeutic protein or antibody formulations must be water-soluble and remain active throughout the shelf life of the product. Reducing agents and oxygen / free radical scavengers work by ablating reactive oxygen species in solution. Chelating agents such as EDTA can be effective by binding trace metal contaminants that promote free radical formation. For example, EDTA is used in liquid formulations of acidic fibroblast growth factor to inhibit metal ion-catalyzed oxidation of cysteine ​​residues. EDTA has been used in commercially available products like and Therefore, the pharmaceutical compositions described herein may further optionally comprise any one or more of these reducing agents, oxygen / free radical scavengers and / or chelating agents.

[0142] However, antioxidants themselves can induce other covalent or physical changes to proteins. Many such cases have been reported in the literature. Reducing agents (like glutathione) can cause the disruption of intramolecular disulfide bonds, which can lead to disulfide reshuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote methionine oxidation in many proteins and polypeptides (Akers MJ and Defelippis MR. Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Sven Frokjaer, Lars Hovgaard, eds. Pharmaceutical Science. Taylor and Francis, UK (1999); Fransson JR, J. Pharm. Sci. 86(9):4046-1050 (1997); Yin J, et al., Pharm Res., 21(12):2377-83 (2004)). Sodium thiosulfate has been reported to reduce the level of light- and temperature-induced methionine oxidation in rhuMabHER2; however, the formation of thiosulfate-protein adducts was also reported in this study (Lam XM, Yang JY et al., J Pharm Sci. [Journal of Pharmaceutical Science] 86(11):1250-5 (1997)). The appropriate antioxidant is selected according to the specific stress and sensitivity of the masked antigen-binding protein.

[0143] Metal ions

[0144] In certain embodiments, the pharmaceutical composition further comprises one or more metal ions. Generally, transition metal ions are undesirable in protein or antibody pharmaceutical compositions or formulations because they can catalyze physical and chemical degradation reactions in proteins. However, when they are cofactors of proteins, specific metal ions are included in formulations where they form coordination complexes (e.g., zinc suspensions of insulin). Recently, it has been proposed to use magnesium ions (10–120 mM) to inhibit aspartate isomerization to isoaspartate (see, e.g., International Patent Publication No. WO 2004 / 039337). Exemplary metal ions include, but are not limited to, Ca +2 Ion, Mg+2 , Mn +2 、Zn +2 , Cu +2 and Fe +2 .

[0145] preservative

[0146] In certain embodiments, the pharmaceutical composition further comprises one or more preservatives. When developing a multiple use parenteral formulation that is repeatedly extracted from the same container, a preservative is essential. Its primary function is to inhibit microbial growth and ensure product sterility during the entire shelf life or service life of the drug product. Commonly used preservatives include phenol, benzyl alcohol, metacresol, alkyl parabens (such as methylparaben or propylparaben), benzalkonium chloride and benzethonium chloride. Other examples of compounds with antimicrobial protection activity include octadecyl dimethyl benzyl ammonium chloride and hexamethonium chloride. Other types of preservatives include aromatic alcohols, such as butanol, phenol, benzyl alcohol, catechol, resorcinol, cyclohexanol and 3-pentanol.

[0147] Multiple use pen presentations optionally include preserved formulations. For example, preserved hGH formulations are currently available on the market. (liquid, Novo Nordisk), Nutropin (liquid, Genentech) and Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while (Eli Lilly) was formulated with m-cresol.

[0148] Several aspects are considered during the formulation development of the preserved dosage form. The optimization of preservative concentration involves testing a given preservative in a dosage form, and its concentration range confers antimicrobial effectiveness without compromising protein stability. For example, three preservatives were successfully screened in a liquid formulation developed for interleukin-1 receptor (type I) using differential scanning calorimetry (DSC). Based on the effect of preservatives on stability at commonly used concentrations in commercial products, preservatives are ranked (Remmele RL Jr. et al., Pharm Res. [Drug Research], 15 (2): 200-8 (1998)).

[0149] Some preservatives can cause injection site reactions, which is another factor to consider when choosing a preservative. In a clinical trial focused on evaluating preservatives and buffers in Norditropin, formulations containing phenol and benzyl alcohol were observed to have reduced pain perception compared to formulations containing metacresol (Kappelgaard AM, Horm Res. [Hormone Research] 62 Suppl 3: 98-103 (2004)). Interestingly, among commonly used preservatives, benzyl alcohol has anesthetic properties (Minogue SC and Sun DA., Anesth Analg. [Anesthesia and Analgesia], 100 (3): 683-6 (2005)).

[0150] The present disclosure also contemplates pharmaceutical compositions that do not contain any preservatives.

[0151] Masked antigen binding protein

[0152] The pharmaceutical compositions or formulations described herein include masked antigen-binding proteins. "Masked antigen-binding proteins" are proteins including a masking domain (MD) coupled to an antigen-binding domain (AB) (e.g., by a covalent bond or a linker), such that the coupling of MD inhibits or reduces the binding of AB to its antigen. MD further includes a protein recognition site (PR), which includes a substrate or binding site for a protein or protease, such that when a protein or protease binds and / or cleaves the protein recognition site, the AB domain binds to the antigen or increases or is induced by the binding of the AB domain to the antigen. Masked antigen-binding proteins have been previously described in, for example, International Publication No. WO 2017 / 040344, U.S. Patent No. 9540440, U.S. Publication No. 20150118254, U.S. Patent No. 9127053, U.S. Patent No. 9517276, and U.S. Patent No. 8563269, each of which is incorporated herein by reference in its entirety. In some embodiments, the masked antigen-binding protein is a Probody (as described in, for example Fig.11 ; or Polu KR and Lowman HB. Expert Opin Biol Ther. 2014 Aug;14(8):1049-53; or Desnoyers LR et al., Sci Transl Med. 2013 Oct 16;5(207)) or ProTIA prodrugs (as described, for example, in Fig.12; or Schellenberger V. Amunix unveils next-generation immuno-oncologic cancer therapy platform. [Amunix company announced the next-generation immuno-oncologic cancer therapy platform] https: / / biopharmadealmakers.nature.com. September 2016 edition, B20; see also http: / / www.amunix.com / technology / pro-tia / ). In some embodiments, the masked antigen-binding protein has the following structural arrangement from N-terminus to C-terminus: MD-AB or AB-MD. In some embodiments, the masked antigen-binding protein includes a connecting peptide (LP), and the masked antigen-binding protein has the following structural arrangement from N-terminus to C-terminus: MD-LP-AB or AB-LP-MD.

[0153] As discussed above, the term "masked antigen-binding protein" refers to the masked antigen-binding protein in the following states: the native state, and the state when a protein or protease binds to a binding site or substrate within the protein recognition site (PR) of the masked antigen-binding protein (which may result in cleavage of the PR or induce a structural conformational change) (i.e., an active state). Therefore, it will be apparent to the skilled person that in some embodiments, the masked antigen-binding protein may lack the MD due to cleavage of the PR by a protease, which results in the release of at least the MD (e.g., wherein the MD is not linked to the masked antigen-binding protein by a covalent bond (e.g., a disulfide bond between cysteine ​​residues)).

[0154] When the masked antigen binding protein is in the presence of antigen in its native state, the binding of AB to the antigen is reduced or inhibited compared to the binding of AB to the antigen when the masked antigen binding protein is in an active state (i.e., when a protein or protease binds to PR and / or removes or displaces MD). The ability of the masked antigen binding protein to bind antigen in its native state is reduced, e.g., by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100%, when compared to the binding of AB not associated with MD (i.e., when the masked antigen binding protein is in an active state) when measured in an in vitro and / or in vivo binding assay.

[0155] In some embodiments, the protein recognition site (PR) acts as a substrate for a protease (preferably an extracellular protease). The PR can be selected based on a protein or protease produced by a cell (including, for example, a tumor cell) close to a cell expressing an antigen and / or produced by a cell (including, for example, a tumor cell) co-localized with the desired antigen of the AB of the masked antigen-binding protein in a tissue. In some embodiments, the protease is u-type plasminogen activator (uPA, also known as urokinase), legumin and / or proteinase (also known as MT-SP1 or MTSP1). In some embodiments, the protease is a matrix metalloproteinase (MMP). In some embodiments, the protease is one of the proteases described in Rawlings, N and Salvesen, G. Handbook of Proteolytic Enzymes (Third Edition). Elsevier, 2013. ISBN: 978-0-12-382219-2 (incorporated herein by reference for all purposes).

[0156] The pharmaceutical compositions described herein may contain masked antigen binding proteins having AB or MD, wherein the pi is acidic (less than pH 7.0), neutral (about pH 7.0), or basic (greater than pH 7.0). In some embodiments, the AB domain in the bispecific or multispecific antigen binding protein may have an acidic, neutral, and / or basic pi, while MD may also have an acidic, neutral, and / or basic pi. In some embodiments, the pharmaceutical compositions described herein contain masked antigen binding proteins having AB and MD, wherein both pis are acidic, neutral, or basic, or AB has an acidic pi and MD has a neutral or basic pi. In other embodiments, AB may have a neutral or basic pi, while MD has an acidic pi.

[0157] In some embodiments, the masked antigen-binding protein comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to an antigen. In some embodiments, the antibody or antigen-binding fragment thereof that binds to an antigen is a monoclonal antibody, a domain antibody, a single-chain antibody, a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a mouse, chimeric, humanized, or fully human monoclonal antibody. In some preferred embodiments, the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, a scFv, or a scAb.

[0158] In some embodiments, the antibody or antigen binding fragment thereof binds to an antigen selected from the group consisting of EGFR, mesothelin, CDH3, CD3, or a combination thereof. In some embodiments, the antibody or antigen binding fragment thereof binds to an antigen selected from the group consisting of hormones, growth factors, cytokines, cell surface receptors, or any ligands thereof. In some embodiments, the antibody or antigen binding fragment binds to an antigen selected from the group consisting of such cytokines, lymphokines, growth factors, or other hematopoietic factors, including but not limited to M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.Additional growth factors for use herein include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein-16, bone morphogenetic protein-17, bone morphogenetic protein-18, bone morphogenetic protein-19, bone morphogenetic protein-20, bone morphogenetic protein-21, bone morphogenetic protein-22, bone morphogenetic protein-23, bone morphogenetic protein-24, bone morphogenetic protein-25, bone morphogenetic protein-26, bone morphogenetic protein-27, bone morphogenetic protein-28, bone morphogenetic protein-29, bone morphogenetic protein-30, bone morphogenetic protein-31, bone morphogenetic protein-32, bone morphogenetic protein-33, bone morphogenetic protein-34, bone morphogenetic protein-35 15. Bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemoattractant 1, cytokine-induced neutrophil chemoattractant 2α, cytokine-induced neutrophil chemoattractant 2β, β-endothelial cell growth factor, endothelin 1, epithelial-derived neutrophil attractant, glial cell line-derived neurotrophic factor receptor α1, glial cell line-derived neurotrophic factor receptor α2, growth-related protein, growth-related protein alpha, growth-related protein beta, growth-related protein gamma, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor alpha, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta 1, transforming growth factor beta 1.2, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, latent transforming growth factor beta 1, transforming growth factor beta binding protein I, transforming growth factor beta binding protein II, transforming growth factor beta binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor and chimeric proteins and biologically or immunologically active fragments thereof.

[0159] In some embodiments, the antibody or antigen-binding fragment binds an antigen selected from the group consisting of: p53, KRAS, NRAS, MAGEA, MAGEB, MAGEC, BAGE, GAGE, LAGE / NY-ESO1, SSX, tyrosinase, gp100 / pmel17, Melan-A / MART-1, gp75 / TRP1, TRP2, CEA, RAGE-1, HER2 / NEU, WT1.

[0160] In exemplary aspects, the antibody or antigen binding fragment binds to an antigen expressed on the surface of an immune cell. In some embodiments, the masked antigen binding protein binds to a differentiation cluster molecule selected from the group consisting of: CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11A, CD11B, CD11C, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31,CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45 RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw 60. CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD9 5. CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD11 6. CD117, CD118, CD119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw 131, CD132, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166, and CD182. ,

[0161] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of muromonab-CD3 (trade name Orthoclone products marketed under the trade names of products), rituximab (marketed under the trade name Commercially available products) (U.S. Patent No. 5,843,439), basiliximab (marketed under the trade name products marketed under the trade name products marketed under the trade name products), infliximab (marketed under the trade name products), trastuzumab (marketed under the trade name Alemtuzumab (marketed under the trade name Campath- Adalimumab (marketed under the trade name Tositumomab-I131 (marketed under the trade name products sold on the market), efalizumab (available under the trade name products), cetuximab (marketed under the trade name Ibritumomab (marketed under the trade name Omalizumab (marketed under the trade name products marketed under the trade name products sold on the market), natalizumab (available under the trade name products marketed under the trade name products marketed under the trade name products marketed under the trade name products marketed under the trade name products marketed under the trade name Canakinumab (marketed under the trade name products marketed under the trade name products sold on the market), Ustekinumab (marketed under the trade name products marketed under the trade name ofatumumab (marketed under the trade name products marketed under the trade name Belimumab (marketed under the trade name products sold under the trade name products) and pertuzumab (available under the brand name Commercially available products). In an exemplary embodiment, the antibody is one of the following: an anti-TNFα antibody, such as adalimumab, infliximab, etanercept, golimumab, and bercezolizumab; an anti-IL1β antibody, such as canakinumab; an anti-IL12 / 23 (p40) antibody, such as ustekinumab and brijinumab; and an anti-IL2R antibody, such as daclizumab. Examples of suitable anti-cancer antibodies include, but are not limited to, anti-BAFF antibodies, such as belimumab; anti-CD20 antibodies, such as rituximab; anti-CD22 antibodies, such as epratuzumab; anti-CD25 antibodies, such as daclizumab; anti-CD30 antibodies, such as israeluzumab; anti-CD33 antibodies, such as gemtuzumab; anti-CD52 antibodies, such as alemtuzumab; anti-CD152 antibodies, such as ipilimumab; anti-EGFR antibodies, such as cetuximab; anti-HER2 antibodies, such as trastuzumab and pertuzumab; anti-IL6 antibodies, such as siltuximab; anti-VEGF antibodies, such as bevacizumab; and anti-IL6 receptor antibodies, such as tocilizumab.

[0162] In some embodiments, the pharmaceutical composition comprises a bispecific or multispecific masked antigen-binding protein. Bispecific or multispecific masked antigen-binding proteins have been previously described. See, for example, International Publication No. WO2017 / 040344; U.S. Patent Publication No. 2015 / 0079088 (incorporated herein by reference). A "bispecific masked antigen-binding protein" is a masked antigen-binding protein that binds two different antigens or epitopes, while a "multispecific masked antigen-binding protein" is a masked antigen-binding protein that recognizes three or more different antigens or epitopes.

[0163] In some embodiments, one antibody or antigen binding fragment thereof (AB1) domain of a bispecific or multispecific antigen binding protein is specific for a target antigen, and another antibody or antigen binding fragment thereof (AB2) domain is specific for another target antigen. In some embodiments, one antibody or antigen binding fragment thereof (AB1) domain of a bispecific or multispecific antigen binding protein is specific for an epitope of a target antigen, and another antibody or antigen binding fragment thereof (AB2) domain is specific for another epitope of the same target antigen.

[0164] Bispecific or multispecific masked antigen-binding proteins include at least one masking domain (MD) comprising a protein recognition site (PR), wherein the MD inhibits or reduces the binding of AB to the antigen. The at least one MD comprises a protein recognition site (PR), which comprises a substrate or binding site for a protein or protease, such that when the protein or protease binds and / or cleaves the protease recognition site, the AB domain binds to the antigen or increases binding. For bispecific masked antigen-binding proteins, the masked antigen-binding protein preferably comprises two MDs (e.g., MD1 and MD2) that reduce the ability of each antigen-binding domain (AB1 and AB2) to bind to its respective antigen or epitope.

[0165] The masked antigen binding proteins and / or bispecific or multispecific masked antigen binding proteins provided herein are stable in circulation and activated at the intended therapy and / or diagnostic site, but not activated in normal (i.e., healthy) tissues. When in an active state, the masked antigen binding proteins and / or bispecific or multispecific masked antigen binding proteins exhibit binding to an antigen that is at least comparable to the binding of a corresponding unmodified antibody or bispecific or multispecific antigen binding protein (i.e., a counterpart that does not comprise a masking domain) to an antigen.

[0166] The term "antibody" refers to a protein having a conventional immunoglobulin form, comprising a heavy chain and a light chain, and comprising a variable region and a constant region. For example, an antibody can be an IgG, which is a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair having a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (typically having a molecular weight of about 50-70 kDa). Antibodies have a variable region and a constant region. In the IgG form, the variable region is typically about 100-110 or more amino acids, comprises three complementary determining regions (CDRs), is primarily responsible for antigen recognition, and is very different from other antibodies that bind to different antigens. See, e.g., Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd. / Garland Publishing, (1999). The antibody may further optionally comprise a constant domain, such that the antibody may be an IgA, IgD, IgE, IgG or IgM antibody, including any of IgG1, IgG2, IgG3 or IgG4. The variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Maryland; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), located within framework regions (designated framework regions 1-4, FR1, FR2, FR3, and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra). The constant region allows the antibody to recruit cells and molecules of the immune system.

[0167] The term "antibody fragment", "antibody fragment thereof" or "antigen-binding antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment" or "antigen-binding fragment thereof" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment may contain the antigen-determining variable regions of an intact antibody. Examples of antibody fragment antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv and single-chain antibodies.

[0168] In many aspects, the antibody is a monoclonal antibody. In some aspects, the antibody is a human antibody. In some aspects, the antibody is chimeric or humanized. The term "chimeric" refers to an antibody containing domains from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species, and a variable domain from a second species, or more generally, may contain segments of amino acid sequences from at least two species. Both "chimeric" and "humanized" generally refer to antigen-binding proteins that combine regions from more than one species. A chimeric antibody may also contain domains from two or more different antibodies within the same species. In one embodiment, a chimeric antibody is a CDR-grafted antibody.

[0169] When used in connection with antigen-binding proteins, the term "humanization" refers to an antigen-binding protein (e.g., an antibody) that has at least a CDR region from a non-human source and is engineered to have a structure and immunological function that is more similar to a true human antibody than the antibody of the original source. For example, humanization may involve grafting CDRs from a non-human antibody (e.g., a mouse antibody) into a human framework region. Typically, in a humanized antibody, the entire antibody except the CDRs is encoded by a polynucleotide of human origin or is identical to such an antibody (except within its CDRs). CDRs (encoded in part or in whole by nucleic acids derived from non-human organisms) are grafted into the β-folded framework of a human antibody variable region to produce an antibody whose specificity is determined by the grafted CDRs. The production of such antibodies is described in, for example, International Patent Publication No. WO 92 / 11018; Jones, 1986, Nature 321:522-525; and Verhoeyen et al., 1988, Science 239:1534-1536, all of which are incorporated by reference in their entirety. "Back mutation" of selected acceptor framework residues to corresponding donor residues is often employed to regain the affinity lost in the initially grafted construct (see, for example, U.S. Pat. Nos. 5530101; 5585089; 5693761; 5693762; 6180370; 5859205; 5821337; 6054297; and 6407213, all of which are incorporated by reference in their entirety). Humanized antibodies optionally also comprise at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region, and thus typically comprise a human Fc region.

[0170] Non-limiting examples of multispecific antibodies include trispecific antibodies, tetraspecific antibodies, etc. Masked antigen binding proteins (including bispecific or multispecific masked antigen binding proteins) are optionally multivalent; multivalency refers to the total number of binding sites on the antibody, whether the binding sites recognize the same or different antigens or epitopes.

[0171] In various embodiments, the bispecific or multispecific masked antigen binding proteins described herein bind to human CD3 (SEQ ID NO: 3). For example, in various aspects, the bispecific or multispecific masked antigen binding proteins activate T cells by engaging CD3ε on T cells. That is, the antibodies excite, stimulate, activate and / or enhance CD3-mediated T cell activation. The biological activities of CD3 include, for example, T cell activation and other signaling through interactions between CD3 and antigen binding subunits of the T cell receptor (TCR).

[0172] The bispecific or multispecific masked antigen binding proteins disclosed herein optionally bind to CD3ε with a binding constant (Kd) ≤ 1 μM, eg, in some embodiments, ≤ 100 nM, ≤ 10 nM, or ≤ 1 nM.

[0173] In various aspects, the masked antigen binding protein (e.g., bispecific or multispecific masked antigen binding protein) binds to epidermal growth factor receptor (EGFR). The sequence of human EGFR is provided as SEQ ID NO: 4. The multispecific masked antigen binding proteins disclosed herein optionally bind to human EGFR with a binding constant (Kd) ≤ 1 μM, e.g., in some embodiments, ≤ 100 nM, ≤ 10 nM, or ≤ 1 nM.

[0174] In preferred embodiments, the masked bispecific or multispecific antigen binding protein binds both CD3 and EGFR.

[0175] In some embodiments, the masked antigen-binding protein is a heterodimer such that it includes an antigen-binding domain that is a Fab (e.g., IgG Fab) and an antigen-binding domain that is a scFv. For example, in an exemplary embodiment, the masked antigen-binding protein comprises a heavy chain and a light chain (e.g., an IgG heavy chain and a light chain) that bind to one target (e.g., EGFR) and an scFv domain that binds to a second target (e.g., a T cell surface antigen such as CD3). Single-chain antibodies (scFv) are antigen-binding proteins in which the VL and VH regions are linked by a linker (e.g., a synthetic sequence of amino acid residues typically about 15 to about 20 amino acids in length) to form a continuous protein chain, wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). An example of a linker suitable for scFv is GGGGSGGGGSGGGGS (SEQ ID NO: 1). Other exemplary linkers contain at least 4-5 amino acids, ranging from about 4 residues to about 20 residues.

[0176] An exemplary form of a masked antigen binding protein comprises (i) two heavy chains containing scFvs, which are operably attached near the N-terminus of one or both heavy chains, and (ii) two light chains, which associate with the heavy chains to form an antigen binding domain, wherein the scFv binds CD3 and the Fab portion binds EGFR. In this example, the antigen binding protein comprises three (or four) antigen binding domains, which typically bind two or more different antigens (or epitopes). An example of a linker suitable for connecting the scFv to the heavy chain variable region is GGGGS (SEQ ID NO: 2). An exemplary linker contains at least one suitable linker with 4-5 amino acids.

[0177] The masked antigen-binding protein comprises a masking domain (MD) coupled to an antibody (AB) (e.g., by a covalent bond or another form of attachment). The masking domain (MD) comprises a masking peptide (or masking polypeptide) (MP) and a protein recognition site (PR). The masking peptide (or masking polypeptide) can be a stretch of amino acids that hinders the binding of the antigen-binding domain to its antigen. Typically, a masking peptide of a short sequence of 5-15 amino acids is used, but shorter and longer sequences (i.e., masking polypeptides) are also contemplated. The masking domain is further described in, for example, International Publication No. Wo 2017 / 040344; U.S. Patent Publication No. 2015 / 0079088 (incorporated by reference in its entirety, and in particular for the disclosure of masking domains used with antibodies that bind to EGFR) and U.S. Patent Publication No. 2016 / 0194399 (incorporated by reference in its entirety, and in particular for the disclosure of masking domains used with antibodies that bind to CD3).

[0178] In some embodiments, the masking peptide (or masking polypeptide) (MP) is attached to the antigen binding domain (AB) via a protein recognition site (PR), which is optionally part of a larger linker sequence (i.e., a stretch of amino acids that connects the MP to the antigen binding protein). The PR acts as a substrate (or binding site) for a protein or protease (preferably an extracellular protease). The PR can be selected based on a protein or protease produced by cells (including, e.g., tumor cells) that are proximal to cells expressing the target and / or produced by cells (including, e.g., tumor cells) that are co-localized in tissues with the desired target of at least one AB of the masked antigen binding protein. In some embodiments, the protease is u-type plasminogen activator (uPA, also known as urokinase), legumin and / or matriptase (also known as MT-SP1 or MTSP1). In some embodiments, the protease is a matrix metalloproteinase (MMP). In some embodiments, the protease is one of the proteases described in Rawlings, N and Salvesen, G. Handbook of Proteolytic Enzymes (Third Edition). Elsevier, 2013. ISBN: 978-0-12-382219-2. Alternatively, the MP is coupled to the antigen binding protein via a non-cleavable protein binding domain. In this regard, PR is optionally an amino acid sequence that changes conformation when interacting with, for example, a protein or protease, such that the position of the MP is adjusted and the AB is free to bind to the target.

[0179] In various aspects of the present disclosure, a bispecific or multispecific masked antigen-binding protein comprises an MD for each antigen-binding portion (e.g., AB1, AB2, AB3, etc.) of the construct. For example, a bispecific or multispecific masked antigen-binding protein comprising two Fab portions and two scFvs may comprise two sets of MDs, which may be independently identical or different. For illustration, a bispecific or multispecific masked antigen-binding protein comprises (i) two scFvs binding to CD3 and MD1 attached to each scFv (optionally the same MD for each scFv), and (ii) two Fab portions binding to EGFR and MD2 attached to each Fab (optionally the same MD for each Fab). In various aspects, the PRs of MD1 and MD2 comprise the same protein recognition sequence, allowing the MD of each binding region to be released in the same environment after engagement with a protease. The MD may be attached to the N-terminus or C-terminus of the antigen-binding domain, as long as the MD is able to interfere with the binding of the antigen-binding domain to the target, and once the MD linker is released, it will not interfere with the binding. When the antigen binding domain is a Fab, the MD can be operably linked to the heavy chain variable region or the light chain variable region. In the case where the bispecific or multispecific masked antigen binding protein comprises a complete antibody having both a Fab antigen binding domain fused to a heavy chain and a scFv (i.e., a "stacked" conformation), the MD associated with the Fab antigen binding domain is preferably fused to the light chain variable region.

[0180] In some embodiments, one AB domain (e.g., AB1) in a bispecific or multispecific masked antigen-binding protein is engaged with a T cell surface ligand (e.g., CD3), and is optionally in the form of a scFv. Exemplary masked antigen-binding proteins engaged with CD3 include, but are not limited to, International Publication No. WO 2017 / 040344, U.S. Patent Publication No. 20160194399 (incorporated by reference in their entirety).

[0181] In some embodiments, one AB domain (e.g., AB2) in the antigen binding protein engages with a tumor antigen (e.g., EGFR). Exemplary masked antigen binding proteins that engage with EGFR include, but are not limited to, those antibodies disclosed in U.S. Patent Publication No. 20150079088 (the disclosure of which is incorporated herein by reference in its entirety).

[0182] In some embodiments, the masked antigen-binding protein (including, for example, bispecific or multispecific masked antigen-binding proteins) is modified to change the isoelectric point of the antibody. For example, in some embodiments, one or more negatively charged pH-sensitive amino acids (e.g., aspartic acid or glutamic acid) in one or both masking domains are replaced with positively charged amino acids (e.g., lysine or arginine). In some embodiments, replacing aspartic acid in one or more masking portions of an antibody can increase the pI. In some embodiments, one or more negatively charged pH-sensitive amino acids (e.g., aspartic acid or glutamic acid) in one or both masking domains are removed or replaced with neutral amino acids.

[0183] Although the masked antigen-binding proteins of the present disclosure (including, for example, bispecific or multispecific masked antigen-binding proteins) are generally described herein as comprising a complete antibody structure, the present disclosure also contemplates the use of antigen-binding antibody fragments lacking at least a portion of the traditional two heavy chains / two light chains structure. The fragment of the antigen-binding protein used as a mask comprises an antigen-binding domain (AB) operably connected to a masking domain (MD) as described above. For example, in some embodiments, the antigen-binding protein comprises two scFvs connected by a suitable linker (e.g., an amino acid segment of sufficient length to allow each scFv to bind to its target). One or two scFvs are attached to MD; when two scFvs are attached to MD, MD can be the same or different (i.e., although MP and / or PR are different in many aspects, PR is preferably the same).

[0184] In some embodiments, the pharmaceutical composition or formulation comprises a masked antigen binding protein (e.g., a bispecific or multispecific masked antigen binding protein) in an amount ranging from about 50 μg to about 200 mg (or from about 500 μg to about 150 mg, or from about 50 mg to about 200 mg, or about 50 mg to about 150 mg, or about 50 mg to about 100 mg, or about 50 mg to about 75 mg). In some embodiments, the pharmaceutical composition comprises a masked antigen binding protein (including a bispecific or multispecific masked antibody binding protein) in an amount of about 50 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 550 μg, about 600 μg, about 650 μg, about 700 μg, about 750 μg, about 800 μg, about 850 μg, about 900 μg, about 950 μg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 50 mg, about 60 mg, about 70 mg, about 750 μg, about 800 μg, about 850 μg, about 900 μg, about 950 μg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 30 mg, about 35 mg, about 35 mg about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg or about 200 mg.

[0185] In some embodiments, the pharmaceutical composition comprises a masked antigen binding protein (e.g., a bispecific or multispecific masked antigen binding protein) at a concentration ranging from about 0.1 mg / mL to about 50 mg / mL (or from about 1 mg / mL to about 10 mg / mL, or from about 10 mg / mL to about 20 mg / mL, or from 20 mg / mL to about 50 mg / mL, or from about 1 mg / mL to about 17 mg / mL, or from about 0.5 mg / mL to about 5 mg / mL, or from about 1 mg / mL to about 5 mg / mL, or from about 3 mg / mL to about 6 mg / mL). In some embodiments, the pharmaceutical composition comprises the masked antigen binding protein at a concentration of about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL or about 50 mg / mL.

[0186] Dosage and therapeutic use of preparations

[0187] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially prevent the disease and its complications in patients already suffering from the disease. The amount or dosage effective for this purpose will depend on the condition to be treated (indications), the antibody construct delivered, the treatment context and goals, the severity of the disease, previous therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the patient's body shape (weight, body surface or organ size) and / or condition (age and general health) and the general state of the patient's autoimmune system. The appropriate dosage can be adjusted according to the judgment of the attending physician so that it can be administered to the patient once or through a series of administrations, and in order to obtain the best therapeutic effect.

[0188] A therapeutically effective amount of a masked antigen binding protein (e.g., a bispecific or multispecific masked antigen binding protein) preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of impairment or disability resulting from disease affliction. For the treatment of cancer, tumors, or malignant cells expressing an antigen recognized by a masked antigen binding protein described herein (including a bispecific or multispecific masked antigen binding protein), a therapeutically effective amount of a masked antigen binding protein (e.g., an anti-tumor cell antigen (e.g., EGFR) / anti-CD3 bispecific or multispecific masked antigen binding protein) preferably inhibits cell division or tumor growth by at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% relative to an untreated patient. The ability of a molecule to inhibit tumor growth can be assessed, for example, in an animal model predictive of efficacy.

[0189] The pharmaceutical compositions and formulations described herein can be used to treat, alleviate and / or prevent pathological medical conditions as described herein in patients in need. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes applying or administering the formulation to the body, separated tissue or cells of a patient suffering from a disease / disorder, having symptoms of a disease / disorder or having a tendency to suffer from a disease / disorder, for the purpose of curing, healing, alleviating, mitigating, changing, remedying, alleviating, improving or affecting the disease, the symptoms of the disease or the tendency to suffer from the disease. The term "disease" refers to any condition that will benefit from treatment with the pharmaceutical compositions described herein. This includes chronic and acute conditions or diseases, including those pathological conditions that make mammals susceptible to the disease under consideration. As used herein, the term "alleviation" refers to any improvement in a disease state (e.g., a disease state of a patient suffering from a tumor or cancer or metastatic cancer) by administering a masked antigen-binding protein to a subject in need. In the case of cancer, this improvement can be considered, for example, to slow down or stop the progression of a patient's tumor or cancer or metastatic cancer. As used herein, the term "prevention" means avoiding the occurrence or recurrence of a disease or disorder or its symptoms. For example, in the case of cancer, "prevention" encompasses avoiding the occurrence or recurrence of a tumor or cancer or metastatic cancer in a subject.

[0190] In some embodiments, the disclosure provides methods for treating a disorder or disease associated with one or more antigens recognized by the masked antigen binding proteins described herein.

[0191] In one embodiment, the present disclosure provides a method for treating a proliferative disease (e.g., cancer or a neoplastic disease), the method comprising administering to a subject in need thereof a step of administering a pharmaceutical composition described herein. Malignant neoplasms (commonly referred to as cancer) typically invade and destroy surrounding tissues, and may form metastases, i.e., they spread to other parts of the body, tissues, or organs. Therefore, the term "metastatic cancer" encompasses metastases to other tissues or organs other than the original tumor. The term "subject in need thereof" or "those in need of treatment" includes those already suffering from the condition and those to be prevented from the condition. Subjects in need thereof or "patients" include people and other mammalian subjects receiving preventive or therapeutic treatment.

[0192] Route of administration

[0193] Exemplary routes of administration include, but are not limited to, topical routes (e.g., epidermal, inhalation, nasal, ocular, ear / ear, vaginal, mucosal); intestinal routes (e.g., oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and parenteral routes (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extra-amniotic, intraarticular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal). Preferably, the pharmaceutical composition is administered parenterally, such as intravenous, subcutaneous, or intramuscularly. Parenteral administration can be achieved by injection, such as bolus injection, or by infusion, such as continuous infusion. Administration can achieve long-term release by a reservoir. In some embodiments, the pharmaceutical composition is administered intravenously by initial bolus injection followed by continuous infusion to maintain therapeutic circulating levels of the drug product. In some embodiments, the pharmaceutical composition is administered in a disposable dose. The pharmaceutical composition can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Pat. Nos. 4,475,196, 4,439,196, 4,447,224, 4,447,233, 4,486,194, 4,487,603, 4,596,556, 4,790,824, 4,941,880, 5,064,413, 5,312,335, 5,312,335, 5,383,851, and 5,399,163.

[0194] The present disclosure contemplates freeze-dried compositions with features described herein. If the pharmaceutical composition has been freeze-dried, the freeze-dried material is first reconstituted in an appropriate liquid before administration. The freeze-dried material can be reconstituted in, for example, bacteriostatic water for injection (BWFI), normal saline, phosphate buffered saline (PBS) or the same formulation in which the protein is before freeze drying. The present disclosure contemplates the reconstitution of the previous freeze-dried composition with features described herein. The pharmaceutical composition can be administered as a single therapeutic agent or in combination with additional therapies (e.g., anticancer therapies as needed, such as other proteins and non-protein drugs). These drugs can be administered simultaneously with the compositions of the present disclosure as defined herein, or administered separately at intervals and doses defined in time before or after the administration of the formulation.

[0195] Medicine Box

[0196] As another aspect, a kit comprising one or more pharmaceutical compositions described herein is provided, the pharmaceutical composition being packaged in a manner conducive to the administration of a subject. In one embodiment, such kits include pharmaceutical compositions described herein (e.g., compositions comprising masked antigen-binding proteins (e.g., bispecific or multispecific masked antigen-binding proteins)), the pharmaceutical composition being packaged in a container, such as in a sealed bottle, a vial, a single-use or multiple-use vial, a pre-filled syringe, or a pre-filled injection device, the kit optionally having a label attached to the container or included in the package, the label describing the use of the compound or composition in practicing the method. In one aspect, the composition is packaged as a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration. Preferably, the kit contains a label describing the use of masked antigen-binding proteins described herein or pharmaceutical compositions described herein.

[0197] In general, a variety of storage and / or dosage forms are contemplated for the pharmaceutical compositions of the invention, depending on the intended route of administration, delivery pattern, and desired dosage (see, e.g., Remington's Pharmaceutical Sciences, 22nd ed., Oslo, A., ed., (2012)). The skilled artisan will appreciate that such selection of a particular dosage form may, for example, affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antibody.

[0198] Examples

[0199] Example 1

[0200] Effects of sugar stabilizers (e.g., sucrose) and low pH on aggregation of masked antigen-binding protein compositions

[0201] A composition comprising a bispecific masked antigen binding protein (1 mg / mL) in 100 mM glycine (pH 4.9) was generated (Formulation A). The liquid composition showed significant aggregation immediately after storage at 2°C-8°C or after storage at 2°C-8°C for four weeks, with approximately 27%-28% high molecular weight (HMW) species (see Figures 6, 7 and 10).

[0202] To improve the clinical utility of the composition by improving stability and reducing the amount of aggregated and HMW species, the composition was buffer exchanged into several formulations ranging from pH 3.6 to pH 7.0 as follows (Formulations BF):

[0203] Formulation B: 10 mM glutamate, 9% sucrose, pH 3.6

[0204] Formulation C: 10 mM glutamate, 9% sucrose, pH 4.2;

[0205] Formulation D: 10 mM acetate, 9% sucrose, pH 5.2;

[0206] Formulation E: 10 mM phosphate, 9% sucrose, pH 6.3; and

[0207] Formulation F: 10 mM phosphate, 9% sucrose, pH 7.0.

[0208] Buffer exchange was performed by dialyzing the starting composition overnight at 4°C in a 7000 kDa cutoff cassette with three buffer changes. The dialyzed material was bottled after the addition of 0.01% polysorbate 80, and the stability of the samples was monitored for one month at 2°C-8°C and 25°C. The final concentration of the masked antigen-binding protein bottled in each formulation was 1 mg / mL. All samples were analyzed by size exclusion ultra-high performance liquid chromatography (SE-UHPLC) at 2°C-8°C and 25°C, respectively, in the second and fourth weeks. The SE-UHPLC analysis results of formulations BF are listed in Figures 1 to 6, respectively. The SE-UHPLC analysis results of formulation A are listed in Figure 10.

[0209] like Fig. 6A As shown, after four weeks of storage at 2°C-8°C, the % high molecular weight (HMW) species was significantly reduced in the lower pH formulations compared to the starting composition (i.e., Formulation A). For example, the masked antigen binding protein formulations described herein are ranked from the highest to the lowest amount of aggregation observed as Formulation A>Formulation F>Formulation E>Formulation D>Formulation C>Formulation B.

[0210] The data strongly suggest that the stability, aggregation, and clinical utility of pharmaceutical compositions containing masked antigen binding proteins are significantly improved by: (1) the addition of sugars or stabilizers (e.g., sucrose) - the inclusion of sugars or stabilizers (e.g., sucrose) reduced aggregation / % HMW species by about 3-fold; and (2) formulating the pharmaceutical compositions at low pH, wherein the pH is below the isoelectric point (pi) of the AB1, AB2, MD1, and MD2 regions of the masked antigen binding protein - which further reduced aggregation / % HMW species by an additional about 3-fold. The pi of the AB1, AB2, MD1, and MD2 regions of the bispecific masked antigen binding protein was determined using the Bjellqvist algorithm to have a pattern comparable to that of the bispecific masked antigen binding protein 2 provided in Table 1. When formulated at a pH greater than 4.54, it appears that the negatively charged MD1 may outweigh the otherwise positively charged protein, potentially leading to solution instability. At a lower pH of 4, all components (e.g., AB1, AB2, MD1, and MD2) are positively charged to varying degrees, resulting in repulsive forces. The data strongly support that the combination of a sugar or stabilizer (e.g., sucrose) and a low pH (where the pH is below the isoelectric point (pi) of the AB1, AB2, MD1, and / or MD2 regions of the masked antigen binding protein) synergistically or dramatically reduces aggregation / % HMW species of pharmaceutical compositions comprising masked antigen binding proteins (including, e.g., bispecific or multispecific masked antigen binding proteins).

[0211] Example 2

[0212] Reversal of aggregation of bispecific masked antigen-binding protein compositions by addition of sugars or stabilizers (e.g., sucrose) and low pH

[0213] As described in Example 1, a bispecific masked antigen binding protein sample with high initial aggregation levels was buffer exchanged into a low pH formulation buffer (Formulation C). Briefly, buffer exchange was performed by dialyzing the starting material overnight in a 7000 kDa cutoff cassette at 4°C with three buffer changes. The dialysis process resulted in a significant reduction in aggregation levels, indicating that the low pH formulation was able to reverse initial aggregation. Comparison Figure 2B (reported 3.91% HMW species for Formulation C) and Fig. 10B (reported 28.49% HMW species for Formulation A) at time zero. This comparison is also shown in Figure 7B The samples were analyzed by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), a technique that uses size exclusion ultra-high performance analytical columns to separate proteins in solution based on their hydrodynamic volumes.

[0214] like Figures 8A-8BAs shown, the 1 mg / mL dialyzed material was stable after storage at 4°C and 25°C for 0, 1, and 2 weeks. There was no increase in aggregation levels after storage. Samples stored at 25°C showed a slight decrease in high molecular species by SEC, suggesting that aggregation may be driven by hydrophobic interactions.

[0215] The low pH formulation (Formulation C) was also effective at stabilizing the bispecific masked antigen-binding protein at higher concentrations. The protein in the low pH formulation buffer (Formulation C) at 17 mg / mL had significantly lower aggregation compared to the starting material (Formulation A) at 11 mg / mL (Figure 9). After storage at 4°C for 0, 2, and 4 weeks, the 17 mg / mL material was stable (Figure 9). Even though Formulation C showed a significant reduction in aggregation / %HMW species and improved stability at 2°C-8°C or 25°C at 0, 2, and 4 weeks (Figure 2) compared to Formulation A (Figure 10), Formulation B at pH 3.6 showed an additional reduction in aggregation / %HMW species compared to Formulation C. See, e.g., Figure 6; see also Figure 1 and compare to Figure 2. The data show that further reducing the pH of the pharmaceutical composition - which is already below the isoelectric point (pI) of the AB1, AB2, MD1, and MD2 regions of the masked antigen-binding protein - can further reduce the aggregation of the masked antigen-binding protein.

[0216] Example 3

[0217] Effects of polyols (e.g., sucrose) and low pH on aggregation of masked antigen-binding protein compositions

[0218] The pI of the antibody domain (AB1 and / or AB2) and the bulking agent / masking domain (MD1 and / or MD2) of the ProTIA prodrug is determined, the ProTIA prodrug comprising an amino acid sequence as described in Table 5, 10 and / or 12 and / or Figure 36 or 37 of International Publication No. WO 2017 / 040344. Next, the ProTIA prodrug is formulated in a suitable buffer (e.g., 10 mM glutamate or 10 mM acetate) together with a suitable polyol (e.g., 9% sucrose) and optionally a surfactant (e.g., 0.01% polysorbate 80), and the pH of the composition is reduced to between about 7.0-3.5, such that the pH of the composition is lower than the pI of the antibody domain (AB1 and / or AB2) and the bulking agent / masking domain (MD1 and / or MD2). As described in Examples 1 and 2, the effects of polyols and low pH on the low pH aggregation of the ProTIA prodrug are evaluated.

Claims

1. A pharmaceutical composition comprising: (a) A masked antigen-binding protein comprising: (i) an antibody or an antigen-binding fragment thereof (AB1) that binds to an antigen; and (ii) a masking domain (MD1) coupled to AB1, wherein the masking domain comprises: a. a masking peptide or masking polypeptide (MP1) that inhibits or reduces the binding of AB1 to the antigen; and b. a protein recognition site (PR1), wherein binding of a protein to the PR1 or cleavage of the PR1 by a protease increases binding of AB1 to the antigen; (b) at least one buffer; and (c) at least one polyol, The pH of the pharmaceutical composition is below the isoelectric point (pi) of the AB1 and MD1 regions of the masked antigen binding protein.

2. The pharmaceutical composition of claim 1, wherein the masked antigen binding protein further comprises: (iii) a second antibody or antigen-binding fragment thereof (AB2) that binds to a second antigen; and (iv) a second masking domain (MD2) coupled to AB2, wherein the second masking domain comprises: a. a masking peptide or masking polypeptide (MP2) that inhibits or reduces the binding of AB2 to the antigen; and b. a protein recognition site (PR2), wherein binding of a protein to the PR2 or cleavage of the PR2 by a protease increases binding of AB2 to the antigen; wherein the pH of the pharmaceutical composition is below the isoelectric point (pi) of the AB1, AB2, MD1 and MD2 regions of the masked antigen binding protein.

3. The pharmaceutical composition of claim 1 or 2, wherein the masked antigen binding protein is a Probody or ProTIA prodrug.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein AB1 and / or AB2 is scFv or Fab.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein AB1 and / or AB2 binds to an antigen expressed on the surface of a cell.

6. The pharmaceutical composition of any one of claims 1-5, wherein the at least one buffer is selected from the group consisting of: acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate and 2-(N-morpholino)ethanesulfonate or a combination thereof.

7. The pharmaceutical composition of claim 6, wherein the at least one buffer is present in a concentration range of about 5 mM to about 200 mM.

8. The pharmaceutical composition of claim 7, wherein the at least one buffer is present in a concentration range of about 10 mM to about 50 mM.

9. The pharmaceutical composition of any one of claims 1-8, wherein the at least one polyol is selected from the group consisting of: Sugars, cyclic polysaccharides, sugar alcohols, linear branched glucans and linear unbranched glucans or a combination thereof.

10. The pharmaceutical composition of claim 9, wherein the sugar is a monosaccharide or a disaccharide.

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