Low ph pharmaceutical antibody formulation

Abstract

To provide low pH formulations comprising, e.g., an antigen-binding protein that binds to CD3, at least one buffer agent, at least one saccharide and at least one surfactant.SOLUTION: In one aspect, described herein is a pharmaceutical composition comprising an antigen binding protein described herein, at least one buffer, at least one surfactant and at least one saccharide, the pH of the pharmaceutical composition ranging from 3.5 to 5. In some embodiments, the antigen-binding protein is an antibody. In some embodiments, the antibody is a bispecific antibody, such as a bispecific antibody that binds to CD3.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 628,267, filed February 8, 2018, and U.S. Provisional Patent Application No. 62 / 799,577, filed January 31, 2019, the disclosures of which are incorporated by reference in their entireties herein.

[0002] Incorporation by Reference This application incorporates by reference WO 2016 / 086196, filed November 25, 2015; U.S. Patent Application Publication No. 20160215063, filed November 25, 2015; WO 2017 / 091656, filed November 23, 2016; and U.S. Patent No. 9,822,186, filed March 30, 2015, which are expressly incorporated by reference herein in their entireties, particularly with respect to any drawings, descriptions and claims therein.

[0003] The computer readable nucleotide / amino acid sequence listing submitted concurrently herewith is incorporated by reference in its entirety, and is identified as follows: an ASCII (text) file named "52588P_Seqlisting.txt" of 756,646 bytes created on February 5, 2019. [Background technology]

[0004] Protein-based drugs are among the fastest growing therapeutic agents in (pre)clinical development and as commercial products. Compared with small molecule chemical drugs, protein drugs have high specificity and activity at relatively low concentrations, and usually provide treatment for high impact diseases such as various cancers, autoimmune diseases and metabolic disorders (Roberts, Trends Biotechnol. 2014 Jul;32(7):372-80, Wang, Int J Pharm. 1999 Aug 20;185(2):129-88).

[0005] With the advancement of commercial-scale purification processes, protein-based pharmaceuticals, such as recombinant proteins, can now be obtained with high purity at the time of initial production. However, proteins are only marginally stable and are highly susceptible to 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 to surfaces, and aggregation. Addressing these physical and chemical instabilities is one of the most difficult challenges in the development of protein pharmaceuticals (Chi et al., Pharm Res, Vol. 20, No. 9, Sept 2003, pp. 1325-1336; Roberts, Trends Biotechnol. 2014 Jul; 32(7): 372-80).

[0006] Protein aggregation represents a major phenomenon of physical instability of proteins and occurs due to their inherent tendency to minimize thermodynamically unfavorable interactions between solvents and hydrophobic protein residues. Protein aggregation is particularly problematic because it occurs routinely during the processes of refolding, purification, sterilization, transportation and storage. Aggregation can occur even under solution conditions where the native state of the protein is highly thermodynamically favorable (e.g., neutral pH and 37°C) and in the absence of stress (Chi et al., Pharm Res, Vol. 20, No. 9, Sept 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.).

[0007] Also, half-life extended antibody constructs such as bispecific T cell engagers that contain half-life extending modalities such as Fc molecules should be protected from protein aggregation and / or other degradation phenomena. Protein aggregation is problematic because it can weaken the biological activity of therapeutic proteins. Furthermore, protein aggregation leads to an undesirable appearance of the drug product and reduces product yields due to the extensive purification steps required to remove aggregates from the final product. More recently, there has been increasing concern and evidence that the presence of aggregated proteins (even humanized or fully human proteins) may significantly increase the risk that patients will mount an immune response against the active protein monomer, resulting in the formation of neutralizing antibodies and drug resistance or other adverse side effects (Mahler J Pharm Sci. 2009 Sep; 98(9): 2909-34).

[0008] Generally, several attempts to minimize protein aggregation by various mechanisms have been reported in the literature. Proteins can be stabilized by modifying their primary structure, thereby increasing the hydrophobicity of the interior and decreasing the hydrophobicity of the exterior, thus protecting them from aggregate formation and other chemical alterations. However, genetic engineering of proteins may result in loss of functionality and / or increased immunogenicity. Another approach focuses on dissociation of aggregates (called "disaggregation") to recover functional native monomers by using various mechanisms such as temperature, pressure, pH and salt. Currently, protein aggregates are mainly removed as impurities in downstream processing steps. However, when the level of high molecular weight (HMW) is high, significant amounts of HMW removal not only result in substantial yield loss but also make the design of robust downstream processes difficult (Chi et al., 2003). et al., Pharm Res, Vol. 20, No. 9, Sept 2003, pp. 1325-1336). Summary of the Invention [Problem to be solved by the invention]

[0009] Maintaining protein stability and activity in biological and biotechnological applications poses serious challenges. There is a need in the art for optimized pharmaceutical compositions that enhance the stabilization of therapeutic proteins and reduce aggregation and denaturation or degradation during formulation, packaging, transportation, storage and administration, thereby preventing loss of function and adverse immunogenic reactions. [Means for solving the problem]

[0010] In one aspect, described herein is a pharmaceutical composition comprising an antigen binding protein as described herein, at least one buffering agent, at least one surfactant, and at least one saccharide, wherein the pharmaceutical composition has a pH in the range of 3.5 to 5.

[0011] In some embodiments, the antigen binding protein is an antibody. In some embodiments, the antibody is a bispecific antibody, such as a bispecific antibody that binds to CD3.

[0012] In some embodiments, the antigen binding protein is a heterodimeric antibody that binds to CD3. In some embodiments, the heterodimeric antibody is an anti-CD3 antibody comprising a) a first Fc domain and (i) an scFv variable light chain domain comprising a vlCDR1 set forth in SEQ ID NO: 15, a vlCDR2 set forth in SEQ ID NO: 16, and a vlCDR3 set forth in SEQ ID NO: 17, and (ii) an scFv variable heavy chain domain comprising a vhCDR1 set forth in SEQ ID NO: 11, a vhCDR2 set forth in SEQ ID NO: 12, and a vhCDR3 set forth in SEQ ID NO: 13. a) a first monomer comprising an scFv, wherein the scFv is covalently linked to the N-terminus of the Fc domain using a domain linker; b) a second monomer comprising i) an anti-CD38 heavy chain variable domain comprising a vhCDR1 set forth in SEQ ID NO: 65, a vhCDR2 set forth in SEQ ID NO: 66, and a vhCDR3 set forth in SEQ ID NO: 67, and ii) a heavy chain constant domain comprising a second Fc domain; and c) a light chain comprising a constant domain and an anti-CD38 variable light chain domain comprising a vlCDR1 set forth in SEQ ID NO: 69, a vlCDR2 set forth in SEQ ID NO: 70, and a vlCDR3 set forth in SEQ ID NO: 71, wherein the pH of the pharmaceutical composition is in the range of 3.5 to 5.

[0013] In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:18.

[0014] In some embodiments, the anti-CD38 variable light chain domain comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:68.

[0015] In some embodiments, the anti-CD38 heavy chain variable domain comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:64.

[0016] In some embodiments, the first monomer comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:335.

[0017] In some embodiments, the second monomer comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:337.

[0018] In some embodiments, the light chain comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:336.

[0019] In various embodiments, the antigen binding protein is a heterodimeric antibody comprising: a) a first monomer comprising: 1) a first variable heavy chain domain; 2) a first constant heavy chain comprising a first CH1 domain and a first Fc domain; and 3) an scFv that binds human CD3 and comprises (i) an scFv variable light chain domain comprising a vlCDR1 set forth in SEQ ID NO: 387, a vlCDR2 set forth in SEQ ID NO: 388, and a vlCDR3 set forth in SEQ ID NO: 389, (ii) an scFv linker, and (iii) an scFv variable heavy chain domain comprising a vhCDR1 set forth in SEQ ID NO: 383, a vhCDR2 set forth in SEQ ID NO: 384, and a vhCDR3 set forth in SEQ ID NO: 385; wherein said scFv is a heterodimeric antibody comprising: a first monomer comprising a first heavy chain comprising an scFv covalently linked between the C-terminus of said CH1 domain and the N-terminus of said first Fc domain using a domain linker. The heterodimeric antibody further comprises: b) a second monomer comprising a second heavy chain comprising a second constant heavy chain comprising a second variable heavy domain and a second Fc domain; and c) a common light chain comprising a variable light domain and a constant light domain. The first variable heavy domain and the variable light domain bind to human STEAP1, and the second variable heavy domain and the variable light domain bind to human STEAP1. The first variable heavy domain and the second variable heavy domain comprise heavy chain CDRs comprising vhCDR1 as set forth in SEQ ID NO: 360, vhCDR2 as set forth in SEQ ID NO: 361 or 363, and vhCDR3 as set forth in SEQ ID NO: 362, and the variable light domain comprises light chain CDRs comprising vlCDR1 as set forth in SEQ ID NO: 357, vlCDR2 as set forth in SEQ ID NO: 358, and vlCDR3 as set forth in SEQ ID NO: 359. Alternatively, the first variable heavy domain and the second variable heavy domain comprise heavy chain CDRs comprising vhCDR1 set forth in SEQ ID NO: 368, vhCDR2 set forth in SEQ ID NO: 369, and vhCDR3 set forth in SEQ ID NO: 370, and the variable light domain comprises light chain CDRs comprising vlCDR1 set forth in SEQ ID NO: 371, vlCDR2 set forth in SEQ ID NO: 372, and vlCDR3 set forth in SEQ ID NO: 373.In various embodiments, the first variable heavy chain domain and the second variable heavy chain domain comprise an amino acid sequence at least 90% identical (e.g., at least 95% identical or 100% identical) to SEQ ID NO: 377 or 379, and / or the variable light chain domain comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical or 100% identical) to SEQ ID NO: 378. Alternatively, the first variable heavy chain domain and the second variable heavy chain domain comprise an amino acid sequence at least 90% identical (e.g., at least 95% identical or 100% identical) to SEQ ID NO: 380, and / or the variable light chain domain comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical or 100% identical) to SEQ ID NO: 381. The scFv optionally comprises the variable heavy chain and variable light chain regions of SEQ ID NO: 382 and SEQ ID NO: 383. The scFv linker optionally comprises SEQ ID NO: 391. In various aspects, the scFv comprises the sequence of SEQ ID NO: 390. In various embodiments, a) the first monomer comprises the sequence of SEQ ID NO: 366 or SEQ ID NO: 367, the second monomer comprises the sequence of SEQ ID NO: 365, and the common light chain comprises the sequence of SEQ ID NO: 364; or b) the first monomer comprises the sequence of SEQ ID NO: 376, the second monomer comprises the sequence of SEQ ID NO: 375, and the common light chain comprises the sequence of SEQ ID NO: 374.

[0020] The pharmaceutical compositions of the present disclosure include at least one buffering agent. In some embodiments, the buffering agent is an acid 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 some embodiments, the at least one buffering agent is present in the composition at a concentration ranging from about 5 mM to about 200 mM (or from about 10 mM to about 50 mM).

[0021] The pharmaceutical composition of the present disclosure comprises at least one saccharide. In some embodiments, the saccharide is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched dextrans, and linear unbranched dextrans. In some embodiments, the saccharide is a sugar alcohol (e.g., sorbitol). In some embodiments, the saccharide is a disaccharide selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol, or combinations thereof. In some embodiments, the at least one saccharide is present in the composition at a concentration ranging from about 1 to about 15% (w / V) (or about 9 to about 12% (w / V) or about 5% to about 12% (w / V) or about 7% to about 12% (w / V)).

[0022] The pharmaceutical composition of the present disclosure comprises at least one surfactant. In some 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, or combinations thereof. In some embodiments, the at least one surfactant is present in the composition at a concentration ranging from 0.004 to about 0.5% (w / V) (or from about 0.001 to about 0.01% (w / V), or from about 0.001% to about 0.5% (w / V), or from about 0.004% to about 0.01% (w / V)).

[0023] In some embodiments, the pH of the composition ranges from 4.0 to 5.0, In some embodiments, the pH of the composition is 4.2.

[0024] In some embodiments, the composition has an osmolality in the range of about 150 to about 500 mOsm.

[0025] The pharmaceutical compositions of the present disclosure may optionally further comprise an excipient selected from the group consisting of a polyol and an amino acid, hi some embodiments, the excipient is present at a concentration ranging from about 0.1 to about 15% (w / V).

[0026] In some embodiments, the pharmaceutical composition comprises 10 mM glutamate, 9% (w / V) sucrose, and 0.01% (w / V) polysorbate 80, and the pH of the liquid pharmaceutical composition is 4.2. In some embodiments, the heterodimeric antibody is present in the composition at a concentration ranging from about 0.1 mg / mL to about 8 mg / mL. In some embodiments, the heterodimeric antibody is present in the composition at a concentration ranging from about 0.1 mg / mL to about 20 mg / mL. In some embodiments, the heterodimeric antibody is present in the composition at a concentration of 1 mg / mL, 5 mg / mL, 10 mg / mL, or 20 mg / mL. In some embodiments, the heterodimeric antibody is present in the composition in an amount ranging from about 50 μg to about 200 mg.

[0027] The pharmaceutical compositions of the present disclosure may be lyophilized compositions or liquid compositions, hi some embodiments, the pharmaceutical compositions are lyophilized compositions or reconstituted lyophilized compositions.

[0028] In another aspect, described herein is a method of treating cancer in a subject, comprising administering to a subject in need thereof a composition of the present disclosure. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is prostate cancer.

[0029] While various embodiments herein are presented using the word "comprising" under various circumstances, it should be understood that related embodiments may also be described using the words "consisting of" or "consisting essentially of." The present disclosure intends that an embodiment described as "comprising" a feature includes embodiments "consisting of" that feature. It should be noted that the terms "a" or "an" refer to one or more, e.g., "immunoglobulin molecule" is understood to refer to one or more immunoglobulin molecules. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0030] When describing a range of values, it should also be understood that the described characteristic may be any individual value found within that range. For example, a "pH of about pH 4 to about pH 6" may be, but is not limited to, pH 4, 4.2, 4.6, 5.1, 5.5, etc., and any value between such values. Furthermore, a "pH of about pH 4 to about pH 6" should not be interpreted to mean that the pH of the formulation of interest will vary by 2 pH units in the range of pH 4 to pH 6 during storage, but rather means that a value may be chosen within that range for the pH of the solution, and the pH will remain buffered at about that pH. In some embodiments, when the term "about" is used, it means to the recited number plus or minus 5%, 10%, 15% or more of the recited number. The actual variation intended can be determined from the context.

[0031] In any of the ranges described herein, the endpoints of the range are included within the range. However, this description also contemplates the same ranges with the lower endpoint and / or the higher endpoint excluded. Additional features and variations of the invention will be apparent to one of ordinary skill in the art from the entirety of this application, including the drawings and detailed description, and all such features are contemplated as aspects of the invention. Similarly, features of the invention described herein can be recombined into additional embodiments that are also contemplated as aspects of the invention, regardless of whether the combination of features is specifically described above as an aspect or embodiment of the invention. Also, only those limitations described herein as essential to the invention should be considered as such; variations of the invention lacking limitations not described herein as essential are contemplated as aspects of the invention.

[0032] All references cited herein are hereby incorporated by reference. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A pharmaceutical composition comprising an antigen-binding protein, at least one buffer, at least one surfactant, and at least one saccharide, wherein the pharmaceutical composition has a pH in the range of 3.5 to 5. (Item 2) 2. The pharmaceutical composition of claim 1, wherein the antigen-binding protein is an antibody. (Item 3) 3. The pharmaceutical composition of item 2, wherein the antibody is a heterodimeric antibody that binds to CD3. (Item 4) The heterodimeric antibody, a) a first Fc domain and a first monomer comprising an anti-CD3 scFv comprising (i) an scFv variable light domain comprising a vlCDR1 set forth in SEQ ID NO: 15, a vlCDR2 set forth in SEQ ID NO: 16, and a vlCDR3 set forth in SEQ ID NO: 17, and (ii) an scFv variable heavy domain comprising a vhCDR1 set forth in SEQ ID NO: 11, a vhCDR2 set forth in SEQ ID NO: 12, and a vhCDR3 set forth in SEQ ID NO: 13, wherein the scFv is covalently linked to the N-terminus of the Fc domain using a domain linker; b) i) an anti-CD38 heavy chain variable domain comprising a vhCDR1 set forth in SEQ ID NO:65, a vhCDR2 set forth in SEQ ID NO:66, and a vhCDR3 set forth in SEQ ID NO:67, and ii) a heavy chain constant domain comprising a second Fc domain; and c) The pharmaceutical composition according to item 3, comprising a light chain comprising an anti-CD38 variable light chain domain comprising a constant domain and a vlCDR1 as set forth in SEQ ID NO: 69, a vlCDR2 as set forth in SEQ ID NO: 70, and a vlCDR3 as set forth in SEQ ID NO: 71. (Item 5) 5. The pharmaceutical composition of item 4, wherein the anti-CD3 scFv comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:18. (Item 6) 5. The pharmaceutical composition according to item 4, wherein the anti-CD3 scFv comprises the amino acid sequence set forth in SEQ ID NO: 18. (Item 7) The pharmaceutical composition according to any one of items 4 to 6, wherein the anti-CD38 variable light chain domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 68. 8. The pharmaceutical composition of item 7, wherein the anti-CD38 variable light chain domain comprises the amino acid sequence set forth in SEQ ID NO:68. (Item 9) The pharmaceutical composition according to any one of items 4 to 8, wherein the anti-CD38 heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 64. 10. The pharmaceutical composition of item 9, wherein the anti-CD38 heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:64. (Item 11) 11. The pharmaceutical composition according to any one of items 4 to 10, wherein the first monomer comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 335. (Item 12) 12. The pharmaceutical composition of claim 11, wherein the first monomer comprises the amino acid sequence set forth in SEQ ID NO: 335. (Item 13) 13. The pharmaceutical composition according to any one of items 4 to 12, wherein the second monomer comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 337. (Item 14) 14. The pharmaceutical composition of claim 13, wherein the second monomer comprises the amino acid sequence set forth in SEQ ID NO: 337. (Item 15) 15. The pharmaceutical composition according to any one of items 4 to 14, wherein the light chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 336. (Item 16) 16. The pharmaceutical composition of item 15, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 336. (Item 17) The heterodimeric antibody, a) 1) a first variable heavy domain; 2) a first constant heavy chain comprising a first CH1 domain and a first Fc domain; ;and 3) binds to human CD3, and (i) an scFv variable light domain comprising a vlCDR1 as set forth in SEQ ID NO: 387, a vlCDR2 as set forth in SEQ ID NO: 388, and a vlCDR3 as set forth in SEQ ID NO: 189; (ii) an scFv linker; and (iii) a first monomer comprising a first heavy chain comprising an scFv comprising an scFv variable heavy chain domain comprising a vhCDR1 set forth in SEQ ID NO: 383, a vhCDR2 set forth in SEQ ID NO: 384, and a vhCDR3 set forth in SEQ ID NO: 385, wherein the scFv is covalently linked using a domain linker between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain; b) a second monomer comprising a second heavy chain comprising a second variable heavy chain domain and a second constant heavy chain comprising a second Fc domain; and c) a common light chain comprising a variable light domain and a constant light domain; the first variable heavy chain domain and the variable light chain domain bind to human STEAP1, the second variable heavy chain domain and the variable light chain domain bind to human STEAP1, and (i) the first variable heavy chain domain and the second variable heavy chain domain comprise heavy chain CDRs comprising a vhCDR1 set forth in SEQ ID NO: 360, a vhCDR2 set forth in SEQ ID NO: 361 or 363, and a vhCDR3 set forth in SEQ ID NO: 362, and the variable light chain domain comprises light chain CDRs comprising a vlCDR1 set forth in SEQ ID NO: 357, a vlCDR2 set forth in SEQ ID NO: 358, and a vlCDR3 set forth in SEQ ID NO: 359; or (ii) The pharmaceutical composition of item 3, wherein the first variable heavy domain and the second variable heavy domain comprise heavy chain CDRs comprising vhCDR1 set forth in SEQ ID NO: 368, vhCDR2 set forth in SEQ ID NO: 369, and vhCDR3 set forth in SEQ ID NO: 370, and the variable light chain domain comprises light chain CDRs comprising vlCDR1 set forth in SEQ ID NO: 371, vlCDR2 set forth in SEQ ID NO: 372, and vlCDR3 set forth in SEQ ID NO: 373. (Item 18) 18. The pharmaceutical composition of item 17, wherein the first variable heavy domain and the second variable heavy domain comprise an amino acid sequence at least 90% identical to SEQ ID NO: 377 or 379. (Item 19) 19. The pharmaceutical composition of item 18, wherein the first variable heavy domain and the second variable heavy domain comprise the amino acid sequence of SEQ ID NO: 377 or 379. (Item 20) 20. The pharmaceutical composition according to any one of items 17 to 19, wherein the variable light chain domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 378. (Item 21) 21. The pharmaceutical composition of item 20, wherein the variable light domain comprises the amino acid sequence of SEQ ID NO: 378. (Item 22) 18. The pharmaceutical composition of item 17, wherein the first variable heavy domain and the second variable heavy domain comprise an amino acid sequence at least 90% identical to SEQ ID NO: 380. (Item 23) 23. The pharmaceutical composition of item 22, wherein the first variable heavy domain and the second variable heavy domain comprise the amino acid sequence of SEQ ID NO: 380. (Item 24) 24. The pharmaceutical composition of claim 22 or 23, wherein the variable light domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 381. (Item 25) 25. The pharmaceutical composition of item 24, wherein the variable light chain domain comprises the amino acid sequence of SEQ ID NO: 381. (Item 26) 26. The pharmaceutical composition according to any one of items 17 to 25, wherein the scFv comprises the variable heavy chain region and the variable light chain region of SEQ ID NO: 382 and SEQ ID NO: 383. (Item 27) 26. The pharmaceutical composition according to any one of items 17 to 25, wherein the scFv linker comprises SEQ ID NO: 391. (Item 28) 26. The pharmaceutical composition according to any one of items 17 to 25, wherein the scFv comprises the sequence of SEQ ID NO: 390. (Item 29) a) the first monomer comprises the sequence of SEQ ID NO: 366 or SEQ ID NO: 367, the second monomer comprises the sequence of SEQ ID NO: 365, and the common light chain comprises the sequence of SEQ ID NO: 364; or b) The pharmaceutical composition according to any one of items 17 to 28, wherein the first monomer comprises the sequence of SEQ ID NO: 376, the second monomer comprises the sequence of SEQ ID NO: 375, and the common light chain comprises the sequence of SEQ ID NO: 374. (Item 30) 30. The liquid pharmaceutical composition according to any one of items 1 to 29, wherein the at least one buffering agent is an acid selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, and 2-(N-morpholino)ethanesulfonate, or a combination thereof. (Item 31) 31. The pharmaceutical composition according to item 30, wherein the at least one buffering agent is present in a concentration range of 5 to 200 mM. (Item 32) 31. The pharmaceutical composition according to item 30, wherein the at least one buffering agent is present in a concentration range of 10 to 50 mM. (Item 33) 33. The pharmaceutical composition according to any one of items 1 to 32, wherein the at least one saccharide is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched dextrans, and linear unbranched dextrans. (Item 34) 34. The pharmaceutical composition according to item 33, wherein the disaccharide is selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol or a combination thereof. (Item 35) 34. The pharmaceutical composition according to claim 33, wherein the sugar alcohol is sorbitol. (Item 36) 36. The pharmaceutical composition according to any one of items 1 to 35, wherein the at least one saccharide is present in a concentration in the range of 1 to 15% (w / V). (Item 37) 36. The pharmaceutical composition according to any one of items 1 to 35, wherein the at least one saccharide is present in a concentration in the range of 5 to 12% (w / V). (Item 38) 38. The pharmaceutical composition according to any one of items 1 to 37, wherein the at least one saccharide is present in a concentration in the range of 7 to 12% (w / V). (Item 39) 39. The pharmaceutical composition according to item 38, wherein the at least one saccharide is present in a concentration ranging from 9 to 12% (w / V). (Item 40) 40. The pharmaceutical composition according to any one of items 1 to 39, 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, or a combination thereof. (Item 41) 41. The pharmaceutical composition according to any one of items 1 to 40, wherein the at least one surfactant is present in a concentration in the range of 0.001 to 0.5% (w / V). (Item 42) 42. The pharmaceutical composition according to any one of items 1 to 41, wherein the at least one surfactant is present in a concentration ranging from 0.004 to 0.5% (w / V). (Item 43) 42. The pharmaceutical composition according to item 41, wherein the at least one surfactant is present in a concentration ranging from 0.001 to 0.01% (w / V). (Item 44) 43. The pharmaceutical composition according to item 42, wherein the at least one surfactant is present in a concentration ranging from 0.004 to 0.01% (w / V). (Item 45) 45. The pharmaceutical composition according to any one of items 1 to 44, wherein the pH of the composition is in the range of 4.0 to 5.0. (Item 46) 46. ​​The pharmaceutical composition according to item 45, wherein the pH of the composition is 4.2. (Item 47) 47. The pharmaceutical composition according to any one of items 1 to 46, having an osmolality in the range of 150 to 500 mOsm. (Item 48) 48. The pharmaceutical composition according to any one of items 1 to 47, further comprising an excipient selected from the group consisting of polyols and amino acids. (Item 48) 45. The pharmaceutical composition according to item 44, wherein the excipient is present in a concentration range of 0.1 to 15% (w / V). (Item 49) 46. ​​The pharmaceutical composition according to any one of items 1 to 45, wherein the composition comprises 10 mM glutamate, 9% (w / V) sucrose and 0.01% (w / V) polysorbate 80, and the pH of the liquid pharmaceutical composition is 4.2. (Item 50) 50. The pharmaceutical composition according to any one of items 4 to 49, wherein the heterodimeric antibody is present in a concentration range of 0.1 to 8 mg / ml. (Item 51) 50. The pharmaceutical composition according to any one of items 4 to 49, wherein the heterodimeric antibody is present in a concentration range of 0.1 to 20 mg / mL. (Item 52) 52. The pharmaceutical composition according to any one of items 4 to 51, wherein the heterodimeric antibody is present in an amount ranging from 50 μg to 200 mg. (Item 53) 53. The pharmaceutical composition according to any one of items 1 to 16 and 30 to 52, wherein the heterodimeric antibody is present at 1 mg / mL. (Item 54) 53. The pharmaceutical composition according to any one of items 1 to 16 and 30 to 52, wherein the heterodimeric antibody is present at 5 mg / mL. (Item 55) 53. The pharmaceutical composition according to any one of items 1 to 16 and 30 to 52, wherein the heterodimeric antibody is present at 20 mg / mL. (Item 56) 53. The pharmaceutical composition according to any one of items 1 to 3 and 17 to 52, wherein the heterodimeric antibody is present at 10 mg / mL. (Item 57) 57. The pharmaceutical composition according to any one of items 1 to 56, which is a lyophilized composition. (Item 58) 58. The pharmaceutical composition according to any one of items 1 to 57, which is a liquid composition. (Item 59) 59. The pharmaceutical composition according to item 58, which is a reconstituted lyophilized composition. (Item 59) 60. A method for treating cancer in a subject in need thereof, comprising administering to the subject the composition according to any one of items 1 to 59. (Item 60) 60. The method of claim 59, wherein the cancer is multiple myeloma. (Item 61) 60. The method of claim 59, wherein the cancer is prostate cancer. [Brief description of the drawings]

[0033] [Figure 1A] Several formats of heterodimeric antibodies are shown. Two forms of "bottle opener" formats are shown, one with an anti-CD3 antigen binding domain containing an scFv and an anti-CD38 antigen binding domain containing a Fab (as an example of an antigen binding domain), and one with the reverse. mAb-Fv, mAb-scFv, central-scFv (or "XmAb2+1 format") and central-Fv formats are all shown. In addition, a "one-arm" format is shown in which one monomer contains only the Fc domain, and both one-arm central-scFv and one-arm central-Fv are shown. A dual-scFv format is also shown. [Figure 1B]Same as above. [Diagram 2] Shown is the sequence of the "high-intermediate #1" anti-CD3_H1.32_L1.47 construct, including the variable heavy and light domains (underlined CDRs), and the individual vl and vhCDRs, as well as the scFv construct with a charged linker (double underlined). As is true for all sequences shown in the figures, this charged linker may be replaced by an uncharged linker or a different charged linker, if desired. [Diagram 3] Intermediate CD38: The OKT10_H1L1.24 construct containing the variable heavy and light domains (underlined CDRs) and the individual vl and vhCDRs, as well as the sequences of the scFv construct with a charged linker (double underlined) are shown. [Figure 4] Low CD38: Shown are the sequences of the OKT10_H1L1 construct containing the variable heavy and light domains (underlined CDRs) and the individual vl and vhCDRs as well as the scFv construct with a charged linker (double underlined). [Diagram 5] The sequence of XENP18971 is shown, with the CDRs underlined. [Figure 6] The sequence of XENP18969 is shown, with the CDRs underlined. [Figure 7] The sequence of human CD3ε is shown (SEQ ID NO: 130). [Figure 8] The full length (SEQ ID NO: 131) and extracellular domain (ECD; SEQ ID NO: 132) of human CD38 protein are shown. [Figure 9A] Useful pairs of heterodimerization mutant sets (including asymmetric and pI mutants) are shown. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 10]A list of antibody constant regions of isosteric variants and their corresponding substitutions is shown. pI_(-) indicates variants with low pI and pI_(+) indicates variants with high pI. These can be optionally and independently combined with heterodimerization variants of the invention (and other variant types as well as outlined herein). [Figure 11] Useful truncation mutants that ablate FcγR binding are shown (sometimes referred to as “knockout” or “KO” mutants). [Figure 12] 1 shows two embodiments of the antibodies of the present disclosure. [Figure 13A] Several charged scFv linkers are shown that find use in raising or lowering the pI of heterodimeric antibodies that utilize one or more scFvs as components. A single prior art scFv linker with a single charge is referred to as "Whitlow" from Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should be noted that this linker was used to reduce aggregation and increase proteolytic stability of scFvs. [Figure 13B] Same as above. [Figure 14] Shown is a list of engineered heterodimer-asymmetric Fc mutants along with heterodimer yield (determined by HPLC-CIEX) and thermal stability (determined by DSC). Thermal stability not determined is indicated by "nd". [Figure 15A] Figure 1 shows a stability-optimized humanized anti-CD3 variant scFv. Substitutions are made to the H1_L1.4 scFv sequence. Amino acid numbering is Kabat numbering. [Figure 15B] Same as above. [Figure 16A] The amino acid sequences of the stability-optimized humanized anti-CD3 variant scFvs are shown. The CDRs are underlined. For each heavy / light chain combination, four sequences are listed: (i) scFv with a C-terminal 6xHis tag, (ii) scFv alone, (iii) VH alone, (iv) VL alone. [Figure 16B] Same as above. [Figure 17] The sequence of XENP18971 is shown, with the CDRs underlined. [Figure 18] The sequence of XENP18969 is shown, with the CDRs underlined. [Figure 19] A matrix of possible combinations of embodiments is shown. "A" means that the CDRs of the CD3 sequence referenced can be combined with the CDRs of the CD38 construct on the left side; i.e., for example, for the top left cell, the vhCDRs of the variable heavy chain CD3H1.30 sequence and the vlCDRs of the variable light chain of the CD3 L1.47 sequence can be combined with the vhCDRs of the CD38 OKT10 H1.77 sequence and the vlCDRs of the OKT10L1.24 sequence. "B" means that the CDRs from the CD3 construct can be combined with the variable heavy and light chain domains from the CD38 construct; i.e., for example, for the top left cell, the vhCDRs of the variable heavy chain CD3H1.30 sequence and the vlCDRs of the variable light chain of the CD3 L1.47 sequence can be combined with the variable heavy chain domains CD38 OKT10 H1.77 sequence and the OKT10L1.24 sequence. "C" is where the variable heavy and light domains from the CD3 sequence are inverted so that they are used with the CDRs of the CD38 sequence; "D" is where both the variable heavy and light chains from each are combined; "E" is where the scFv of CD3 is used with the CDRs of the CD38 antigen binding domain construct, and "F" is where the scFv of CD3 is used with the variable heavy and light domains of the CD38 antigen binding domain. [Figure 20] The sequence of XmAb18968, also referred to herein as Antibody A, is shown with the CDRs underlined. [Figure 21-1]Table relating various CDR sequences, variable region sequences, heavy and light chain sequences, scFv sequences, scaffold sequences, etc., with sequence identifiers as set out in the sequence listing attached to this application. For some bottle opener format scaffolds described (SEQ ID NOs: 347-354), the sequences are provided without Fv sequences (e.g., scFv and Fab vh and vl). As will be appreciated by the skilled artisan and as outlined below, these sequences can be used with any of the vh and vl pairs outlined herein, where one monomer comprises an scFv (optionally including a charged scFv linker) and the other monomer comprises a Fab sequence (e.g., a vh attached to the "Fab heavy chain" and a vl attached to the "constant light chain"). The scFv can be anti-CD3 or anti-CD38, and the Fab the other. ("Fab" refers to the portion comprising the VH, CH1, VL, and CL immunoglobulin domains.) That is, for example, any of the Fv sequences outlined herein for CD3 and CD38 can be incorporated into these frameworks in any combination. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 21-4] Same as above. [Figure 21-5] Same as above. [Figure 21-6] Same as above. [Figure 21-7] Same as above. [Figure 21-8] Same as above. [Figure 21-9] Same as above. [Figure 21-10] Same as above. [Figure 21-11] Same as above. [Figure 21-12] Same as above. [Figure 21-13] Same as above. [Figure 21-14] Same as above. [Figure 21-15] Same as above. [Figure 21-16] Same as above. [Figure 21-17] Same as above. [Figure 21-18] Same as above. [Figure 21-19] Same as above. [Figure 21-20] Same as above. [Figure 21-21] Same as above. [Figure 21-22] Same as above. [Figure 21-23] Same as above. [Figure 22A] 22A and 22B show the reduction of the main peak of antibody A when formulated in formulation A (FIG. 22A) and formulation B (FIG. 22B) as determined by SE-UHPLC. [Figure 22B] Same as above. [Figure 23] 1 shows the reduction of the main peak of antibody A when formulated in formulation C as determined by SE-UHPLC. [Figure 24A] 24A and 24B show the reduction of the main peak of antibody A when formulated in formulation A (FIG. 24A) and formulation B (FIG. 24B) as determined by CE-HPLC. [Figure 24B] Same as above. [Diagram 25] 1 shows the reduction of the main peak of antibody A when formulated in formulation C as determined by CE-HPLC. [Figure 26] 1 shows the decrease in the main peak of Antibody A at -30°C when formulated in Formulation A as determined by rCE. [Figure 27] 1 shows the reduction of the main peak of Antibody A at 40° C. when formulated in Formulation B as determined by rCE. [Figure 28] 1 shows the reduction of the main peak of antibody A when formulated in formulation C as determined by rCE. [Figure 29] 1 shows the percent deamidation of antibody A at 4° C., 2° C., and 40° C. over a three month period when formulated in formulations B and C, as determined by MAM. [Figure 30-1] 4 shows the percent deamidation of antibody A at 40° C. at 0, 2 and 4 weeks when formulated in formulation D, as determined by MAM. [Figure 30-2] Same as above. [Diagram 31]1 shows the percent deamidation of antibody A at 40° C. over one month when formulated in formulations B and C, as determined by MAM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present disclosure describes low pH formulations comprising antigen binding proteins that bind to CD3.

[0035] In some embodiments, the antigen binding protein is an antibody, such as a bispecific antibody (e.g., a bispecific antibody that binds CD3). In some embodiments, the antigen binding protein is a heterodimeric antibody that co-binds CD3 and CD38 in such a manner as to transiently bind malignant cells to T cells, thereby inducing T cell-mediated killing of the bound malignant cells. In other embodiments, the antigen binding protein is a heterodimeric antibody that co-binds CD3 and STEAP1.

[0036] Certain protein-based pharmaceuticals are not stable in liquid formulations for extended periods of time, particularly at refrigerated temperatures above 4° C. The general concept underlying the present invention is the discovery that the colloidal stability of liquid pharmaceutical compositions comprising an antigen-binding protein according to the invention is enhanced at low pH.

[0037] Various aspects of the formulation are described below. The use of section headings is merely for the convenience of reading and is not intended to be limiting per se. It should be understood that the entire specification is intended to be considered as a unified disclosure, and that all combinations of the features described herein are contemplated.

[0038] In one aspect, described herein is a pharmaceutical composition comprising an antigen binding protein as described herein, at least one buffering agent, at least one surfactant, and at least one saccharide, wherein the pharmaceutical composition has a pH in the range of 3.5 to 5.

[0039] Buffer The pharmaceutical compositions of the present invention comprise a buffering agent that may be optionally selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate, potassium phosphate, acetic acid / sodium acetate, citric acid / sodium citrate, succinic acid / sodium succinate, tartaric acid / sodium tartrate, histidine / histidine HCl, glycine, Tris, glutamate, and combinations thereof. In some embodiments, the pharmaceutical compositions comprise at least one buffering agent selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate, and combinations thereof.

[0040] Buffers are often utilized in formulations to control pH. In some embodiments, buffers are added at concentrations that maintain the pH of the formulation at about 3.5-5, or about 4-5, or about 4.2. The effect of pH on a formulation can be characterized using any one or more of several techniques, such as accelerated stability studies and calorimetric screening studies (Remmele RL Jr., et al., Biochemistry, 38(16):5241-7(1999)).

[0041] Organic acids, phosphates, and Tris are preferred buffers in protein formulations (Table 1). The buffering capacity of a buffer species is greatest when the pH is equal to the pKa and decreases as the pH increases or decreases from this value. Ninety percent of the buffering capacity is within one pH unit of its pKa. Buffering capacity also increases proportionally with increasing buffer concentration.

[0042] When selecting a buffer, several factors are usually considered. For example, the buffer type 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 side chain residues of proteins. A third aspect to be considered is the sensation of stinging and irritation that the buffer may induce. For example, citrate is known to cause stinging upon injection (Laursen T, et al., Basic Clin Pharmacol Toxicol., 98(2):218-21(2006)). The possibility of stinging and irritation is greater for drugs administered via SC or IM routes, where the drug solution remains at the site for a relatively long period of time, than when administered via IV routes, where the formulation is rapidly diluted into the blood upon administration. For formulations administered by direct IV infusion, the total amount of buffer (and any other formulation components) needs to be monitored. For example, it has been reported that potassium ions administered in the form of potassium phosphate buffer can induce cardiovascular effects in patients (Hollander-Rodriguez JC, et al., Am. Fam. Physician., 73(2):283-90(2006)).

[0043] [Table 1]

[0044] The buffer system present in the formulation is selected to be physiologically compatible and to maintain the desired pH.

[0045] The buffering agent may be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. The buffering agent may be present at a concentration between about 0.1 mM and about 1000 mM (1M), or between about 5 mM and about 200 mM, or between about 5 mM and about 100 mM, or between about 10 mM and about 50 mM. Suitable buffering agent concentrations include concentrations of about 200 mM or less. In some embodiments, the buffering agent in the formulation is present at a concentration of about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, about 10 mM, or about 5 mM. In some embodiments, the concentration of the buffering agent is at least 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 buffering agent is between 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 buffering agent is between 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 buffering agent is about 10 mM.

[0046] Other exemplary pH buffering agents used to buffer the formulations as described herein include, but are not limited to, glycine, glutamate, succinate, phosphate, acetate, and aspartate. Amino acids such as histidine and glutamic acid can also be used as buffering agents.

[0047] Surfactants The pharmaceutical compositions described herein 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 outcompete proteins for interfacial positions. The hydrophobic portion of the surfactant molecule occupies the interfacial positions (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (typically near the detergent's critical micelle concentration), the surface layer of the surfactant molecule serves to prevent protein molecules from adsorbing at the interface. This minimizes surface-induced degradation. Surfactants include, for example, fatty acid esters of sorbitan polyethoxylates, namely polysorbate 20 and polysorbate 80 (see, for example, Avonex®, Neupogen®, Neulasta®). The two differ only in the length of the aliphatic chain that confers hydrophobic properties to the molecules C-12 and C-18, respectively. Thus, 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 commercial liquid products such as Gonal-F®, Norditropin®, and Ovidrel®.

[0048] Detergents can also affect the thermodynamic conformational stability of proteins. Again, the effect of a given excipient is protein specific. For example, polysorbates have been shown to decrease the stability of some proteins and increase the stability of others. Detergent protein destabilization makes sense in terms of the hydrophobic tails of the detergent molecules that can be involved in specific binding to partially or totally unfolded protein states. These types of interactions can cause a shift in the conformational equilibrium towards more extended protein states (i.e., increasing the exposure of hydrophobic parts of the protein molecule, complementing the binding polysorbates). Alternatively, if the native state of a protein exhibits several hydrophobic surfaces, detergents binding to the native state can stabilize that conformation.

[0049] Another aspect of polysorbates is that they are inherently prone to oxidative degradation. They often contain sufficient amounts of peroxide as raw material to cause oxidation of protein residue side chains, especially methionine. Due to the possibility of oxidative damage resulting from the addition of stabilizers, it is emphasized that the lowest effective concentration of excipients should be used in the formulation. In the case of surfactants, the effective concentration for a given protein is determined by the stabilization mechanism. If the stabilization mechanism of the surfactant is related to the prevention of surface denaturation, it is assumed that the effective concentration will be near the critical micelle concentration of the detergent. Conversely, if the stabilization mechanism is related to a specific protein-detergent interaction, the effective surfactant concentration will be related to the protein concentration and the stoichiometry of the interaction (Randolph TW, et al., Pharm Biotechnol., 13:159-75 (2002)).

[0050] Surfactants may also be added in appropriate amounts to prevent surface-related aggregation phenomena during freezing and drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Exemplary surfactants include anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, including those derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic 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 hexadecyltrimethylammonium 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; polyoxyl 40 stearate; polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60; glycerol monostearate; polysorbate 40, 60, 65, and 80; soy lecithin and other phospholipids such as DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid esters; methylcellulose, and carboxymethylcellulose.

[0051] The pharmaceutical compositions described herein contain at least one surfactant, either individually or as a mixture in various ratios. In some embodiments, the compositions contain a surfactant at a concentration of about 0.001% to about 5% w / v (or about 0.004 to about 0.5% w / v, or about 0.001 to about 0.01% w / v, or about 0.004 to about 0.01% w / v). In some embodiments, the composition comprises a surfactant at a concentration of at least 0.001, at least 0.002, at least 0.003, at least 0.004, at least 0.005, at least 0.007, at least 0.01, at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 4.5% w / v. In some embodiments, the composition comprises a surfactant at a concentration of about 0.004% to about 0.5% w / v. In some embodiments, the composition comprises a surfactant at a concentration of about 0.004 to about 0.5% w / v. In some embodiments, the composition comprises a surfactant at a concentration of about 0.001 to about 0.01% w / v. In some embodiments, the composition comprises a surfactant at a concentration of about 0.004 to about 0.01% w / v. In some embodiments, the composition comprises a surfactant at a concentration of about 0.004, about 0.005, about 0.007, about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, 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% 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.

[0052] Sugars The pharmaceutical compositions described herein include at least one sugar. Sugars may be added as stabilizers or bulking agents. As used herein, the term "stabilizer" refers to an excipient that can prevent aggregation or other physical and chemical deterioration (e.g., autolysis, deamidation, oxidation, etc.) in aqueous and solid states. Stabilizers used in pharmaceutical compositions include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, polysaccharides such as cellulose and hyaluronic acid, and sodium chloride (Carpenter et al., 2001). al.,Develop.Biol.Standard 74:225,(1991)).

[0053] In some embodiments, the at least one saccharide is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched dextrans, and linear unbranched dextrans, and combinations thereof. In some embodiments, the at least one saccharide is a disaccharide selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol, or combinations thereof.

[0054] In some embodiments, the pharmaceutical composition comprises at least one saccharide at a concentration of about 0.01% to about 40% w / v, or about 00.1% to about 20% w / v, or about 1% to about 15% w / v. In some embodiments, the pharmaceutical composition comprises at least one saccharide at a concentration of at least 0.5, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, or at least 40% w / v. In some embodiments, the pharmaceutical composition comprises at least one saccharide at a concentration of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14% to about 15% w / v. In some embodiments, the pharmaceutical composition comprises at least one saccharide at a concentration of about 1% to about 15% w / v. In further embodiments, the pharmaceutical composition comprises at least one saccharide at a concentration of about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, or about 12% w / v. In some embodiments, the pharmaceutical composition comprises at least one saccharide at a concentration of about 9% to about 12% w / v. In some embodiments, the at least one saccharide is in the composition at a concentration of about 9% w / v. In some embodiments, the at least one saccharide is selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol or a combination thereof. In some embodiments, the saccharide is sorbitol and is present in the composition in a range of about 9% to about 12% w / v.

[0055] If necessary, the formulation also includes appropriate amounts of bulking agents and osmolality adjusting agents, such as sugars suitable for forming a lyophilization "cake."

[0056] In a preferred embodiment, the pharmaceutical composition comprises 10 mM glutamate, 9% (w / V) sucrose and 0.01% (w / V) polysorbate 80, and the pH of the pharmaceutical composition is 4.2.

[0057] Other considerations As used herein, the term "pharmaceutical composition" refers to a composition suitable for administration to a patient 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 which administration of the pharmaceutical composition of the present invention is desired. Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc., with humans being preferred. The pharmaceutical composition of the present invention is stable and pharmaceutical acceptable, i.e., capable of eliciting the desired therapeutic effect without causing significant undesirable local or systemic effects in the subject to which the pharmaceutical composition is administered. The pharmaceutical acceptable composition of the present invention may be sterile and / or pharmaceutical inactive. In particular, the term "pharmaceutical acceptable" may mean approved by a regulatory agency or other generally recognized pharmacopoeias for use in animals and more particularly for use in humans.

[0058] Formulations provided by the present disclosure include antigen binding proteins (e.g., heterodimeric antibodies) described herein. In some embodiments, the heterodimeric antibodies are provided in a therapeutically effective amount. By "therapeutically effective amount" is meant the amount of said heterodimeric antibody that elicits the desired therapeutic effect. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal in 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio ED50 / LD50. Formulations that exhibit large therapeutic indices are generally preferred.

[0059] Protein preparations are generally administered parenterally. When given parenterally, they must be sterile. Sterile diluents include liquids that are pharma- ceutically acceptable (safe and non-toxic for human administration) and useful for preparing liquid preparations, such as preparations that are reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution or dextrose solution. Diluents may include aqueous solutions of salts and / or buffers.

[0060] Excipients are additives that are included in formulations because they provide or enhance the stability, delivery and manufacturability of drug products. Regardless of the reason for including excipients, they are essential components of drug products and therefore need to be safe and well tolerated by patients. For protein drugs, the selection of excipients is particularly important because they can affect the efficacy and immunogenicity of drugs. Therefore, protein formulations need to be developed with the appropriate selection of excipients that provide suitable stability, safety and marketability.

[0061] The excipients described herein are organized by either their chemical species or their functional role in the formulation. A brief description of the mode of stabilization is provided when discussing each excipient type. With the teachings and guidance provided herein, one of skill in the art will be able to easily vary the amount or range of excipients without increasing the viscosity to undesirable levels. Excipients may be selected to achieve the desired osmolality (i.e., isotonic, hypotonic or hypertonic), pH, desired stability, resistance to aggregation or degradation or precipitation, protection under freezing, lyophilization or high temperature conditions, or other properties of the final solution. Various types of excipients are known in the art. Exemplary excipients include salts, amino acids, other isotonicity agents, surfactants, stabilizers, bulking agents, cryoprotectants, lyoprotectants, antioxidants, metal ions, chelating agents and / or preservatives.

[0062] Additionally, when a particular excipient is reported in a formulation, for example, as a percentage (%) w / v, one of skill in the art will recognize that the equimolar concentration of that excipient is also intended.

[0063] Other stabilizers and fillers Stabilizers include a class of compounds that can act as cryoprotectants, lyoprotectants, and glass formers. Cryoprotectants act to stabilize proteins during freezing or in the frozen state at low temperatures. Lyoprotectants stabilize proteins in freeze-dried solid dosage forms by preserving the native-like conformational properties of the protein during the dehydration stage of freeze-drying. Glass state properties have been classified as "robust" or "fragile" depending on their relaxation properties as a function of temperature. To provide stability, it is important that cryoprotectants, lyoprotectants, and glass formers remain in the same phase as the protein. Sugars, polymers, and polyols fall into this category and can in some cases fulfill all three roles.

[0064] Polyols encompass a class of excipients that include sugars (e.g., mannitol, sucrose, or sorbitol) and other polyhydric alcohols (e.g., glycerol and propylene glycol). Polyethylene glycol (PEG) polymers fall into this category. Polyols are commonly used as stabilizing excipients and / or tonicity agents in both liquid and lyophilized parenteral protein formulations. Polyols can protect proteins from both physical and chemical degradation pathways.

[0065] Exemplary C3-C6 polyols include propylene glycol, glycerin (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, and the like. Higher sugars include dextran, propylene glycol, or polyethylene glycol. Reducing sugars such as fructose, maltose, or galactose are more easily oxidized than non-reducing sugars. Further examples of sugar alcohols are glucitol, maltitol, lactitol, or iso-maltulose. Further exemplary lyoprotectants include glycerin and gelatin, and the sugars melibiose, melezitose, raffinose, mannotriose and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, iso-maltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyalcohols. Monoglycosides include compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose.

[0066] amino acid In some embodiments, the pharmaceutical compositions described herein further comprise one or more amino acids. Amino acids are versatilely used in protein formulations as buffers, bulking agents, stabilizers, and antioxidants. Histidine and glutamic acid are utilized to buffer protein formulations in the pH ranges of 5.5-6.5 and 4.0-5.5, respectively. The imidazole group of histidine has a pKa=6.0, and the carboxyl group of the glutamic acid side chain has a pKa of 4.3, making them suitable for buffering in their corresponding pH ranges. Glutamic acid is found in some formulations (e.g., Stemgen®). Histidine is commonly found in commercial protein formulations (e.g., Xolair®, Herceptin®, Recombinate®). It offers a good alternative to citrate, a buffering agent known for its pain upon injection. Interestingly, histidine has also been reported to have a stabilizing effect when used at high concentrations in both liquid and lyophilized formats (Chen B, et al., Pharm Res., 20(12):1952-60(2003)). Histidine (up to 60 mM) was also observed to reduce the viscosity of highly concentrated formulations of antibodies. However, in the same study, the authors observed increased aggregation and discoloration in formulations containing histidine during freeze-thaw testing of antibodies in stainless steel containers. The authors attributed this to the effect of iron ions leached from corrosion of the steel containers. Another caution when using histidine is that it undergoes photo-oxidation in the presence of metal ions (Tomita M, et al., 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 some oxidative stresses (Lam XM, et al., J Pharm Sci., 86(11):1250-5(1997)).

[0067] The amino acids glycine, proline, serine and alanine stabilize proteins. Glycine is also a commonly used bulking agent in lyophilized formulations (e.g., Neumega®, Genotropin®, Humatrope®). Arginine has been shown to be an effective agent in inhibiting aggregation and has been used in both liquid and lyophilized formulations (e.g., Activase®, Avonex®, Enbrel® liquid).

[0068] Antioxidants In some embodiments, the pharmaceutical compositions described herein further comprise one or more antioxidants. Oxidation of protein residues arises from several different sources. In addition to the addition of specific antioxidants, prevention of oxidative protein damage requires careful control of several factors during the manufacturing process and storage of the product, such as atmospheric oxygen, temperature, exposure to light, and chemical contamination. The most commonly used pharmaceutical antioxidants are reducing agents, oxygen / free radical scavengers, or chelating agents. Antioxidants in therapeutic protein formulations must be water soluble and must remain active throughout the shelf life of the product. Reducing agents and oxygen / free radical scavengers function by cleaving reactive oxygen species in solution. Chelating agents such as EDTA may exert their effect by binding trace metal contaminants that promote free radical formation. For example, EDTA has been utilized in liquid formulations of acidic fibroblast growth factor to inhibit metal ion catalyzed oxidation of cysteine ​​residues. EDTA is used in commercial products such as Kineret® and Ontak®.

[0069] However, antioxidants themselves may induce other covalent or physical changes to proteins. Several such cases have been reported in the literature. Reducing agents (such as glutathione) can result in the disruption of intramolecular disulfide bonds that can lead to disulfide shuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote methionine oxidation in some proteins and peptides (Akers MJ, and Defelippis MR. Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Sven Frokjaer, Lars Hovgaard, editors. 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 rhuMab HER2; however, the formation of thiosulfate-protein adducts was also reported in this study (Lam XM, Yang JY, et al., J Pharm Sci. 86(11):1250-5(1997)). The selection of an appropriate antioxidant depends on the specific stress and sensitivity of the protein.

[0070] Metal ions In some embodiments, the pharmaceutical composition further comprises one or more metal ions. In general, transition metal ions in protein formulations are undesirable because they can catalyze physical and chemical decomposition reactions in proteins. However, certain metal ions are included in the formulation when they are cofactors for proteins and in suspension formulations of proteins where they form coordination compounds (e.g., zinc suspensions of insulin). Recently, the use of magnesium ions (10-120 mM) to inhibit the isomerization of aspartic acid to isoaspartic acid has been proposed (WO 2004 / 039337).

[0071] Two examples where metal ions confer increased stability or activity to proteins are human deoxyribonuclease (rhDNase, Pulmozyme®) and Factor VIII. In the case of rhDNase, Ca +2 ions (up to 100 mM) increased the stability of the enzyme through specific binding sites (Chen B, et al., J Pharm Sci., 88(4):477-82 (1999)). Indeed, removal of calcium ions from solution by EGTA led to increased deamidation and aggregation. However, this effect was not observed in the presence of Ca +2 ions; other divalent cations (Mg +2 , Mn +2 , and Zn +2 ) was observed to destabilize rhDNase. A similar effect was observed with factor VIII. +2 and Sr +2 ions stabilized the protein, while Mg +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 Others, such as , destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm., 155, 121-131 (1997). In another study with factor VIII, Al +3A significant increase in the rate of aggregation was observed in the presence of ions (Derrick TS, et al., J. Pharm. Sci., 93(10):2549-57(2004)). The authors found that other excipients, such as buffer salts, did not. +3 They point out that ionic contamination is common, illustrating the need to use excipients of appropriate quality in formulated products.

[0072] Preservatives In some embodiments, the pharmaceutical composition further comprises one or more preservatives. Preservatives are necessary when developing a multiple-use parenteral formulation with more than one withdrawal from the same container. Its main function is to inhibit microbial growth and ensure sterility of the product over the shelf life or use period of the drug product. Commonly used preservatives include phenol, benzyl alcohol, meta-cresol, alkyl parabens such as methyl paraben or propyl paraben, benzalkonium chloride, and benzethonium chloride. Other examples of compounds with antimicrobial preservative activity include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride. Other types of preservatives include aromatic alcohols such as butyl alcohol, phenol, benzyl alcohol; atechol, resorcinol, cyclohexanol, 3-pentanol. Although preservatives have a long history of use, developing protein formulations containing preservatives can be difficult. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations (Roy S, et al., J Pharm Sci., 94(2):382-96 (2005)).

[0073] Multi-use injection pen formats include preserved formulations. For example, preserved formulations of hGH are currently available on the market. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech) and Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope® (Eli Lilly) is formulated with m-cresol.

[0074] During formulation development of preserved dosage forms, several aspects are considered. Optimization of preservative concentration requires testing a given preservative in a dosage form with a concentration range that provides antimicrobial efficacy without compromising protein stability. For example, three preservatives were successfully screened using differential scanning calorimetry (DSC) in the development of a liquid formulation for interleukin-1 receptor (type I). The preservatives were ranked based on their impact on stability at concentrations commonly used in commercial products (Remmele RL Jr., et al., Pharm Res., 15(2):200-8 (1998)).

[0075] Some preservatives may cause injection site reactions, another factor to consider when selecting a preservative. In a clinical trial focused on the evaluation of preservatives and buffers in Norditropin, lower pain perception was observed in formulations containing phenol and benzyl alcohol compared to formulations containing m-cresol (Kappelgaard AM, Horm Res. 62 Suppl 3:98-103 (2004)). Interestingly, of the commonly used preservatives, benzyl alcohol has anesthetic properties (Minogue SC, and Sun DA., Anesth Analg., 100(3):683-6 (2005)).

[0076] However, the present disclosure also contemplates pharmaceutical compositions that do not contain any preservatives.

[0077] Antigen-binding proteins An "antigen binding protein" is a protein that includes a portion that binds to a designated target antigen (such as CD3 and / or CD38). An antigen binding protein includes a scaffold or framework portion that enables the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to an antigen. In exemplary aspects, the antigen binding protein is an antibody or immunoglobulin, or an antigen-binding antibody fragment, or an antibody protein product.

[0078] The term "antibody" refers to an intact antigen-binding immunoglobulin. An "antibody" is a type of antigen-binding protein. An antibody can be an IgA, IgD, IgE, IgG, or IgM antibody, including any one of IgG1, IgG2, IgG3, or IgG4. In various embodiments, an intact antibody comprises two full-length heavy chains and two full-length light chains. An antibody has a variable region and a constant region. In the IgG format, the variable region is generally about 100-110 or more amino acids, contains three complementarity determining regions (CDRs), and is primarily responsible for antigen recognition and varies substantially among other antibodies that bind different antigens. The variable regions usually contain at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), which are flanked by framework regions (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra). The constant regions enable the antibody to recruit cells and molecules of the immune system.

[0079] In various aspects, the antibody is a monoclonal antibody. In certain aspects, the antibody is a human antibody. In certain aspects, the antibody (or other antigen binding protein) is chimeric or humanized. The term "chimeric" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody can, for example, contain a constant domain from one species and a variable domain from a second species, or more commonly, contain a stretch of amino acid sequences from at least two species. Both "chimeric" and "humanized" often refer to antigen binding proteins that combine regions from two or more species. A chimeric antibody can also contain domains from two or more different antibodies within the same species. In one embodiment, a chimeric antibody is a CDR-grafted antibody.

[0080] The term "humanized" when used in reference to an antigen-binding protein refers to an antigen-binding protein (e.g., an antibody) having at least a CDR region derived from a non-human source that has been engineered to have a structure and immune function more similar to a true human antibody than the original source antibody. For example, humanization can involve grafting CDRs derived from a non-human antibody, such as a mouse antibody, into human framework regions. Generally, in a humanized antibody, the entire antibody except for the CDRs is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. CDRs encoded by nucleic acids partially or entirely derived from a non-human organism are grafted into the beta-sheet framework of a human antibody variable region to generate an antibody whose specificity is determined by the grafted CDRs. The generation of such antibodies is described, for example, in 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. "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often utilized to restore affinity lost in the initial grafted construct (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205; 5,821,337; 6,054,297; and 6,407,213, all of which are incorporated by reference in their entirety). The humanized antibody optimally also will contain at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus typically will contain a human Fc region.

[0081] Optionally, the antibody of the composition is a bispecific antibody, i.e., an antibody that binds to two different targets (e.g., CD3 and a second different target). In various embodiments, the antibody of the composition is a heterodimeric antibody.

[0082] In some embodiments, the compositions described herein comprise a heterodimeric antibody comprising a first monomer comprising a first Fc domain and an anti-CD3 scFv. The heterodimeric antibody further comprises a second monomer comprising a heavy chain constant domain comprising an anti-CD38 heavy chain variable domain and a second Fc domain. The heterodimeric antibody also comprises a light chain comprising a constant domain and an anti-CD38 variable light chain domain. Characteristics of the monomers are further described below.

[0083] An scFv comprises a variable heavy chain, an scFv linker, and a variable light chain domain. Optionally, the C-terminus of the variable light chain is attached to the N-terminus of the scFv linker, which in turn is attached to the N-terminus of the variable heavy chain (N-vh-linker-vl-C), although the configuration can be switched (N-vl-linker-vh-C). Thus, in depicting and describing scFvs, either orientation of the scFv is specifically included. In various embodiments, the scFv domain linker is a charged linker. Several suitable scFv linkers may be used, many of which are described in the figures. Charged scFv linkers can be utilized to facilitate separation in pI between the first and second monomers. That is, the incorporation of either positively or negatively charged scFv linkers (or both, in the case of scaffolds that use scFvs on different monomers) allows the monomers containing the charged linker to change pI without creating additional charges in the Fc domain.

[0084] The scFv is covalently linked to the N-terminus of the Fc domain using a domain linker. The "domain linker" links any two domains outlined herein together. If necessary, a charged domain linker can be used. A charged domain linker can also increase the pI separation of the monomers of the present disclosure, for example, and therefore those included in the drawings can be used in any embodiment of the present specification in which a linker is utilized.

[0085] The linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of sufficient length to link two molecules such that they assume the correct conformation relative to one another and thus retain the desired activity. In one embodiment, the linker is about 1-50 amino acids long, preferably about 1-30 amino acids long. In one embodiment, linkers of 1-20 amino acids long may be used, with about 5 to about 10 amino acids finding use in some embodiments. Useful linkers include glycine-serine polymers, such as (GS), (GSGGS) (SEQ ID NO: 332), (GGGGS) (SEQ ID NO: 333), and (GGGS) (SEQ ID NO: 334), where n is an integer of at least 1 (and usually 3-4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of non-proteinaceous polymers may find use as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0086] Other linker sequences may include any sequence of any length of the CL / CH1 domain, but not all residues of the CL / CH1 domain; for example, the first 5-12 amino acid residues of the CL / CH1 domain. Linkers may be derived from immunoglobulin light chains, such as Cκ or Cλ. Linkers may be derived from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), sequences from hinge regions, and other naturally occurring sequences from other proteins.

[0087] The anti-CD3 scFv comprises (i) an scFv variable light chain domain comprising a vlCDR1 set forth in SEQ ID NO: 15, a vlCDR2 set forth in SEQ ID NO: 16, and a vlCDR3 set forth in SEQ ID NO: 17, and (ii) an scFv variable heavy chain domain comprising a vhCDR1 set forth in SEQ ID NO: 11, a vhCDR2 set forth in SEQ ID NO: 12, and a vhCDR3 set forth in SEQ ID NO: 13. Optionally, the anti-CD3 scFv comprises a variable heavy chain domain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 10. Also optionally, the anti-CD3 scFv comprises a variable light chain domain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In this regard, the anti-CD3 scFv, in various embodiments, comprises a variable heavy chain domain of SEQ ID NO: 10 and a variable light chain domain of SEQ ID NO: 14. Optionally, the variable heavy and variable light chain domains are linked by an scFv domain linker comprising the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 158). In this regard, the anti-CD3 scFv, in various embodiments, comprises an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18 (scFv of antibody A). In various embodiments, sequence variations resulting in less than 100% percent identity to the reference sequence represent modifications outside the CDR sequences. In various embodiments, the scFv comprises a sequence described herein as belonging to anti-CD3_H1.32_L1.47 (corresponding to antibody A).

[0088] "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and optionally a portion of the hinge. Thus, "Fc domain" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, the Fc domain includes immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). The heterodimeric antibody is preferably an IgG antibody (including several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226, or P230, to the carboxyl terminus, where numbering is according to the EU index as described in Kabat. In some embodiments, amino acid modifications are made to the Fc region to, for example, alter binding to one or more FcγR or FcRn receptors.

[0089] In various embodiments, the first monomer (i.e., the first Fc domain and the anti-CD3 scFv) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:335 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:335 (corresponding to Antibody A)).

[0090] The heterodimeric antibody optionally further comprises a second monomer comprising i) an anti-CD38 heavy chain variable domain and ii) a heavy chain constant domain comprising a second Fc domain. The anti-CD38 heavy chain variable domain comprises the following CDR sequences: variable heavy chain (vh) CDR1 set forth in SEQ ID NO:65, vhCDR2 set forth in SEQ ID NO:66, and vhCDR3 set forth in SEQ ID NO:67. Optionally, the anti-CD38 heavy chain variable domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:64 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:64). In various embodiments, the second monomer (i.e., the heavy chain constant domain comprising an anti-38 heavy chain variable domain and a second Fc domain) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:82 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:82 (corresponding to Antibody A)).

[0091] In various embodiments, the heterodimeric antibody further comprises a light chain comprising a constant domain and an anti-CD38 variable light (vl) domain. The anti-CD38 variable light domain comprises the following CDRs: vlCDR1 set forth in SEQ ID NO:69, vlCDR2 set forth in SEQ ID NO:70, and vlCDR3 set forth in SEQ ID NO:71. Optionally, the anti-CD38 variable light domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:68 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:68 (corresponding to antibody A)). In some embodiments, the light chain (including the constant domain and the anti-CD38 variable light domain) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 84 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 84 (corresponding to Antibody A)).

[0092] In a preferred embodiment, the heterodimeric antibody is antibody A and comprises a first monomer comprising an anti-CD3 scFv comprising an anti-CD3 variable light domain comprising the amino acid sequence of SEQ ID NO: 14 and an anti-CD3 variable heavy domain comprising the amino acid sequence of SEQ ID NO: 10, a second monomer comprising an anti-CD38 variable heavy domain comprising the amino acid sequence of SEQ ID NO: 64, and a light chain comprising a variable light domain comprising the amino acid sequence of SEQ ID NO: 68. For example, in one embodiment, the heterodimeric antibody comprises a first monomer comprising the amino acid sequence of SEQ ID NO: 335, a second monomer comprising the amino acid sequence of SEQ ID NO: 82, and a light chain comprising the amino acid sequence of SEQ ID NO: 84.

[0093] In some embodiments, the compositions described herein comprise heterodimeric antibodies that bind to CD3 and STEAP1. STEAP1 is a 339 amino acid protein that contains six transmembrane domains resulting in three extracellular loops and two intracellular loops. The amino acid sequence of human STEAP1 is set forth herein as SEQ ID NO: 356. The predicted locations of the extracellular loops are amino acids 92-118 (extracellular loop 1), amino acids 185-217 (extracellular loop 2), and amino acids 279-290 (extracellular loop 3). STEAP1 is differentially expressed in prostate cancer compared to normal tissues, and increased expression has been observed in bone and lymph node prostate cancer metastases compared to primary prostate cancer samples. STEAP1 is an ideal target for diagnostics and antibody-based therapeutics, such as, for example, bispecific anti-STEAP1 / anti-CD3 T cell recruiting antibodies to induce T cell-dependent cytotoxicity or redirected lysis of prostate cancer cells. The antigen binding proteins of the present disclosure optionally bind STEAP1 at a region outside the second extracellular loop. The antigen binding protein, in at least one embodiment, binds to a region of STEAP1 within amino acids 92-118 (extracellular loop 1) and / or amino acids 279-290 (extracellular loop 3). Optionally, the antigen binding protein does not bind to STEAP2 (UniProtKB No. Q8NFT2; SEQ ID NO: 177). The present disclosure provides antigen binding proteins in the formats described herein, optionally in the "bottle opener" format of FIG. 1A or the central-scFv format of FIG. 1B ("XmAb 2+1 The present invention provides compositions comprising antigen binding proteins that bind to STEAP1 and CD3 in either an immunoglobulin or immunoglobulin (also referred to as an immunoglobulin or immunoglobulin) format.

[0094] In some embodiments, the compositions described herein comprise a heterodimeric antibody comprising a first monomer comprising a first Fc domain and an anti-CD3 scFv and a second monomer further comprising a heavy chain constant region comprising an anti-STEAP1 heavy chain variable domain and a second Fc domain. The heterodimeric antibody also comprises a light chain comprising a constant domain and an anti-STEAP1 variable light chain domain. Exemplary aspects of the scaffolding monomers are described above. In this embodiment, the heterodimeric antibody further comprises a second monomer comprising i) an anti-STEAP1 heavy chain variable domain and ii) a heavy chain constant domain comprising a second Fc domain. The anti-STEAP1 heavy chain variable domain optionally comprises a variable heavy chain (vh) CDR1 as set forth in SEQ ID NO: 360, a vhCDR2 as set forth in SEQ ID NO: 361 or SEQ ID NO: 363, and a vhCDR3 as set forth in SEQ ID NO: 362. The anti-STEAP1 variable light domain of the light chain comprises a vlCDR1 set forth in SEQ ID NO: 357, a vlCDR2 set forth in SEQ ID NO: 358, and a vlCDR3 set forth in SEQ ID NO: 359. Alternatively, the anti-STEAP1 variable heavy domain comprises a vhCDR1 set forth in SEQ ID NO: 368, a vhCDR2 set forth in SEQ ID NO: 369, and a vhCDR3 set forth in SEQ ID NO: 370; and the variable light domain comprises a vlCDR1 set forth in SEQ ID NO: 371, a vlCDR2 set forth in SEQ ID NO: 372, and a vlCDR3 set forth in SEQ ID NO: 373.

[0095] Optionally, the anti-STEAP1 heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 377 or SEQ ID NO: 379 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 377 or SEQ ID NO: 379). Optionally, the anti-STEAP1 variable light chain domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 378 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 378). In a preferred embodiment, the anti-STEAP1 variable heavy chain domain comprises SEQ ID NO: 380 or SEQ ID NO: 379 and the anti-STEAP1 variable light chain domain comprises SEQ ID NO: 378.

[0096] Also, optionally, the anti-STEAP1 heavy chain variable domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 380 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 380). Optionally, the anti-STEAP1 variable light chain domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 381 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 381). In a preferred embodiment, the anti-STEAP1 variable heavy chain domain comprises SEQ ID NO: 380 and the anti-STEAP1 variable light chain domain comprises SEQ ID NO: 381.

[0097] In some embodiments, such as those embodiments, the antigen binding protein is a heterodimeric antibody that binds to CD3 and STEAP1, and the CD3 binding domain (optionally an scFv as described above) comprises a variable heavy chain domain comprising heavy chain CDRs comprising vhCDR1 set forth in SEQ ID NO:383, vhCDR2 set forth in SEQ ID NO:384, and vhCDR3 set forth in SEQ ID NO:385, and a variable light chain domain comprising light chain CDRs comprising vlCDR1 set forth in SEQ ID NO:387, vlCDR2 set forth in SEQ ID NO:388, and vlCDR3 set forth in SEQ ID NO:389. For example, the disclosure provides compositions comprising multispecific (e.g., bispecific) constructs comprising an anti-CD3 variable heavy chain domain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 382 and / or an anti-CD3 variable light chain domain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 386. In various embodiments, the heterodimeric antibody comprises an anti-CD3 scFv comprising SEQ ID NO: 390. The scFv is described in more detail above, and the above scFv characteristics also apply herein.

[0098] In various embodiments, the antigen binding protein is the central-scFv shown in FIG. 2+1" format of heterodimeric antibodies. The format relies on the use of an inserted scFv domain that forms a third antigen-binding domain, where the Fab portions of the two monomers bind one target and the "additional" scFv domain binds another target. The scFv domain is inserted between the Fc domain and the CH1-Fv region of one of the monomers, thereby resulting in a third antigen-binding domain. In this embodiment, one monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain (and optional linker / hinge) and an Fc domain, together with an scFv comprising an scFv variable light domain, an scFv linker and an scFv variable heavy domain. The scFv is covalently linked using an optional domain linker between the C-terminus of the CH1 domain of the heavy chain constant domain and the N-terminus of the first Fc domain (VH1-CH1-[optional domain linker]-VH2-scFv linker-VL2-[optional domain linker including hinge]-CH2-CH3, or in the opposite orientation with respect to the scFv, VH1-CH1-[optional domain linker]-VL2-scFv linker-VH2-[optional domain linker including hinge]-CH2-CH3). In some embodiments, the first monomer is VH1-CH1-domain linker-VH2-scFv linker-VL2 domain linker-CH2-CH3. The other monomer is a standard Fab side (i.e., VH1-CH1-domain linker (e.g., hinge)-CH2-CH3). This embodiment further utilizes a common light chain that includes a variable light domain and a constant light domain associated with the heavy chain that form two identical Fabs that bind to the target. For many of the embodiments herein, these constructs include asymmetric mutants, pI mutants, truncation mutants, additional Fc mutants, etc. as desired and described herein and in WO 2017 / 21870.

[0099] In some embodiments, the antigen binding protein is "XmAb 2+1" format, and the CD3 binding domain (optionally an scFv as described above) comprises a variable heavy chain domain comprising heavy chain CDRs comprising vhCDR1 set forth in SEQ ID NO: 383, vhCDR2 set forth in SEQ ID NO: 384, and vhCDR3 set forth in SEQ ID NO: 385, and a variable light chain domain comprising light chain CDRs comprising vlCDR1 set forth in SEQ ID NO: 387, vlCDR2 set forth in SEQ ID NO: 388, and vlCDR3 set forth in SEQ ID NO: 389. For example, the construct optionally comprises an anti-CD3 variable heavy chain domain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 380 and / or an anti-CD3 variable light chain domain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 381. In various embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO: 391. In various embodiments, the heterodimeric antibody comprises an anti-CD3 scFv comprising SEQ ID NO: 390.

[0100] In various embodiments, the antigen binding protein is XmAb, which comprises two Fabs that bind to STEAP1. 2+1In this regard, in some embodiments, the first and second variable heavy chain domains of the heterodimeric antibody comprise a vhCDR1 as set forth in SEQ ID NO: 360, a vhCDR2 as set forth in SEQ ID NO: 361 or SEQ ID NO: 363, and a vhCDR3 as set forth in SEQ ID NO: 362; and the variable light chain domain comprises a vlCDR1 as set forth in SEQ ID NO: 357, a vlCDR2 as set forth in SEQ ID NO: 358, and a vlCDR3 as set forth in SEQ ID NO: 359. Alternatively, the first and second variable heavy chain domains comprise a vhCDR1 as set forth in SEQ ID NO: 368, a vhCDR2 as set forth in SEQ ID NO: 369, and a vlCDR3 as set forth in SEQ ID NO: 370; and the variable light chain domain comprises a vlCDR1 as set forth in SEQ ID NO: 371, a vlCDR2 as set forth in SEQ ID NO: 372, and a vlCDR3 as set forth in SEQ ID NO: 373. In a preferred embodiment, the first variable heavy chain domain and the second variable heavy chain domain comprise an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 377 (corresponding to antibody B) or SEQ ID NO: 379, and / or the variable light chain domain comprises an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 378 (corresponding to antibody B). Alternatively, the first variable heavy chain domain and the second variable heavy chain domain comprise an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to SEQ ID NO: 380, and / or the variable light chain domain comprises an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to SEQ ID NO: 381. As noted above, mutations in any variable domain sequence (or full length monomer sequence) described herein that result in less than 100% sequence identity compared to a reference sequence are preferably located outside of the CDR regions.

[0101] Thus, the present disclosure provides a pharmaceutical composition comprising a heterodimeric antibody comprising: (a) a first monomer comprising: 1) a first variable heavy chain domain; 2) a first constant heavy chain comprising a first CH1 domain and a first Fc domain; and 3) a first heavy chain comprising an scFv that binds human CD3. The scFv comprises: (i) an scFv variable light chain domain comprising a vlCDR1 as set forth in SEQ ID NO: 387, a vlCDR2 as set forth in SEQ ID NO: 388, and a vlCDR3 as set forth in SEQ ID NO: 389, (ii) an scFv linker, and (iii) an scFv variable heavy chain domain comprising a vhCDR1 as set forth in SEQ ID NO: 383, a vhCDR2 as set forth in SEQ ID NO: 384, and a vhCDR3 as set forth in SEQ ID NO: 385. The scFv is covalently linked using a domain linker between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain. The heterodimeric antibody further comprises: b) a second monomer comprising a second heavy chain comprising a second constant heavy chain comprising a second variable heavy domain and a second Fc domain; and c) a common light chain comprising a variable light domain and a constant light domain; the first variable heavy domain and the variable light domain bind to human STEAP1, and the second variable heavy domain and the variable light domain bind to human STEAP1. In some embodiments, the first variable heavy domain and the second variable heavy domain comprise heavy chain CDRs comprising vhCDR1 set forth in SEQ ID NO: 360, vhCDR2 set forth in SEQ ID NO: 361 or SEQ ID NO: 363, and vhCDR3 set forth in SEQ ID NO: 362. The variable light domain optionally comprises light chain CDRs comprising vlCDR1 set forth in SEQ ID NO: 357, vlCDR2 set forth in SEQ ID NO: 358, and vlCDR3 set forth in SEQ ID NO: 359. Alternatively, the first variable heavy domain and the second variable heavy domain comprise heavy chain CDRs comprising vhCDR1 set forth in SEQ ID NO: 368, vhCDR2 set forth in SEQ ID NO: 369, and vhCDR3 set forth in SEQ ID NO: 370. The variable light domain optionally comprises light chain CDRs comprising vlCDR1 set forth in SEQ ID NO: 371, vlCDR2 set forth in SEQ ID NO: 372, and vlCDR3 set forth in SEQ ID NO: 373.Optionally, the first variable heavy chain domain and the second variable heavy chain domain comprise an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 377 (corresponding to antibody B) or 379, and / or the variable light chain domain comprises an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 378 (corresponding to antibody B). The scFv optionally comprises a variable heavy chain region and a variable light chain region having amino acid sequences at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 382 and SEQ ID NO: 386, respectively, and the scFv linker optionally comprises SEQ ID NO: 391. In various embodiments, the scFv comprises the sequence of SEQ ID NO: 390.

[0102] In various embodiments of the disclosure, the anti-CD3 / anti-STEAP1 antigen binding protein is an XmAb comprising a first monomer comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 366 or 367, a second monomer comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 365, and a common light chain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 364. 2+1In some embodiments, the anti-CD3 / anti-STEAP1 antigen binding protein is a heterodimeric antibody of the format XmAb, which comprises a first monomer comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 366, a second monomer comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 365, and a common light chain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 364 (corresponding to antibody B). 2+1 Alternatively, the anti-CD3 / anti-STEAP1 antigen binding protein is a heterodimeric antibody of the format XmAb, comprising a first monomer comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:376, a second monomer comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:375, and a common light chain comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:374. 2+1 It is a heterodimeric antibody of the above format.

[0103] In various embodiments, the antigen binding protein comprises a first heavy chain comprising VH1-CH1-[domain linker]-VH2-scFv linker-VL2-[domain linker (optionally including a hinge)]-CH2-CH3; a second heavy chain comprising VH1-CH1-domain linker-CH2-CH3; and a common light chain comprising VL1; VH1 and VL1 bind to STEAP1, and VH2 and VL2 bind to CD3. In this format, VH2 optionally comprises the CDR sequences of SEQ ID NO:383 (CDR1), SEQ ID NO:384 (CDR2), and SEQ ID NO:385 (CDR3), while VL2 comprises the CDR sequences of SEQ ID NO:387 (CDR1), SEQ ID NO:388 (CDR2), and SEQ ID NO:389 (CDR3). VH1 comprises the CDR sequences of SEQ ID NO:360 (CDR1), SEQ ID NO:361 or 363 (CDR2), and SEQ ID NO:362 (CDR3); and VL1 comprises the CDR sequences of SEQ ID NO:357 (CDR1), SEQ ID NO:358 (CDR2), and SEQ ID NO:359 (CDR3). Alternatively, VH1 comprises the CDR sequences of SEQ ID NO:368 (CDR1), SEQ ID NO:369 (CDR2), and SEQ ID NO:370 (CDR3); and VL1 comprises the CDR sequences of SEQ ID NO:371 (CDR1), SEQ ID NO:372 (CDR2), and SEQ ID NO:373 (CDR3). Optionally, the antigen binding protein comprises modifications in a first heavy chain including but not limited to E233P, delL234, L235V, G236A, S267K, r292c, n297g, v302c, E357Q, and S364K (EU numbering, lower case letters refer to SEFL2 substitutions further described herein), and the second heavy chain comprises modifications including but not limited to N208D, E233P, delL234, L235V, G236A, S267K, r292c Q295E, n297g, v302c, L368D, K370S, N384D, Q418E, and N421D (EU numbering, lower case letters refer to SEFL2 substitutions further described herein). A linker for use in the context of this embodiment is optionally GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:391).

[0104] The Fc domain of the central scFv format optionally comprises an asymmetric mutant (e.g., selected from the group consisting of S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L, K370S:S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C), optionally a truncation mutant, optionally a charged scFv linker, and the heavy chain comprises a pI mutant. In some embodiments, the central scFv format comprises asymmetric mutants, pI mutants, and truncation mutants. Thus, some embodiments comprise a) a first monomer comprising an asymmetric mutant S364K / E357Q, a truncation mutant E233P / L234V / L235A / G236del / S267K, and a first variable heavy chain domain that together with a first variable light chain domain of the light chain constitutes an Fv that binds to a first target, and a second variable heavy chain domain; b) a first monomer comprising an asymmetric mutant L368D / K370S, a pI mutant N208D / Q295E / N3 a second monomer comprising a first variable heavy chain domain which, together with the first variable light chain domain, constitutes an Fv that binds to a first target, and a second variable light chain which, together with the second variable heavy chain, forms an Fv that binds to a second target; and c) a light chain comprising the first variable light chain domain and a constant light chain domain.

[0105] The different binding regions of the multispecific antigen-binding protein can be independently -4 M or less, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less, or 10 -13 M or less (e.g., 10 -7 M~10 -12M) for their corresponding antigens (e.g., CD3 and STEAP1 or CD3 and CD38), where KD refers to the off-rate of a specific antibody-antigen interaction. A STEAP1-binding region (or a CD38-binding region) need not bind STEAP1 (or CD38) with the same affinity as, for example, a CD3-binding region binds CD3.

[0106] In some embodiments, the formulation comprises an antigen binding protein (e.g., an antibody) described herein in an amount ranging from about 50 μg to about 200 mg (or about 500 μg to about 150 mg, or 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 formulation comprises 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 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 of the antibody.

[0107] In some embodiments, the formulation comprises an antigen binding protein (e.g., an antibody) at a concentration in the range of about 0.1 to about 20 mg / mL (or about 0.5 to about 10 mg / mL, or about 1 to about 10 mg / mL, or about 1 to about 20 mg / mL, or about 10 to about 20 mg / mL). In some embodiments, the formulation comprises an antigen binding protein (e.g., an antibody such as any of the heterodimeric antibodies described herein) 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, or about 20 mg / mL.

[0108] In some embodiments, the formulation comprises an antigen binding protein (e.g., an antibody) at a concentration ranging from about 0.1 to about 8 mg / mL (or about 0.5 to about 5 mg / mL or about 1 to about 5 mg / mL, or about 3 to about 6 mg / mL). In some embodiments, the formulation comprises an antigen binding protein (e.g., an antibody, such as any of the heterodimeric antibodies described herein) 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, or about 8 mg / mL.

[0109] Heterodimeric antibody formats are further described in WO 2017 / 218707, which is incorporated herein by reference in its entirety and in particular with respect to the figures and figure legends. In a preferred embodiment, the heterodimeric antibody takes the structure referred to as "bottle opener" in FIG. 1A. One heavy chain of the "bottle opener" format contains an scFv, while the other heavy chain is a "conventional" Fab format that includes conventional heavy and light chains. The two heavy chains are linked by the use of amino acid variants in the constant regions (e.g., Fc domain, CH1 domain and / or hinge region) that promote the formation of heterodimeric antibodies. The "bottle opener" format has several distinct advantages. Antibody analogs that rely on two scFv constructs often have stability and aggregation problems that are mitigated in the present disclosure by the addition of "conventional" heavy and light chain pairing. Additionally, in contrast to formats that rely on two heavy and two light chains, there are no problems with inappropriate pairing of heavy and light chains (eg, heavy chain 1 pairing with light chain 2).

[0110] Heterodimeric antibodies, in various embodiments, include modifications that promote heterodimeric antibody formation (i.e., reduce homodimerization), adjust the functionality of the antibody, etc., compared to the wild-type antibody domain sequence. The modifications are generally centered in the Fc domain (although this is not required). The modifications are referred to by the amino acid position of the substitution, deletion, or insertion relative to the native sequence. For example, N434S or 434S is an Fc domain substitution of serine at position 434 relative to the parent Fc polypeptide, where the numbering is according to the EU index. Similarly, M428L / N434S defines an Fc modification having substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the wild-type amino acids may not be specified, in which case the aforementioned variant is referred to as 428L / 434S. The order in which the substitutions are made is arbitrary, i.e., for example, 428L / 434S is the same as M428L / N434S, etc. For all positions discussed in the antibody, unless otherwise noted, the numbering of amino acid positions is according to the EU index. The EU index or EU index as Kabat or EU numbering scheme refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). Modifications can be additions, deletions, or substitutions. Substitutions may include naturally occurring amino acids and, optionally, synthetic amino acids.See, for example, U.S. Pat. No. 6,586,207; U.S. Patent Application Publication No. 2004-0214988A1; WO 98 / 48032; WO 03 / 073238; WO 05 / 35727A2; WO 05 / 74524A2; WO 17 / 218707; JW Hin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JW Hin, & P.G. Schultz, (2002), ChemBioChem 11:1135-1137; JW Hin, et al., (2002), PICAS United States of America, all of which are incorporated by reference in their entirety. 99:11020-11024; and L. Wang, & P. ​​G. Schultz, (2002), Chem. 1-10.

[0111] There are several mechanisms that can be used to generate heterodimeric proteins. Amino acid mutants that result in the generation of heterodimers are called "heterodimerization mutants." Heterodimerization mutants can include conformational mutants (e.g., "knob-and-hole" or "asymmetric" mutants, described below, and "charge pair" mutants, described below) as well as "pI mutants," which allow purification of homodimers from heterodimers. As generally described in WO2014 / 145806 and WO2017 / 218707, which are incorporated by reference herein in their entirety and specifically for their discussion of "heterodimerization mutants", useful mechanisms for heterodimerization include "knobs and holes" ("KIH"; sometimes referred to herein as "asymmetric" mutants), "electrostatic steering" or "charge pairing" as described in WO2014 / 145806, pI mutants as described in WO2014 / 145806, and additional Fc mutants generally as outlined in WO2014 / 145806 and herein.

[0112] There are several basic mechanisms that can facilitate the purification of heterodimeric antibodies; some rely on the use of pI variants, so that each monomer has a different pI, thereby allowing isoelectric purification of AA, AB, and BB dimeric proteins. Alternatively, some backbone formats, such as the "bottle opener" format, also allow size-based separation. It is also possible to "make asymmetric" so that heterodimer formation predominates over homodimers. Thus, the combination of stereodimerization variants and pI or charge pair variants finds particular use in the present invention.

[0113] A.pI (isoelectric point) mutant With respect to pI variants, amino acid modifications can be introduced into one or both of the monomeric polypeptides; i.e., the pI of one of the monomers (referred to herein for simplicity as "monomer A") can be engineered away from monomer B, or both monomers A and B can be altered, increasing the pI of monomer A and decreasing the pI of monomer B. The pI change of one or both monomers can be made by removing or adding a charged residue (e.g., replacing a neutral amino acid with a positively or negatively charged amino acid residue, e.g., replacing glycine with glutamic acid), changing a charged residue from a positive or negative charge to the opposite charge (aspartic acid to lysine), or changing a charged residue to a neutral residue (e.g., loss of charge; lysine to serine). Some of these variants are shown in the figures. These modifications result in a sufficient change in pI in at least one of the monomers such that the heterodimer can separate from the homodimer. As will be appreciated by one of skill in the art, this can be accomplished by using a "wild type" heavy chain constant region and mutated regions engineered to raise or lower its pI (wtA-+B or wtA--B), or by raising one region and lowering the other (A+-B- or A-B+).

[0114] Thus, in various embodiments, the heterodimeric antibody contains one or more modifications in the constant regions that alter the isoelectric point (pI) of at least one if not both monomers of the dimeric protein by incorporating amino acid substitutions ("pI variants" or "pI substitutions") into one or both monomers to form a "pI antibody." Separation of the heterodimer from the two homodimers can be achieved if the pI of the two monomers differs by as little as 0.1 pH units, with differences of 0.2, 0.3, 0.4, and 0.5 or more being all suitable.

[0115] The number of pI variants to be included in each or both monomers to obtain good separation will depend in part on the starting pI of the components, e.g., the starting pI of the anti-CD3 scFv and anti-CD38 Fab. That is, to determine which monomers to engineer or in which "direction" (e.g., positive or negative), the Fv sequences of the two domains are calculated and a decision is made from there. Different Fvs will have different starting pIs that can be utilized. In some embodiments, the pI changes are calculated based on the variant heavy chain constant domains using the chart in Figure 19 of US Patent Application Publication No. 2014 / 0370013. Alternatively, the pI of each monomer can be compared. Generally, the pI is engineered to result in a total pI difference of at least about 0.1 log for each monomer, with 0.2-0.5 being preferred.

[0116] A preferred combination of pI mutants is shown in Figure 10. These changes are shown for IgG1, but all isotypes, not just isotype hybrids, can be modified in this manner. When the heavy chain constant domain is IgG2-4, R133E and R133Q can also be used.

[0117] In one embodiment, the Fab monomer (negative side) comprises the substitutions 208D / 295E / 384D / 418E / 421D (N208D / Q295E / N384D / Q418E / N421D (relative to human IgG1) and the scFv monomer (positive side) comprises a positively charged scFv linker comprising (GKPGS)4.

[0118] Modifications to adjust the pI can also be made in the light chain. Amino acid substitutions to lower the pI of the light chain include, but are not limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E, K207E, and the addition of the peptide DEDE at the C-terminus of the light chain. Changes in this category based on the constant lambda light chain include one or more substitutions at R108Q, Q124E, K126Q, N138D, K145T, and Q199E. In addition, the pI of the light chain can be increased.

[0119] B. Asymmetric / Stereovariants There are several suitable pairs of sets of heterodimerizing asymmetric mutants. These mutants are provided in "pairs" of "sets"; i.e., one set of the pair is incorporated into the first monomer and the other set of the pair is incorporated into the second monomer. It should be noted that these sets do not necessarily behave as "knobs-in-holes" mutants with a one-to-one correspondence between residues on one monomer and residues on the other; i.e., these sets of pairs form an interface between the two monomers that promotes heterodimer formation and prevents homodimer formation, allowing the percentage of heterodimers that form spontaneously under biological conditions to exceed 90% rather than the expected 50% (25% homodimer A / A: 50% heterodimer A / B: 25% homodimer B / B).

[0120] In some embodiments, heterodimer formation is promoted by the addition of steric variants, i.e., by varying the amino acids in each heavy chain, different heavy chains are more likely to associate to form heterodimeric structures than to form homodimers with the same Fc amino acid sequence. Suitable examples of steric variants are included in FIG. 9.

[0121] One mechanism is commonly referred to in the art as "knobs and holes," which refers to amino acid manipulations that create steric effects to favor heterodimer formation and discourage homodimer formation, and may be optionally used. This is further described in U.S. Patent Publication No. 20130205756; Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Patent No. 8,216,805, all of which are incorporated by reference in their entirety. The figures identify several "monomer A-monomer B" pairs that rely on "knobs and holes." In addition, these "knobs and holes" mutations can be combined with disulfide bonds to asymmetrically dissociate heterodimer formation, as described in Merchant et al., Nature Biotech. 16:677 (1998).

[0122] A further mechanism that finds use in the generation of heterodimers may be referred to as "electrostatic steering", as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), which is incorporated herein by reference in its entirety. This may be referred to herein as "charge pairing". In this embodiment, electrostatics are used to asymmetrically drive the formation towards heterodimerization. As one skilled in the art will appreciate, these may also have an effect on pI and therefore purification, and therefore may in some cases also be considered to be pI mutants. However, because these were generated to force heterodimerization and were not used as purification tools, they are classified as "steric mutants". These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R (i.e., these are corresponding sets of monomers) and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0123] Additional monomer A and monomer B variants that can be optionally and independently combined in any amount with other variants such as the pI variants outlined herein or other stereovariants shown in Figure 37 of US Patent Publication No. 2012 / 0149876, the figures and legends and sequence numbers of which are expressly incorporated herein by reference.

[0124] In some embodiments, the steric variants outlined herein may be optionally and independently incorporated into one or both monomers along with any pI variants (or other variants such as Fc variants, FcRn variants, etc.) and may independently and optionally be included or excluded from the proteins of the invention.

[0125] A list of suitable asymmetric mutants can be found in Figures 9 and 12. Set pairs including, but not limited to, S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411T / E360E / Q362E:D401K;L368D / K370S:S364K / E357L and K370S:S364K / E357Q are particularly useful in many embodiments. In nomenclature terms, the pair "S364K / E357Q:L368D / K370S" means that one of the monomers has the double mutant set S364K / E357Q and the other has the double mutant set L368D / K370S.

[0126] C. Additional Fc variants to modulate functionality There are a number of useful Fc amino acid modifications that can be made for a variety of reasons, including, but not limited to, altering binding to one or more FcγR receptors, altered binding to the FcRn receptor, and the like.

[0127] There are several useful Fc substitutions that can be made to alter binding to one or more of the FcγR receptors. Substitutions that result in increased and decreased binding can be useful. For example, increased binding to FcγRIIIa generally results in increased ADCC (antibody-dependent cellular cytotoxicity; a cellular reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause lysis of the target cells). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can be beneficial in some situations as well. Amino acid substitutions that find use in the present invention include those listed in US Patent Publication Nos. 2006 / 0024298 (particularly FIG. 41), 2006 / 0121032, 2006 / 0235208, and 2007 / 0148170, all of which are expressly incorporated herein by reference in their entirety and specifically with respect to the variants disclosed. Particular mutants that find use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243A, 243L, 264A, 264V and 299T.

[0128] Additionally, there are additional Fc substitutions that find use in increasing binding to the FcRn receptor and increasing serum half-life, as specifically disclosed in U.S. Patent Application Publication No. 2009 / 0163699, incorporated herein by reference in its entirety, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L and 259I / 308F / 428L.

[0129] Another category of functional variants are "FcγR truncation variants" or "Fc knockout (FcKO or KO) variants. For some therapeutic applications, it is desirable to reduce or eliminate the normal binding of the Fc domain to one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. Thus, for example, particularly in the use of bispecific antibodies that bind monovalently to CD3, it may be desirable to ablate FcγRIIIa binding to eliminate or significantly reduce ADCC activity. Any level of reduction is contemplated (e.g., a 50%, 60%, 70%, 80%, 90%, or 100% reduction in binding or activity). Abbreviation Examples of mutant modifications are shown in Figure 11, each of which may be independently and optionally included or excluded, with preferred embodiments utilizing truncation mutants selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235A / G236del. It should be noted that the truncation mutants referred to herein ablate FcγR binding, but generally do not ablate FcRn binding.

[0130] D. Additional Antibody Considerations The present disclosure contemplates the use of other heterodimeric antibodies in the formulations described herein. For example, the variable heavy and light chain sequences described above, as well as scFv sequences (and Fab sequences including these variable heavy and light chain sequences), may be used in other formats, such as those shown in FIG. 2 of WO 2014 / 145806 or FIG. 1 of WO 2017 / 218707, and FIG. 1A and 1B, whose figures, formats and legends are expressly incorporated herein by reference. Additionally, the amino acid sequences of the CD3 binding region and the CD38 binding region (e.g., CDR sequences, variable light and variable heavy chain sequences, and / or full-length heavy and light chain sequences) are provided in the sequence listing provided herewith and summarized in FIG. 21. Any combination of the sequences referenced in FIG. 21 is contemplated herein, so long as the resulting heterodimeric antibody binds to both CD3 and CD38. Anti-CD3 / anti-CD38 antibodies are further described in references WO 2016 / 086196; U.S. Patent Application Publication No. 20160215063; WO 2017 / 091656; and U.S. Pat. No. 9,822,186, which are incorporated by reference herein in their entirety and in particular for the description of anti-CD3 / anti-CD38 antibodies and their amino acid and nucleic acid sequences, sequence listing, and figures.

[0131] With respect to CD3 binding, the heterodimeric antibody may comprise an anti-CD3 antigen binding domain that has intermediate or "moderate" affinity for CD3. In this regard, the heterodimeric antibody binds to CD3 with an affinity (KD) of about 15-50 nM (e.g., 16-50 nM, 15-45 nM, about 20-40 nM, about 25-40 nM, or about 30-40 nM), optionally measured using the assays described in U.S. Patent Publication No. 20160215063 and WO 2017 / 091656, which are incorporated herein by reference.

[0132] In another aspect, the heterodimeric antibody of the method comprises an anti-CD3 antigen binding domain that is a "strong" or "high affinity" binder for CD3 (e.g., one example is the heavy and light chain variable domains designated as H1.30_L1.47 (optionally including a charged linker as appropriate)). In various embodiments, the antibody construct binds to CD3 with an affinity (KD) of about 3-15 nM (e.g., 3-10 nM or about 4-7 nM), optionally measured using the assays described in U.S. Patent Publication No. 20160215063 and WO 2017 / 091656, which are incorporated herein by reference. In other embodiments, the method utilizes a heterodimeric antibody that comprises an anti-CD3 antigen binding domain that is a "light" or "low affinity" binder for CD3. In this regard, the heterodimeric antibody optionally binds to CD3 with an affinity (KD) of about 51 nM or greater (e.g., 51-100 nM), optionally measured using the assays described in U.S. Patent Publication No. 20160215063 and WO 2017 / 091656, which are incorporated by reference herein. The heterodimeric antibody also binds, for example, to CD38 or STEAP1.

[0133] The affinity of a bispecific antibody for CD38 also influences the efficacy of the antibody in targeting cells expressing CD38: bispecific antibodies with "moderate" or "low" affinity for CD38 can efficiently kill target cells in vitro and in vivo with a reduced toxicity profile. In various embodiments, a bispecific antibody exhibiting a "high" affinity for CD38, e.g., binds to CD38 with an affinity (KD) of less than 1 nM; a bispecific antibody exhibiting a "moderate" or "intermediate" affinity for CD38, e.g., binds to CD38 with an affinity (KD) of about 1-10 nM (e.g., 2-8 nM or 3-7 nM); and a bispecific antibody exhibiting a "low" or "light" affinity for CD38, e.g., binds to CD38 with an affinity (KD) of about 11 nM or greater (e.g., 11-100 nM), all optionally measured using the methods described in U.S. Patent Publication No. 20160215063 and WO 2017 / 091656, which are incorporated herein by reference.

[0134] Generally, specific binding is, for example, at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12Specific binding to a particular antigen may also be demonstrated by an antibody having a KD of at least M, where KD refers to the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold or more than a control molecule for the antigen. Specific binding to a particular antigen may also be demonstrated by an antibody having a K or Ka for an antigen or epitope that is, for example, at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold or more than a control molecule for the antigen, where K or Ka refers to the association rate of a particular antibody-antigen interaction. It will be understood that the disclosure regarding antibodies also applies to antigen-binding proteins.

[0135] Optionally, the heterodimeric antibody comprises a substitution of cysteine ​​at position 220 with serine; typically this is on the "scFv monomer" side of the heterodimeric antibody, but it may also be on the "Fab monomer" side, or both, to reduce disulfide formation. One or both of these substituted cysteines (C220S) are specifically included within the sequences herein.

[0136] E. Fragment The present disclosure also contemplates the use of antibody fragments (as distinguished from full-length antibodies, which constitute the natural biological form of an antibody comprising variable and constant regions, generally including Fab and Fc domains along with optional additional antigen-binding domains, such as scFv). Antibody fragments contain at least one constant domain that can be engineered, such as by pI engineering, to generate heterodimers. Other antibody fragments that can be used include fragments containing one or more of the CH1, CH2, CH3, hinge and CL domains of the invention that have been pI engineered.

[0137] F. Chimeric / Humanized Heterodimeric antibodies may be a mixture of different species, e.g., chimeric and / or humanized antibodies. In general, both "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions from two or more species. For example, "chimeric antibodies" conventionally contain variable regions from a mouse (or occasionally a rat) and constant regions from a human. "Humanized antibodies" generally refer to non-human antibodies in which the framework regions of the variable domains have been replaced with sequences found in human antibodies. Generally, in humanized antibodies, the entire antibody, except for the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody, except within its CDRs. CDRs encoded by nucleic acids partially or entirely originating from a non-human organism are grafted onto the beta-sheet framework of human antibody variable regions to generate an antibody whose specificity is determined by the grafted CDRs. The production of such antibodies is described, for example, in 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. "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often necessary to restore affinity lost in the initial grafted construct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205; 5,821,337; 6,054,297; and 6,407,213, all of which are incorporated by reference in their entirety). A humanized antibody may also comprise at least a portion of an immunoglobulin constant region, which is typically that of a human immunoglobulin, and thus typically will comprise a human Fc region. Humanized antibodies can also be produced using mice with genetically engineered immune systems. Roque et al., 2004, Biotechnol. Prog. 20:639-654, incorporated by reference in its entirety.Various techniques and methods for humanizing and reconstituting non-human antibodies are well known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein, all of which are incorporated by reference in their entirety). Methods for humanization include those described in Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160:1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1997, Cancer Res. 57(20):4593-9, all of which are incorporated by reference in their entirety. Humanization or other methods for reducing the immunogenicity of non-human antibody variable regions may include, but are not limited to, the methods described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, which is incorporated by reference in its entirety.

[0138] Dosage The term "effective dose" or "effective administration amount" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The amount or dose effective for this use will depend on the condition (indication) being treated, the antibody construct being delivered, the nature and purpose of the treatment, the severity of the disease, previous treatments, the patient's medical history and responsiveness to the therapeutic agent, the route of administration, the size (weight, body surface area or organ size) and / or condition (age and general health) of the patient, and the general condition of the patient's own immune system. The appropriate dose can be administered to the patient in a single dose or multiple doses, and can be adjusted according to the judgment of the attending physician to obtain the optimal therapeutic effect.

[0139] A therapeutically effective amount of an antigen binding protein (e.g., an antibody) preferably reduces the severity of disease symptoms, increases the frequency or duration of disease symptom-free periods, or prevents impaired function or disability due to disease affliction. For the treatment of antigen-expressing tumors of target cells, a therapeutically effective amount of an antigen binding protein (e.g., an antibody), such as an anti-target cell antigen / anti-CD3 antibody construct, preferably inhibits cell proliferation 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% compared to untreated patients. The ability of the molecule to inhibit tumor growth can be evaluated in animal models predictive of efficacy.

[0140] The term "effective and non-toxic dose" refers to a tolerated dose of an antigen binding protein (e.g., an antibody) that is high enough to result in depletion of pathological cells, elimination of tumors, shrinkage of tumors, or stabilization of disease without or essentially without causing significant toxic effects. Such an effective and non-toxic dose may be determined, for example, by dose escalation studies as described in the art, and should be below the dose that induces serious adverse side events (dose-limiting toxicity, DLT).

[0141] As used herein, the term "toxicity" refers to the toxic effects of a drug that manifest as adverse events or serious adverse events. These side events may refer to a lack of systemic drug tolerance and / or a lack of local tolerance after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

[0142] The terms "safety", "in vivo safety" or "tolerability" are defined as the administration of a drug that does not induce serious adverse events immediately after administration (local tolerance) and during longer drug application periods. "Safety", "in vivo safety" or "tolerability" can be evaluated, for example, during treatment and periodically during follow-up periods. Measurements include clinical evaluations, such as organ findings and screening for laboratory abnormalities. Clinical evaluations can be performed and deviations from normal findings can be recorded / coded according to NCI-CTC and / or MedDRA standards. Organ findings can include criteria such as allergy / immunology, blood / bone marrow, cardiac arrhythmias, coagulation, etc., as indicated in the Common Terminology Criteria for adverse events v3.0 (CTCAE). Laboratory parameters that can be tested include, for example, hematology, clinical chemistry, coagulation profile and urinalysis, as well as tests of other body fluids, such as serum, plasma, lymph or spinal fluid. Thus, safety can be assessed, for example, by physical examination, imaging techniques (i.e. ultrasound, x-ray, CT scan, magnetic resonance imaging (MRI)), other measurements using technical devices (i.e. electrocardiogram), vital signs, measuring laboratory parameters and recording adverse events. For example, in the uses and methods according to the invention, adverse events in non-chimpanzee primates can be examined by histopathological and / or histochemical methods.

[0143] The above terms are also referenced, for example, in Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6; ICH Harmonised Tripartite Guideline; ICH Steering Committee meeting of July 16, 1997.

[0144] Typical dosages can range from about 0.1 μg / kg up to about 30 mg / kg or more depending on the factors mentioned above. In certain embodiments, dosages can range from 1.0 μg / kg up to about 20 mg / kg, optionally 10 μg / kg up to about 10 mg / kg or 100 μg / kg up to about 5 mg / kg. The formulations can be provided such that the heterodimeric antibody is provided in a unit dose to achieve a dose of, for example, 0.1 to 50 mg of antibody (calculating the mass of the protein alone without chemical modifications) per kilogram of body weight.

[0145] Therapeutic Use of the Preparation The formulations described herein are useful as pharmaceutical compositions for treating, ameliorating and / or preventing the pathological medical conditions described herein in patients in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes application or administration of the formulation to the body, isolated tissues or cells of a patient having a disease / disorder, a symptom of a disease / disorder or a predisposition to a disease / disorder with the intent to cure, remedy, relieve, alleviate, alter, correct, ameliorate, improve or affect the disease, symptom of a disease or predisposition to a disease.

[0146] As used herein, the term "amelioration" refers to any improvement in the disease state of a patient having a tumor or cancer or metastatic cancer as set forth herein below, by administration to a subject in need thereof of a composition comprising an antigen binding protein as described herein. Such improvement may be seen as a slowing or halting of progression of the patient's tumor or cancer or metastatic cancer. As used herein, the term "prevention" refers to the avoidance of the onset or recurrence of a patient having a tumor or cancer or metastatic cancer as set forth herein below, by administration to a subject in need thereof of a composition comprising an antigen binding protein as described herein (i.e., an antibody construct).

[0147] In one embodiment, the present invention provides a method for the treatment or amelioration of a proliferative disease, a neoplastic disease, a viral disease or an immune disorder, comprising administering to a subject in need thereof a formulation as described herein. The term "disease" refers to any condition that would benefit from treatment with the pharmaceutical compositions described herein. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disease in question. The term "viral disease" refers to a disease that is the result of viral infection of a subject. The term "immune disorder" is used herein in line with the general definition of the term, which includes immune disorders such as autoimmune diseases, hypersensitivity, and immune deficiencies.

[0148] A "neoplasm" is an abnormal growth of tissue that usually, but not necessarily, forms a mass. When it also forms a mass, it is commonly referred to as a "tumor." A neoplasm or tumor can be benign, potentially malignant (precancerous), or malignant. Malignant neoplasms are commonly called cancers. They usually invade and destroy surrounding tissues and can form metastases, i.e., they spread to other parts, tissues, or organs of the body. Thus, the term "metastatic cancer" encompasses metastases to other tissues or organs other than that of the primary tumor. Lymphomas and leukemias are lymphatic neoplasms. For purposes of the present invention, they are also encompassed by the term "tumor" or "cancer." The term "subject in need" or "subject in need of treatment" includes subjects already with the disorder as well as those in whom the disorder is to be prevented. A subject in need or "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0149] Route of administration Exemplary routes of administration include, but are not limited to, topical routes (e.g., epicutaneous, inhalation, nasal, ocular, auricular / auricular, vaginal, mucosal); enteral routes (e.g., oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and parenteral routes (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extraamniotic, intraarticular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal). Preferably, the pharmaceutical formulation is administered parenterally, e.g., intravenously, subcutaneously, or intramuscularly. Parenteral administration may be achieved by injection, such as a bolus injection, or by infusion, such as continuous infusion. Administration may be achieved via a depot for extended release. In some embodiments, the formulation is administered intravenously by continuous infusion after an initial bolus to maintain therapeutic circulating levels of the drug product. In some embodiments, the formulation is administered as a single dose. Pharmaceutical composition can be administered using medical device.Examples of medical device for administering pharmaceutical composition are described in U.S. Patent No. 4,475,196; U.S. Patent No. 4,439,196; U.S. Patent No. 4,447,224; U.S. Patent No. 4,447,233; U.S. Patent No. 4,486,194; U.S. Patent No. 4,487,603; U.S. Patent No. 4,596,556; U.S. Patent No. 4,790,824; U.S. Patent No. 4,941,880; U.S. Patent No. 5,064,413; U.S. Patent No. 5,312,335; U.S. Patent No. 5,312,335; U.S. Patent No. 5,383,851; and U.S. Patent No. 5,399,163.

[0150] In particular, the present invention provides for uninterrupted administration of suitable compositions. As a non-limiting example, uninterrupted or substantially uninterrupted, i.e. continuous administration, can be achieved by a miniature pump system worn by the patient to regulate the inflow of therapeutic agent into the patient's body. The pharmaceutical composition can be administered by using said pump system. Such pump systems are generally known in the art and typically rely on periodic replacement of a cartridge containing the therapeutic agent to be infused. When replacing the cartridge in such a pump system, a temporary interruption in the otherwise uninterrupted inflow of therapeutic agent into the patient's body may result. Even in such cases, the administration step before the cartridge replacement and the administration step after the cartridge replacement will still be considered within the meaning of the pharmaceutical means and methods of the present invention, which together constitute the "uninterrupted administration" of such therapeutic agent.

[0151] The continuous or uninterrupted administration of the formulation may be intravenous or subcutaneous administration by a fluid delivery device or miniature pump system, which includes a fluid delivery mechanism for pumping the fluid from a reservoir and a drive mechanism for driving the delivery mechanism. The pump system for subcutaneous administration may include a needle or cannula for penetrating the patient's skin and delivering the suitable composition into the patient's body. The pump system may be fixed or attached directly to the patient's skin, whether vein, artery or blood vessel, allowing direct contact between the pump system and the patient's skin. The pump system may be attached to the patient's skin for 24 hours to several days. In some cases, the pump system may be a miniature pump system with a small reservoir volume. As a non-limiting example, the reservoir volume for the suitable pharmaceutical composition to be administered may be 0.1 to 50 ml.

[0152] Continuous administration can also be transdermal, by a patch that is attached to the skin and replaced from time to time.Those skilled in the art are aware of suitable patch systems for drug delivery for this purpose.It is noted that transdermal administration is particularly suitable for uninterrupted administration, since it has the advantage that, for example, a new second patch can be attached to the skin surface directly next to the first used patch, and the replacement of the first used patch can be completed at the same time just before removing the first used patch.There is no problem of interruption of inflow or battery failure.

[0153] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with a suitable liquid before administration. The lyophilized material may be reconstituted, for example, with bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS) or the same formulation in which the protein was present before lyophilization. The pharmaceutical composition may be administered in a single treatment or, if necessary, in combination with additional therapies, such as anticancer therapies, for example, other proteinaceous and non-proteinaceous drugs. These drugs may be administered simultaneously with the composition of the present invention as defined herein, or may be administered separately at a predefined time interval and dose before or after administration of the formulation.

[0154] kit In a further aspect, described herein is a kit comprising one or more pharmaceutical compositions described herein packaged in a manner that facilitates their use for administration to a subject.In one embodiment, such a kit comprises a formulation described herein (e.g., a composition comprising an antibody described herein) packaged in a container, such as a sealed bottle, container, single-use or multiple-use vial, pre-filled syringe, or pre-filled injection device, optionally with a label attached to the container, or contained in a package that describes the use of the compound or composition in practicing the method.In one aspect, the composition is packaged in a unit dosage form.The kit may further comprise a device suitable for administering the composition according to a specific route of administration.Preferably, the kit contains a label that describes the use of the antibody described herein or the formulation described herein.

[0155] The pharmaceutical compositions described herein can be formulated in various forms, such as solid, liquid, frozen, gaseous or lyophilized forms, and may be in the form of an ointment, cream, transdermal patch, gel, powder, tablet, solution, aerosol, granules, pills, suspension, emulsion, capsule, syrup, liquid, elixir, extract, tincture or liquid extract, among others.

[0156] Generally, various storage and / or dosage forms are contemplated for the pharmaceutical compositions of the invention, i.e., depending on the intended route of administration, delivery format, and desired dosage (see, e.g., Remington's Pharmaceutical Sciences, 22nd edition, Oslo, A., Ed., (2012)). One of skill in the art will recognize that the selection of such a particular dosage form can affect, for example, the physical state, stability, in vivo release rate, and in vivo clearance rate of the antibody.

[0157] For example, the primary vehicle or carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature.Suitable vehicles or carriers may be water for injection, saline solution or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration.Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. EXAMPLES

[0158] Materials and Methods SE-UHPLC: Size-exclusion ultra-performance liquid chromatography is a method for quantitative analysis of recombinant monoclonal antibodies (mAbs) or X-mAbs. SE-UHPLC separates proteins based on differences in hydrodynamic volume. Molecules with larger hydrodynamic volumes elute earlier than molecules with smaller volumes. Samples are loaded onto an SE-UHPLC column (BEH200, 4.6x300mm, (Waters Corporation, 186005226)) and separated isocratically, and the eluate is monitored by UV absorbance. Purity is determined by calculating the percentage of each separated component compared to the total integrated area. SE-UHPLC settings are as follows: flow rate: 0.4mL / min, run time: 12min, UV detection: 280nm, column temperature: ambient, target protein load: 6μg, protein compatible flow cell: 5mm.

[0159] Cation exchange high performance liquid chromatography (CEX) is a method for quantitative purity analysis on charge variant distribution. Mobile phase A: 1x CX-1 pH gradient buffer A, pH 5.6 (10x CX-1 pH gradient buffer A, pH 5.6, 250 mL) and mobile phase B: 1x CX-1 pH gradient buffer, pH 10.2.

[0160] CE-HPLC: Cation exchange high performance liquid chromatography is a method for quantitative purity analysis on charge variant distribution. Mobile phase A: 25 mM sodium phosphate, 10% acetonitrile, pH 6.7 and mobile phase B: 25 mM sodium phosphate, 500 mM sodium chloride, 10% acetonitrile, pH 6.7. The column used for CE-HPLC of antibody A is Bio Mab NP-5, 4.6x250mm, 5μm (Agilent Technology, 5190-2407). The CE-HPLC method settings are as follows: flow rate: 0.75mL / min, run time: 60min, column temperature set point: 30℃±5℃, detection wavelength: 280nm, target protein load: 20μg. The column used for the CE-HPLC method of antibody B is YMC BioPro SP-F, 4.6x100mm, 5μm (YMC Co., Ltd., SF00S05-1046WP). The settings for the CE-HPLC method are as follows: flow rate: 1.0mL / min, run time: 45min, autosampler temperature set point: 5±3°C, column temperature set point: 30±2°C, detection wavelength: 280nm, target protein load: 70±10ug.

[0161] rCE-SDS: Reduced capillary electrophoresis-sodium dodecyl sulfate is a method for quantitative purity analysis under denaturing and reducing conditions.

[0162] MAM: Multi-attribute method is a method for multiple product quality attributes (PQA) (oxidation, isomerization, deamidation and glycation) using Thermo Scientific Orbitrap mass spectrometer and Chromeleaon software. Chemical modifications upon heat stress (incubation at 40°C) were measured using peptide mapping. Proteins were enzymatically digested and the resulting peptides were separated using reversed-phase chromatography. Proteins were denatured with guanidine HCl and subsequently reduced with dithiothreitol (DTT). After incubation in DTT, free cysteine ​​residues were alkylated by addition of iodoacetic acid. Samples were then buffer exchanged into 50 mM Tris-HCl, 20 mM methionine, pH 7.8 for digestion. Trypsin and elastase were added to separate reaction tubes (enzyme to protein ratio 1:20). Samples were digested for 60 min at 37°C and 30 min at 37°C, respectively. Digestion was quenched by adding guanidine HCl, 250 mM acetate, pH 4.7.

[0163] The moisture content of the lyophilized drug product was determined by calorimetric titration with an oven. The moisture limit for lyophilized drug products is 2%. The principle of the Karl Fischer method is based on the moisture content in the sample determined by calorimetric titration. Water is released by heating the sample in an oven. Dry air or an inert gas such as nitrogen carried the evaporated moisture to the titrator. The amount of water present is determined by measuring the amount of coulombs (current / time) produced during the titration. When all the water has been consumed by the titration, excess iodine is generated. The end point is indicated in terms of volume by applying an alternating current of constant strength to two Pt electrodes. This results in a voltage difference between the Pt wires of the indicator electrodes that drops significantly in the presence of minimal amounts of free iodine. This voltage difference is used to determine the end point of the titration.

[0164] Example 1 - Antibody stability in low pH formulations The following examples describe assays to validate the stability of the heterodimeric antibodies described herein in either liquid or lyophilized formulations at various protein concentrations (i.e., 1 mg / mL and 5 mg / mL) at a variety of different temperatures (4°C, 25°C, 40°C, -30°C and -40°C) for up to 3 years. The stability of the heterodimeric antibodies was analyzed using the following assays: appearance (via visual inspection of 20 vials at each time point), pH, osmolality, CE-HPLC, rCE, MAM (multi-attribute method) and Karl Fischer (water content). Samples for validation studies were 1.3 mL filled in 5 cc vials. The heterodimeric antibody DS (material without polysorbate 80) was 10.6 mg / mL and was diluted with buffer (G42Su) to reach 5 mg / mL and 1 mg / mL. The formulation was isotonic in the G42SuT formulation with an osmolality value of 326 mOsm / kg.

[0165] The stability of the heterodimeric antibody was evaluated in the following formulations: Formulation A: lyophilized formulation of 1 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; Formulation B: lyophilized formulation of heterodimeric antibody at 5 mg / mL in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; Formulation C: A liquid formulation of 5 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; and Formulation D: Liquid formulation of 1 mg / mL heterodimeric antibody in 10 mM acetate, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 5.2.

[0166] result: Antibody A, when formulated in formulation C, was determined to have a 13.4% main peak loss via SE-UHPLC after 3 months at 40° C. See FIG. 23. In contrast, antibody A, when formulated in formulation A (FIG. 22A) and formulation B (FIG. 22B), was determined to have a 0.2% and 0.0% main peak loss via SE-UHPLC under the same conditions, respectively. Antibody A, when formulated in formulation C, was determined to have a 58.2% main peak loss via CE-HPLC after 3 months at 40° C. See FIG. 25. In contrast, antibody A, when formulated in formulation A (FIG. 24A) and formulation B (FIG. 24B), was determined to have a 10.5% and 0.5% main peak loss via CE-HPLC under the same conditions, respectively.

[0167] Antibody A, when formulated in formulation A, showed a 2.9% main peak loss via rCE after 3 months at -30°C (Figure 26). Antibody A, when formulated in formulation C, showed a 19.9% ​​main peak loss via rCE after 3 months at 40°C. See Figure 28. However, Antibody A, in formulation A (Figure 26), showed a 0.6% main peak loss via rCE after 3 months at 40°C, and when formulated in formulation B, showed a 0.0% main peak loss via rCE. See Figure 27.

[0168] Antibody A, when formulated in formulation C, showed 9.1% deamidation at N103 (CD3 scFv-FC) via MAM after 3 months at 40° C. See the last column in FIG. 29. In contrast, antibody A, when formulated in formulation B, showed 0.3% deamidation via MAM. See the second column from the right in FIG. 29. Antibody A, when formulated in formulation D, showed 13.8% deamidation at N103 (CD3 scFv-FC) via MAM after 4 weeks at 40° C. (see FIG. 30), and when formulated in formulation C, showed 3.7% deamidation at N103 (CD3 scFv-FC) via MAM after 1 month at 40° C. See FIG. 31. In contrast, antibody A, when formulated in formulation B, showed 0.4% deamidation via MAM after 1 month at 40° C. See FIG. 31.

[0169] The data provided herein demonstrates that lyophilized formulations A and B are more stable than liquid formulations C and D due to the increased risk of deamidation in the liquid formulations.

[0170] Example 2 - Antibody stability in low pH formulations The following examples describe assays to verify the stability of the heterodimeric antibodies described herein in liquid or lyophilized formulations at various protein concentrations (e.g., 1 mg / mL, 5 mg / mL, and 20 mg / mL) at various different temperatures (4° C., 25° C., 40° C., −30° C., and −40° C.) and at various time points. The stability of the heterodimeric antibodies was evaluated in the following formulations: Formulation E: lyophilized formulation of 1 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; Formulation F: lyophilized formulation of heterodimeric antibody at 5 mg / mL in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; Formulation G: lyophilized formulation of 20 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; Formulation H: liquid formulation of 1 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; Formulation I: A liquid formulation of 5 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; and Formulation J: Liquid formulation of 20 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2.

[0171] The stability of lyophilized formulations E–G was compared with that of liquid formulations H–J.

[0172] The stability of the heterodimeric antibody was analyzed using the following assays: appearance (via visual inspection of 20 vials at each time point), pH, osmolality, CE-HPLC, rCE, MAM (multi-attribute method) and Karl Fischer (water content). Samples for validation studies were 1.3 mL filled in 5 cc vials. Formulations E-G were isotonic with osmolality values ​​of 314 mOsm / kg and 311 mOsm / kg, respectively. No proteinaceous particles were observed in any of the formulations tested.

[0173] The stability of the heterodimeric antibody was evaluated in the following formulations: result: Antibody A, when formulated in formulation J, was determined to have a 34.7% main peak loss via CEX after 3 months at 40° C. In contrast, antibody A, when formulated in formulation G, was determined to have a 2.8% main peak loss via CEX under the same conditions. See Table 2.

[0174] [Table 2]

[0175] Antibody A, when formulated in formulation J, was determined to have a 15.3% main peak loss via SE-UHPLC after 3 months at 40° C. In contrast, Antibody A, when formulated in formulation G, was determined to have a 1.6% main peak loss via SE-UHPLC under the same conditions. See Table 3.

[0176] [Table 3]

[0177] Antibody A, when formulated in formulation J, showed a 3.7% main peak loss via rCE after 3 months at 40° C. However, antibody A showed a 0.0% main peak loss via rCE after 3 months at 40° C. in formulation G. See Table 4.

[0178] [Table 4]

[0179] When formulated in formulation G, antibody A is expected to exhibit the same % deamidation as formulation B described above in Example 1.

[0180] The data provided herein demonstrates that lyophilized formulation G is more stable than liquid formulation J as demonstrated by the % reduction in main peak reduction observed in formulation G at the time tested. It was also determined that formulation J, having a heterodimeric antibody concentration of 20 mg / mL, had a similar % reduction in main peak reduction as formulations A (heterodimeric antibody 1 mg / mL) and B (heterodimeric antibody 5 mg / mL), which was determined to be surprising due to the heterodimeric antibody present at a higher concentration in formulation J (20 mg / mL).

[0181] Example 3 - Antibody stability in low pH formulations The following examples describe assays to verify the stability of the heterodimeric antibodies described herein in liquid or lyophilized formulations at a protein concentration of 10 mg / mL for up to 3 years at a variety of different temperatures (4° C., 25° C., 40° C., −30° C., and −40° C.). The stability of the heterodimeric antibodies was evaluated in the following formulations: Formulation K: a lyophilized formulation of a 10 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2; and Formulation L: Liquid formulation of 10 mg / mL heterodimeric antibody in 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2.

[0182] The stability of lyophilized formulation K was compared to that of liquid formulation L. In this study, antibody B was utilized.

[0183] The stability of the heterodimeric antibody was analyzed using the following assays: appearance (via visual inspection of 20 vials at each time point), pH, osmolality, SE-HPLC, rCE, MAM (multi-attribute method) and Karl Fischer (water content). Samples for validation studies were 1.3 mL filled in 5 cc vials. Formulation K was isotonic with an osmolality value of 305 mOsm / kg. No proteinaceous particles were observed in any of the formulations tested.

[0184] The stability of the heterodimeric antibody was evaluated in the following formulations: result: Antibody B, when formulated in formulation L, was determined to have a main peak loss of 34.5 via CEX after 3 months at 40° C. In contrast, antibody B, when formulated in formulation K, was determined to have a main peak loss of 0.7% via CEX under the same conditions. See Table 5.

[0185] [Table 5]

[0186] Antibody B, when formulated in formulation L, was determined to have a 22.5% main peak loss via SE-HPLC after 3 months at 40° C. In contrast, antibody B, when formulated in formulation K, was determined to have a 0.7% main peak loss via CEX under the same conditions. See Table 6 for SE-HPLC data.

[0187] [Table 6]

[0188] Antibody B, when formulated in formulation L, exhibited 6.8% deamidation via the MAM after 3 months at 40° C. See Table 7. In contrast, antibody B, when formulated in formulation K, exhibited <0.6% deamidation via the MAM.

[0189] [Table 7]

[0190] The data provided herein demonstrate that lyophilized formulation K is more stable than liquid formulation L due to the increased risk of deamidation in the liquid formulation.

Claims

[Claim 1] An object, method or system as described in this specification and drawings.

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