PHARMACEUTICAL COMPOSITIONS AND DOSAGE REGIMENS CONTAINING ANTI-ALPHA(V)BETA(6) ANTIBODIES

Abstract

Formulations and dosage regimens of an anti-αvβ6 antibody or αvβ6-binding fragment thereof are provided. These formulations find use in the treatment of e.g., fibrosis (e.g., idiopathic pulmonary fibrosis), acute lung injury, and acute kidney injury.

Claims

1. A pharmaceutical composition comprising an anti-αvβ6 antibody or αvβ6-binding fragment thereof, and arginine hydrochloride (Arg.HCl), wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), the VH and VL, respectively, comprising: (a) VH complementarity determining regions (CDRs), wherein VH-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:1; VH-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:2; and VH-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:3; and (b) VL CDRs, wherein VL-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:4; VL-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:5; and VL-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:6, and wherein the composition has a pH of 5.2 to 5.7.

2. The pharmaceutical composition of claim 1, wherein the composition comprises the anti-αvβ6 antibody or αvβ6-binding fragment thereof at a concentration of 50 mg/ml to 200 mg/ml.

3. The pharmaceutical composition of claim 1, wherein the composition comprises the anti-αvβ6 antibody or αvβ6-binding fragment thereof at a concentration of 100 mg/ml to 175 mg/ml.

4. The pharmaceutical composition of claim 1, wherein the composition comprises the anti-αvβ6 antibody or αvβ6-binding fragment thereof at a concentration of 150 mg/ml.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the composition comprises Arg.HCl at a concentration of 50 mM to 250 mM.

6. The pharmaceutical composition of any one of claims 1 to 4, wherein the composition comprises Arg.HCl at a concentration of 100 mM to 200 mM.

7. The pharmaceutical composition of any one of claims 1 to 4, wherein the composition comprises Arg.HCl at a concentration of 150 mM.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the composition comprises methionine.

9. The pharmaceutical composition of claim 8, wherein the composition comprises methionine at a concentration of 0.5 mM to 30 mM.

10. The pharmaceutical composition of claim 8, wherein the composition comprises methionine at a concentration of 1 mM to 10 mM.

11. The pharmaceutical composition of claim 8, wherein the composition comprises methionine at a concentration of 5 mM.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the composition comprises Polysorbate-80 (PS80).

13. The pharmaceutical composition of claim 12, wherein the composition comprises PS80 at a concentration of 0.01% to 0.1%.

14. The pharmaceutical composition of claim 12, wherein the composition comprises PS80 at a concentration of 0.03% to 0.08%.

15. The pharmaceutical composition of claim 12, wherein the composition comprises PS80 at a concentration of 0.05%.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the composition comprises sodium citrate and citric acid.

17. The pharmaceutical composition of claim 16, wherein the composition comprises sodium citrate and citric acid at a concentration of 5 mM to 30 mM.

18. The pharmaceutical composition of claim 16, wherein the composition comprises sodium citrate and citric acid at a concentration of 15 mM to 25 mM.

19. The pharmaceutical composition of claim 16, wherein the composition comprises sodium citrate and citric acid at a concentration of 20 mM.

20. The pharmaceutical composition of any one of claims 1 to 19, wherein the composition has a pH of 5.3 to 5.6.

21. The pharmaceutical composition of any one of claims 1 to 19, wherein the composition has a pH of 5.5.

22. The pharmaceutical composition of any one of claims 1 to 21, wherein the composition comprises a thiol-containing antioxidant.

23. The pharmaceutical composition of claim 22, wherein the thiol-containing antioxidant is selected from the group consisting of GSH, GSSG, the combination of GSH and GSSG, cystine, cysteine, and the combination of cysteine and cystine.

24. The pharmaceutical composition of claim 22, wherein the thiol-containing antioxidant is GSH.

25. The pharmaceutical composition of claim 22, wherein the thiol-containing antioxidant is GSSG.

26. The pharmaceutical composition of claim 22, wherein the thiol-containing antioxidant is the combination of GSH and GSSG.

27. The pharmaceutical composition of any one of claims 22 to 26, wherein the thiol-containing antioxidant is at a concentration of 0.02 mM to 2 mM.

28. The pharmaceutical composition of any one of claims 22 to 26, wherein the thiol-containing antioxidant is at a concentration of 0.2 mM.

29. The pharmaceutical composition of any one of claims 22 to 26, wherein the thiol-containing antioxidant is at a concentration of 0.4 mM.

30. The pharmaceutical composition of any one of claims 22 to 26, wherein the thiol-containing antioxidant is at a concentration of 1 mM.

31. The pharmaceutical composition of claim 26, wherein the GSH is at a concentration of 0.4 mM and the GSSG is at a concentration of 0.2 mM.

32. The pharmaceutical composition of claim 1, comprising: the anti-αvβ6 antibody or the αvβ6-binding fragment thereof at a concentration of 125 mg/ml to 175 mg/ml; Arg.HCl at a concentration of 125 mM to 175 mM; methionine at a concentration of 1 mM to 10 mM; sodium citrate and citric acid at a concentration of 15 mM to 25 mM; and PS80 at a concentration of 0.03% to 0.08%, wherein the composition has a pH of 5.3 to 5.7.

33. The pharmaceutical composition of claim 1, comprising: the anti-αvβ6 antibody or the αvβ6-binding fragment thereof at a concentration of 125 mg/ml to 175 mg/ml; Arg.HCl at a concentration of 125 mM to 175 mM; methionine at a concentration of 1 mM to 10 mM; sodium citrate and citric acid at a concentration of 15 mM to 25 mM; a thiol-containing antioxidant is a concentration of 0.02 mM to 2 mM; and PS80 at a concentration of 0.03% to 0.08%, wherein the composition has a pH of 5.3 to 5.7.

34. The pharmaceutical composition of claim 1, comprising: the anti-αvβ6 antibody or the αvβ6-binding fragment thereof at a concentration of 150 mg/ml; Arg.HCl at a concentration of 150 mM; methionine at a concentration of 5 mM; sodium citrate and citric acid at a concentration of 20 mM; and PS80 at a concentration of 0.05%, wherein the composition has a pH of 5.5.

35. The pharmaceutical composition of claim 1, comprising: the anti-αvβ6 antibody or the αvβ6-binding fragment thereof at a concentration of 150 mg/ml; Arg.HCl at a concentration of 150 mM; methionine at a concentration of 5 mM; sodium citrate and citric acid at a concentration of 20 mM; a thiol-containing antioxidant selected from the group consisting of GSH at a concentration of 0.4 mM, cysteine at a concentration of 0.4 mM, GSSG at a concentration of 0.2 mM, cystine at a concentration of 0.2 mM, GSSH at a concentration of 0.2 mM and GSSG at a concentration of 0.4 mM, and cysteine at a concentration of 0.4 mM and cystine at a concentration of 0.2 mM; and PS80 at a concentration of 0.05%, wherein the composition has a pH of 5.5.

36. The pharmaceutical composition of any one of claims 1 to 35, wherein: (i) the VH consists of a sequence at least 80% identical to SEQ ID NO:7 and the VL consists of a sequence at least 80% identical to SEQ ID NO:8; (ii) the VH consists of a sequence at least 90% identical to SEQ ID NO:7 and the VL consists of a sequence at least 90% identical to SEQ ID NO:8; or (iii) the VH consists of the amino acid sequence set forth in SEQ ID NO:7 and the VL consists of the amino acid sequence set forth in SEQ ID NO:8.

37. The pharmaceutical composition of any one of claims 1 to 36, wherein the anti-αvβ6 antibody comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein: (i) the heavy chain consists of a sequence at least 80% identical to SEQ ID NO:9 and the light chain consists of a sequence at least 80% identical to SEQ ID NO:10; (ii) the heavy chain consists of a sequence at least 90% identical to SEQ ID NO:9 and the light chain consists of a sequence at least 90% identical to SEQ ID NO:10; or (iii) the heavy chain consists of the amino acid sequence set forth in SEQ ID NO:9 and the light chain consists of the amino acid sequence set forth in SEQ ID NO:10.

38. A method of treating a condition selected from the group consisting of fibrosis, acute lung injury, and acute kidney injury in a human subject in need thereof, the method comprising administering to the human subject the pharmaceutical composition of any one of claims 1 to 37.

39. The method of claim 38, wherein the condition is fibrosis.

40. The method of claim 39, wherein the fibrosis is lung fibrosis.

41. The method of claim 40, wherein the lung fibrosis is idiopathic pulmonary fibrosis.

42. The method of any one of claims 38 to 41, wherein the pharmaceutical composition is administered subcutaneously to the human subject.

43. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 40 mg once weekly.

44. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 48 mg once weekly.

45. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 56 mg once weekly.

46. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 64 mg once weekly.

47. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 0.5 mg/kg to 0.8 mg/kg once weekly.

48. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 0.5 mg/kg once weekly.

49. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 0.6 mg/kg once weekly.

50. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 0.7 mg/kg once weekly.

51. The method of any one of claims 38 to 42, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof of the pharmaceutical composition is administered to the human subject at a dose of 0.8 mg/kg once weekly.

52. A method of treating a condition selected from the group consisting of fibrosis, acute lung injury, and acute kidney injury in a human subject in need thereof, the method comprising administering subcutaneously to the human subject an anti-αvβ6 antibody or αvβ6-binding fragment thereof at a dose of 40 mg once every week, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), the VH and VL, respectively, comprising: (a) VH complementarity determining regions (CDRs), wherein VH-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:1; VH-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:2; and VH-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:3; and (b) VL CDRs, wherein VL-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:4; VL-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:5; and VL-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:6.

53. A method of treating a condition selected from the group consisting of fibrosis, acute lung injury, and acute kidney injury in a human subject in need thereof, the method comprising administering subcutaneously to the human subject an anti-αvβ6 antibody or αvβ6-binding fragment thereof at a dose of 48 mg once every week, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), the VH and VL, respectively, comprising: (a) VH complementarity determining regions (CDRs), wherein VH-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:1; VH-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:2; and VH-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:3; and (b) VL CDRs, wherein VL-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:4; VL-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:5; and VL-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:6.

54. A method of treating a condition selected from the group consisting of fibrosis, acute lung injury, and acute kidney injury in a human subject in need thereof, the method comprising administering subcutaneously to the human subject an anti-αvβ6 antibody or αvβ6-binding fragment thereof at a dose of 56 mg once every week, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), the VH and VL, respectively, comprising: (a) VH complementarity determining regions (CDRs), wherein VH-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:1; VH-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:2; and VH-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:3; and (b) VL CDRs, wherein VL-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:4; VL-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:5; and VL-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:6.

55. A method of treating a condition selected from the group consisting of fibrosis, acute lung injury, and acute kidney injury in a human subject in need thereof, the method comprising administering subcutaneously to the human subject an anti-αvβ6 antibody or αvβ6-binding fragment thereof at a dose of 64 mg once every week, wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), the VH and VL, respectively, comprising: (a) VH complementarity determining regions (CDRs), wherein VH-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:1; VH-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:2; and VH-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:3; and (b) VL CDRs, wherein VL-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:4; VL-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:5; and VL-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:6.

56. The method of any one of claims 52 to 55, wherein the human subject is administered at least 4 doses of the anti-αvβ6 antibody or antigen-binding fragment thereof.

57. The method of any one of claims 52 to 55, wherein the human subject is administered at least 7 doses of the anti-αvβ6 antibody or antigen-binding fragment thereof.

58. The method of any one of claims 52 to 55, wherein the human subject is administered at least 10 doses of the anti-αvβ6 antibody or antigen-binding fragment thereof.

59. The method of any one of claims 52 to 58, wherein: (i) the VH consists of a sequence at least 80% identical to SEQ ID NO:7 and the VL consists of a sequence at least 80% identical to SEQ ID NO:8; (ii) the VH consists of a sequence at least 90% identical to SEQ ID NO:7 and the VL consists of a sequence at least 90% identical to SEQ ID NO:8; or (iii) the VH consists of the amino acid sequence set forth in SEQ ID NO:7 and the VL consists of the amino acid sequence set forth in SEQ ID NO:8.

60. The method of any one of claims 52 to 59, wherein the anti-αvβ6 antibody comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein: (i) the heavy chain consists of a sequence at least 80% identical to SEQ ID NO:9 and the light chain consists of a sequence at least 80% identical to SEQ ID NO:10; (ii) the heavy chain consists of a sequence at least 90% identical to SEQ ID NO:9 and the light chain consists of a sequence at least 90% identical to SEQ ID NO:10; or (iii) the heavy chain consists of the amino acid sequence set forth in SEQ ID NO:9 and the light chain consists of the amino acid sequence set forth in SEQ ID NO:10.

61. The method of any one of claims 52 to 60, wherein the condition is fibrosis.

62. The method of claim 61, wherein the fibrosis is lung fibrosis.

63. The method of claim 62, wherein the lung fibrosis is idiopathic pulmonary fibrosis.

64. A syringe or pump comprising a sterile preparation of the pharmaceutical composition of any one of claims 1 to 37 adapted for subcutaneous administration of the anti-αvβ6 antibody or αvβ6-binding fragment thereof at a fixed dose of 40 mg, 48 mg, 56 mg, or 64 mg.

65. A syringe or pump comprising 0.5 to 5.0 mL of a sterile preparation of the pharmaceutical composition of any one of claims 1 to 37.

66. A syringe or pump comprising a sterile preparation of an anti-αvβ6 antibody or αvβ6-binding fragment thereof, wherein the syringe or pump is adapted for subcutaneous administration of the anti-αvβ6 antibody or αvβ6-binding fragment thereof at a fixed dose of 40 mg, 48 mg, 56 mg, or 64 mg, and wherein the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), the VH and VL, respectively, comprising: (a) VH complementarity determining regions (CDRs), wherein VH-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:1; VH-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:2; and VH-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:3; and (b) VL CDRs, wherein VL-CDR1 consists of the amino acid sequence set forth in SEQ ID NO:4; VL-CDR2 consists of the amino acid sequence set forth in SEQ ID NO:5; and VL-CDR3 consists of the amino acid sequence set forth in SEQ ID NO:6.

67. The syringe or pump of claim 66, wherein: (i) the VH consists of a sequence at least 80% identical to SEQ ID NO:7 and the VL consists of a sequence at least 80% identical to SEQ ID NO:8; (ii) the VH consists of a sequence at least 90% identical to SEQ ID NO:7 and the VL consists of a sequence at least 90% identical to SEQ ID NO:8; or (iii) the VH consists of the amino acid sequence set forth in SEQ ID NO:7 and the VL consists of the amino acid sequence set forth in SEQ ID NO:8.

68. The syringe or pump of claim 66 or claim 67, wherein the anti-αvβ6 antibody comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein: (i) the heavy chain consists of a sequence at least 80% identical to SEQ ID NO:9 and the light chain consists of a sequence at least 80% identical to SEQ ID NO:10; (ii) the heavy chain consists of a sequence at least 90% identical to SEQ ID NO:9 and the light chain consists of a sequence at least 90% identical to SEQ ID NO:10; or (iii) the heavy chain consists of the amino acid sequence set forth in SEQ ID NO:9 and the light chain consists of the amino acid sequence set forth in SEQ ID NO:10.

69. The method of any one of claims 38 to 63, wherein the method further comprises administering to the human subject a therapeutically effective amount of prifenidone or nintedanib.

70. The method of claim 69, wherein the human subject is administered prifenidone as follows: TABLE-US-00026 Treatment days Dosage Days 1 through 7 267 mg three times daily (801 mg/day) Days 8 through 14 534 mg three times daily (1602 mg/day) Days 15 onward 801 mg three times daily (2403 mg/day).

71. The method of claim 69, wherein the human subject is administered nintedanib at a fixed dose of 150 mg twice daily.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1A is a graph depicting the % total aggregation as determined by size exclusion chromatography (SEC) of 150 g/L STX-100 formulations with different excipients.

[0033] FIG. 1B is a bar graph showing the total sub-visible particles per mL of 150 g/L STX-100 formulations with different excipients. For each formulation tested, T=0 is depicted as the left bar and T=4 wk is depicted as the right bar.

[0034] FIG. 1C is a bar graph showing the viscosity at ambient temperature of 150 g/L STX-100 formulations with different excipients.

[0035] FIG. 2 is a bar graph showing the results of a pH-arginine screening study. For each formulation tested, T=0 is depicted as the left bar and T=1M 40° C. is depicted as the right bar.

[0036] FIG. 3 is a graph showing the effect of GSH on the aggregation of STX-100 formulations at 25° C. (top) and 40° C. (bottom). The STX-100 formulations contain 150 mg/ml of STX-100, 20 mM citrate/citric acid, 150 mM arginine hydrochloride, 5 mM methionine, 0.05% PS80 and a pH of 5.5, and either no GSH or 0.4 mM GSH.

[0037] FIG. 4 provides graphs depicting the percentage of HMW species of SB4 (BENEPALI®, an etanercept biosimilar referencing Enbrel®) formulation (50 mg/ml SB4; 10 mM sodium phosphate; 140 mM NaCl; 1% sucrose, pH 6.2) with or without GSH (0.4 mM) at 25° C. and 40° C.

[0038] FIG. 5 provides graphs depicting the percentage of HMW species of an anti-αvβ5 integrin antibody (STX200) formulation (50 mg/ml antibody; 20 mM histidine; 5% sorbitol; 0.05% PS80, pH 6.5) with or without GSH (0.4 mM) at 25° C. and 40° C.

DETAILED DESCRIPTION

[0039] This application provides pharmaceutical compositions and dosage regimens of anti-αvβ6 antibodies and αvβ6-binding fragments thereof and their use in the treatment of diseases such as, but not limited to, fibrosis, acute lung injury, acute kidney injury, and cancer.

αvβ6

[0040] αvβ6 is an integrin that is expressed on epithelial cells. It can bind to several ligands including fibronectin, vitronectin, cytotactin, tenascin, and the latency associated peptide-1 and -3 (LAP1 and LAP3)—the N-terminal 278 amino acids of the latent precursor form of TGF-β1—through a direct interaction with an arginine-glycine-aspartate (“RGD”) motif. The TGF-β cytokine is synthesized as a latent complex which has the N-terminal LAP noncovalently associated with the mature active C-terminal TGF-β cytokine. The latent TGF-β complex cannot bind to its cognate receptor and thus is not biologically active until converted to an active form. αvβ6 binding to LAP1 or LAP3 leads to activation of the latent precursor form of TGF-β1 and TGF-β3 as a result of a conformational change in the latent complex allowing TGF-β to bind to its receptor. Thus, upregulated expression of αvβ6 can lead to local activation of TGF-β, which in turn can activate a cascade of events downstream events. The TGF-β1 cytokine is a pleiotropic growth factor that regulates cell proliferation, differentiation, and immune responses.

[0041] The amino acid sequence of human integrin αv (UniProtKB—P06756 (ITAV HUMAN) is shown below (the 30 aa signal peptide sequence is underlined):

TABLE-US-00001 (SEQ ID NO: 12)         10         20         30         40 MAFPPRRRLR LGPRGLPLLL SGLLLPLCRA FNLDVDSPAE         50         60         70         80 YSGPEGSYFG FAVDFFVPSA SSRMFLLVGA PKANTTQPGI         90        100        110        120 VEGGQVLKCD WSSTRRCQPI EFDATGNRDY AKDDPLEFKS        130        140        150        160 HQWFGASVRS KQDKILACAP LYHWRTEMKQ EREPVGTCFL        170        180        190        200 QDGTKTVEYA PCRSQDIDAD GQGFCQGGFS IDFTKADRVL        210        220        230        240  LGGPGSFYWQ GQLISDQVAE IVSKYDPNVY SIKYNNQLAT        250        260        270        280 RTAQAIFDDS YLGYSVAVGD FNGDGIDDFV SGVPRAARTL        290        300        310        320 GMVYIYDGKN MSSLYNFTGE QMAAYFGFSV AATDINGDDY        330        340        350        360 ADVFIGAPLF MDRGSDGKLQ EVGQVSVSLQ RASGDFQTTK        370        380        390        400 LNGFEVFARF GSAIAPLGDL DQDGFNDIAI AAPYGGEDKK        410        420        430        440 GIVYIFNGRS TGLNAVPSQI LEGQWAARSM PPSFGYSMKG        450        460        470        480 ATDIDKNGYP DLIVGAFGVD RAILYRARPV ITVNAGLEVY        490        500        510        520 PSILNQDNKT CSLPGTALKV SCFNVRFCLK ADGKGVLPRK        530        540        550        560 LNFQVELLLD KLKQKGAIRR ALFLYSRSPS HSKNMTISRG        570        580        590        600 GLMQCEELIA YLRDESEFRD KLTPITIFME YRLDYRTAAD        610        620        630        640  TTGLQPILNQ FTPANISRQA HILLDCGEDN VCKPKLEVSV        650        660        670        680 DSDQKKIYIG DDNPLTLIVK AQNQGEGAYE AELIVSIPLQ        690        700        710        720 ADFIGVVRNN EALARLSCAF KTENQTRQVV CDLGNPMKAG        730        740        750        760 TQLLAGLRFS VHQQSEMDTS VKFDLQIQSS NLFDKVSPVV        770        780        790        800 SHKVDLAVLA AVEIRGVSSP DHVFLPIPNW EHKENPETEE        810        820        830        840  DVGPVVQHIY ELRNNGPSSF SKAMLHLQWP YKYNNNTLLY        850        860        870        880 ILHYDIDGPM NCTSDMEINP LRIKISSLQT TEKNDTVAGQ        890        900        910        920 GERDHLITKR DLALSEGDIH TLGCGVAQCL KIVCQVGRLD        930        940        950        960 RGKSAILYVK SLLWTETFMN KENQNHSYSL KSSASFNVIE        970        980        990        1000 FPYKNLPIED ITNSTLVTTN VTWGIQPAPM PVPVWVIILA       1010       1020       1030       1040  VLAGLLLLAV LVFVMYRMGF FKRVRPPQEE QEREQLQPHE NGEGNSET
The mature αv protein corresponds to amino acids 31-1048 of SEQ ID NO:12.

[0042] The amino acid sequence of human integrin β6 (UniProtKB—P18564 (ITB6 HUMAN) is provided below (the 21 aa signal peptide sequence is underlined):

TABLE-US-00002 (SEQ ID NO: 13)         10         20         30         40 MGIELLCLFF LFLGRNDHVQ GGCALGGAET CEDCLLIGPQ         50         60         70         80 CAWCAQENFT HPSGVGERCD TPANLLAKGC QLNFIENPVS         90        100        110        120 QVEILKNKPL SVGRQKNSSD IVQIAPQSLI LKLRPGGAQT        130        140        150        160 LQVHVRQTED YPVDLYYLMD LSASMDDDLN TIKELGSRLS        170        180        190        200 KEMSKLTSNF RLGFGSFVEK PVSPFVKTTP EEIANPCSSI        210        220        230        240 PYFCLPTFGF KHILPLTNDA ERFNEIVKNQ KISANIDTPE        250        260        270        280 GGFDAIMQAA VCKEKIGWRN DSLHLLVFVS DADSHFGMDS        290        300        310        320 KLAGIVIPND GLCHLDSKNE YSMSTVLEYP TIGQLIDKLV        330        340        350        360 QNNVLLIFAV TQEQVHLYEN YAKLIPGATV GLLQKDSGNI        370        380        390        400 LQLIISAYEE LRSEVELEVL GDTEGLNLSF TAICNNGTLF        410        420        430        440 QHQKKCSHMK VGDTASFSVT VNIPHCERRS RHIIIKPVGL        450        460        470        480 GDALELLVSP ECNCDCQKEV EVNSSKCHHG NGSFQCGVCA        490        500        510        520 CHPGHMGPRC ECGEDMLSTD SCKEAPDHPS CSGRGDCYCG        530        540        550        560 QCICHLSPYG NIYGPYCQCD NFSCVRHKGL LCGGNGDCDC        570        580        590        600 GECVCRSGWT GEYCNCTTST DSCVSEDGVL CSGRGDCVCG        610        620        630        640 KCVCTNPGAS GPTCERCPTC GDPCNSKRSC IECHLSAAGQ        650        660        670        680 AREECVDKCK LAGATISEEE DFSKDGSVSC SLQGENECLI        690        700        710        720 TFLITTDNEG KTIIHSINEK DCPKPPNIPM IMLGVSLAIL        730        740        750        760 LIGVVLLCIW KLLVSFHDRK EVAKFEAERS KAKWQTGTNP        770        780 LYRGSTSTFK NVTYKHREKQ KVDLSTDC

[0043] The mature β6 protein corresponds to amino acids 22-788 of SEQ ID NO:13.

[0044] The antibodies described herein can bind specifically to the αvβ6 protein having the amino acid sequence set forth in positions 31-1048 of SEQ ID NO:12 and the amino acid sequence set forth in positions 22-788 of SEQ ID NO:13. In some embodiments, the antibodies described herein can bind specifically to the β6 protein having the amino acid sequence set forth in positions 22-788 of SEQ ID NO:13.

Anti-αvβ6 Antibodies

[0045] In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof used in the compositions and methods described herein comprises the three heavy chain variable domain complementarity determining regions (CDRs) of an antibody referred to as “STX-100”. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises the three light chain variable domain CDRs of STX-100. In still other embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises the three heavy chain variable domain CDRs and the three light chain variable domain CDRs of STX-100. The CDRs can be based on any CDR definition known in the art, e.g., the definitions of Kabat, Chothia, Chothia from Abysis, enhanced Chothia/AbM, or based on the contact definition. Exemplary CDR sequences of STX-100 (according to Kabat) are provided in Table 1 below.

TABLE-US-00003 TABLE 1 Sequences of the Kabat CDRs of STX-100 Domain CDR VH CDR1 RYVMS (SEQ ID NO: 1) VH CDR2 SISSGGRMYYPDTVKG (SEQ ID NO: 2) VH CDR3 GSIYDGYYVFPY (SEQ ID NO: 3) VL CDR1 SASSSVSSSYLY (SEQ ID NO: 4) VL CDR2 STSNLAS (SEQ ID NO: 5) VL CDR3 HQWSTYPPT (SEQ ID NO: 6)

[0046] In some aspects, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises of a VH CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:1 or GFTFSRYVMS (SEQ ID NO:11), a VH CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2; and a VH CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a VL CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:4, a VL CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5; and a VL CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:6.

[0047] In certain aspects, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises the CDRs comprising the amino acid sequences set forth in SEQ ID NOs:1 to 6. In certain aspects, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises the CDRs comprising the amino acid sequences set forth in SEQ ID NOs:11, 2, 3, 4, 5, and 6. In certain aspects, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises the CDRs consisting of the amino acid sequences set forth in SEQ ID NOs:1 to 6. In certain aspects, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises the CDRs consisting of the amino acid sequences set forth in SEQ ID NOs:11, 2, 3, 4, 5, and 6.

[0048] STX-100 is a humanized humanIgG1/human kappa monoclonal antibody that specifically binds to the integrin αvβ6.

[0049] The heavy chain variable domain (VH) of STX-100 comprises or consists of the following amino acid sequence (VH CDRs (Kabat definition) bolded):

TABLE-US-00004 (SEQ ID NO: 7) 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYVMSWVRQA PGKGLEWVAS 51 ISSGGRMYYP DTVKGRFTIS RDNAKNSLYL QMNSLRAEDT AVYYCARGSI 101 YDGYYVFPYW GQGTLVTVSS 

[0050] The light chain variable domain (VL) of STX-100 comprises or consists of the following amino acid sequence (VL CDRs (Kabat definition) bolded):

TABLE-US-00005 (SEQ ID NO: 8) 1 EIVLTQSPAT LSLSPGERAT LSCSASSSVS SSYLYWYQQK PGQAPRLLIY 51 STSNLASGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCH QWSTYPPTFG 101 GGTKVEIK 

[0051] In certain embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof selectively binds to αvβ6 and comprises a VH domain that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the VH domain of STX-100 (SEQ ID NO:7), or differs at least at 1 to 5 amino acid residues, but at fewer than 40, 30, 20, 15, or 10, residues, from SEQ ID NO:7. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof blocks the binding of αvβ6 to its ligand, latency associated peptide (LAP), as determined by blocking of ligand binding either to purified hsαvβ6 or to β6-expressing cells. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof have one or more (e.g., one, two, three, four) of these properties: (i) specifically bind with high affinity to αvβ6; (ii) inhibit the binding of αvβ6 to LAP, fibronectin, vitronectin, or tenascin with an IC50 value lower than that of the 10D5 antibody (WO 99/07405); (iii) block or inhibit activation of TGF-0; (iv) specifically bind to the β6 subunit; and (v) recognize αvβ6 in immunostaining procedures such as immunostaining of paraffin-embedded tissues.

[0052] In certain embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof selectively binds to αvβ6 and comprises a VL domain that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the VL domain of STX-100 (SEQ ID NO:8), or differs at least at 1 to 5 amino acid residues, but at fewer than 40, 30, 20, 15, or 10, residues, from SEQ ID NO:8. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof blocks the binding of αvβ6 to its ligand, latency associated peptide (LAP), as determined by blocking of ligand binding either to purified hsαvβ6 or to β6-expressing cells. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof have one or more (e.g., one, two, three, four) of these properties: (i) specifically bind with high affinity to αvβ6; (ii) inhibit the binding of αvβ6 to LAP, fibronectin, vitronectin, or tenascin with an IC50 value lower than that of the 10D5 antibody (WO 99/07405); (iii) block or inhibit activation of TGF-β; (iv) specifically bind to the β6 subunit; and (v) recognize αvβ6 in immunostaining procedures such as immunostaining of paraffin-embedded tissues.

[0053] In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a VH having the amino acid sequence set forth in SEQ ID NO:7 and a VL having the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof selectively binds to αvβ6 and comprises (i) a VH domain that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the VH domain of STX-100 (SEQ ID NO:7), and (ii) a VL domain that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the VL domain of STX-100 (SEQ ID NO:8); or differs at least at 1 to 5 amino acid residues, but at fewer than 40, 30, 20, 15, or 10, residues, from SEQ ID NO:7 and/or SEQ ID NO:8. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof blocks the binding of αvβ6 to its ligand, latency associated peptide (LAP), as determined by blocking of ligand binding either to purified hsαvβ6 or to β6-expressing cells. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof have one or more (e.g., one, two, three, four) of these properties: (i) specifically bind with high affinity to αvβ6; (ii) inhibit the binding of αvβ6 to LAP, fibronectin, vitronectin, or tenascin with an IC50 value lower than that of the 10D5 antibody (WO 99/07405); (iii) block or inhibit activation of TGF-β; (iv) specifically bind to the β6 subunit; and (v) recognize αvβ6 in immunostaining procedures such as immunostaining of paraffin-embedded tissues.

[0054] An antibody consisting of the mature heavy chain (SEQ ID NO:9) and the mature light chain (SEQ ID NO:10) listed below is termed “STX-100” or “BG00011” or “BG11”. STX-100 is an IgG1/kappa antibody.

Mature STX-100 Heavy Chain (HC) [H-CDR1, H-CDR2, and H-CDR3 are bolded; constant region underlined; N-linked glycosylation site bolded & underlined]

TABLE-US-00006 (SEQ ID NO: 9) 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYVMSWVRQA PGKGLEWVAS 51 ISSGGRMYYP DTVKGRFTIS RDNAKNSLYL QMNSLRAEDT AVYYCARGSI 101 YDGYYVFPYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK 151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT 201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP 251 KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 301 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 351 VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG
Mature STX-100 Light Chain (LC) [L-CDR1, L-CDR2, and L-CDR3 are bolded; constant region underlined]

TABLE-US-00007 (SEQ ID NO: 10) 1 EIVLTQSPAT LSLSPGERAT LSCSASSSVS SSYLYWYQQK PGQAPRLLIY 51 STSNLASGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCH QWSTYPPTFG 101 GGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK 151 VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ 201 GLSSPVTKSF NRGEC

[0055] In certain embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a HC having the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof selectively binds to αvβ6 and comprises a HC that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:9, or differs at least at 1 to 5 amino acid residues, but at fewer than 40, 30, 20, 15, or 10, residues, from SEQ ID NO:9. In certain embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a heavy chain set forth in SEQ ID NO:9, except for 1 to 5 amino acid substitutions in the heavy chain constant region. In some embodiments, these anti-αvβ6 antibodies or αvβ6-binding fragments thereof block the binding of αvβ6 to its ligand, latency associated peptide (LAP), as determined by blocking of ligand binding either to purified hsαvβ6 or to β6-expressing cells. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof have one or more (e.g., one, two, three, four) of these properties: (i) specifically bind with high affinity to αvβ6; (ii) inhibit the binding of αvβ6 to LAP, fibronectin, vitronectin, or tenascin with an IC50 value lower than that of the 10D5 antibody (WO 99/07405); (iii) block or inhibit activation of TGF-β; (iv) specifically bind to the β6 subunit; and (v) recognize αvβ6 in immunostaining procedures such as immunostaining of paraffin-embedded tissues.

[0056] In certain embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a LC having the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof selectively binds to αvβ6 and comprises a LC that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:10, or differs at least at 1 to 5 amino acid residues, but at fewer than 40, 30, 20, 15, or 10, residues, from SEQ ID NO:10. In certain embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a light chain set forth in SEQ ID NO:10, except for 1 to 5 amino acid substitutions in the light chain constant region. In some embodiments, these anti-αvβ6 antibodies or αvβ6-binding fragments thereof block the binding of αvβ6 to its ligand, latency associated peptide (LAP), as determined by blocking of ligand binding either to purified hsαvβ6 or to β6-expressing cells. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof have one or more (e.g., one, two, three, four) of these properties: (i) specifically bind with high affinity to αvβ6; (ii) inhibit the binding of αvβ6 to LAP, fibronectin, vitronectin, or tenascin with an IC50 value lower than that of the 10D5 antibody (WO 99/07405); (iii) block or inhibit activation of TGF-β; (iv) specifically bind to the β6 subunit; and (v) recognize αvβ6 in immunostaining procedures such as immunostaining of paraffin-embedded tissues.

[0057] In certain embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof comprises a HC having the amino acid sequence set forth in SEQ ID NO:9 and a LC having the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof selectively binds to human αvβ6 and comprises (i) a HC that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:9, or differs at least at 1 to 5 amino acid residues, but at fewer than 40, 30, 20, 15, or 10, residues, from SEQ ID NO:9; and (ii) a LC that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:10, or differs at least at 1 to 5 amino acid residues, but at fewer than 40, 30, 20, 15, or 10, residues, from SEQ ID NO:10. In some embodiments, these anti-αvβ6 antibodies or αvβ6-binding fragments thereof block the binding of αvβ6 to its ligand, latency associated peptide (LAP), as determined by blocking of ligand binding either to purified hsαvβ6 or to β6-expressing cells. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof have one or more (e.g., one, two, three, four) of these properties: (i) specifically bind with high affinity to αvβ6; (ii) inhibit the binding of αvβ6 to LAP, fibronectin, vitronectin, or tenascin with an IC50 value lower than that of the 10D5 antibody (WO 99/07405); (iii) block or inhibit activation of TGF-β; (iv) specifically bind to the β6 subunit; and (v) recognize αvβ6 in immunostaining procedures such as immunostaining of paraffin-embedded tissues.

[0058] In certain embodiments, the anti-αvβ6 antibody is an IgG antibody. In specific embodiments, the anti-αvβ6 antibody has heavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In one embodiment, the anti-αvβ6 antibody is of the human IgG1 isotype. In another embodiment, the anti-αvβ6 antibody is of the human IgG2 isotype. In yet another embodiment, the anti-αvβ6 antibody is of the human IgG3 isotype. In yet another embodiment, the anti-αvβ6 antibody is of the human IgG4 isotype. In further embodiments, the antibody has a light chain constant region chosen from, e.g., a human kappa or human lambda light chain. In a certain embodiment, the anti-αvβ6 antibody is a human IgG1/human kappa antibody. In some cases, the heavy chain constant region is human or a modified form of a human constant region. In certain instances, the human constant region may include at least 1 and up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions. In a particular embodiment, the modified human Fc region is a modified human IgG1 Fc region. In some cases, the constant region of an anti-αvβ6 antibody is modified by mutation of one or more amino acid residues to impart a desired functional property (e.g., altered effector function or half-life, reduced glycosylation). For example, the N-linked glycosylation site may be substituted to prevent or reduce N-linked glycosylation of Fc region (e.g., human IgG1 Fc region).

[0059] In some embodiments, the anti-αvβ6 antibody is a full-length (whole) antibody or substantially full-length. The protein can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains. In some embodiments, the anti-αvβ6 antibody is an αvβ6-binding fragment. In some instances, the αvβ6-binding fragment is a Fab, a Fab′, an F(ab′)2, a Facb, an Fv, a single chain Fv (scFv), a sc(Fv)2, or a diabody.

[0060] Antibodies, such as STX-100, or αvβ6-binding fragments thereof can be made, for example, by preparing and expressing synthetic genes that encode the recited amino acid sequences or by mutating human germline genes to provide a gene that encodes the recited amino acid sequences. Moreover, this antibody and other anti-αvβ6 antibodies can be produced, e.g., using one or more of the following methods.

[0061] Methods of Producing Antibodies Anti-αvβ6 antibodies or αvβ6-binding fragments can be produced in bacterial or eukaryotic cells. Some antibodies, e.g., Fab's, can be produced in bacterial cells, e.g., E. coli cells. Antibodies can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, COS). In addition, antibodies (e.g., scFv's) can be expressed in a yeast cell such as Pichia (see, e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces. To produce the antibody of interest, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in suitable host cells. Polynucleotides encoding an anti-αvβ6 antibody comprising the VH and/or VL, HC and/or LC of the αvβ6 antibodies described herein would be readily envisioned by the ordinarily skilled artisan. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody.

[0062] If the anti-αvβ6 antibodies or αvβ6-binding fragments is to be expressed in bacterial cells (e.g., E. coli), the expression vector should have characteristics that permit amplification of the vector in the bacterial cells. Additionally, when E. coli such as JM109, DHSα, HB101, or XL1-Blue is used as a host, the vector must have a promoter, for example, a lacZ promoter (Ward et al., 341:544-546 (1989), araB promoter (Better et al., Science, 240:1041-1043 (1988)), or T7 promoter that can allow efficient expression in E. coli. Examples of such vectors include, for example, M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), “QIAexpress system” (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al., J Bacteriol., 169:4379 (1987)) may be used as the signal sequence for antibody secretion. For bacterial expression, calcium chloride methods or electroporation methods may be used to introduce the expression vector into the bacterial cell.

[0063] If the antibody is to be expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, for example, an SV40 promoter (Mulligan et al., Nature, 277:108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or domain thereof, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0064] In one embodiment, antibodies are produced in mammalian cells. Exemplary mammalian host cells for expressing an antibody include Chinese Hamster Ovary (CHO cells) (including dhfr.sup.− CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.

[0065] In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain of an anti-αvβ6 antibody (e.g., STX-100) is introduced into dhfr.sup.− CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and the antibody is recovered from the culture medium.

[0066] Antibodies can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest. The antibody can be purified from the milk, or for some applications, used directly. Animals are also provided comprising one or more of the nucleic acids described herein.

[0067] The antibodies of the present disclosure can be isolated from inside or outside (such as medium) of the host cell and purified as substantially pure and homogenous antibodies. Methods for isolation and purification commonly used for antibody purification may be used for the isolation and purification of antibodies, and are not limited to any particular method. Antibodies may be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be carried out using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A column and protein G column. Examples of columns using protein A column include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that are highly purified using these purification methods.

Anti-αvβ6 Antibody Compositions

[0068] This disclosure also provides compositions (e.g., pharmaceutical compositions) comprising the anti-αvβ6 antibodies or αvβ6-binding fragments thereof described herein. For example, the anti-αvβ6 antibody compositions comprise an anti-αvβ6 antibody or αvβ6-binding fragment thereof comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises the H-CDRs and the VL comprises the L-CDRs of STX-100. In certain instances, the heavy chain CDRs (H-CDRs) comprise or consist of the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and the light chain CDRs (L-CDRs) comprise or consist of the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In certain instances, the heavy chain CDRs (H-CDRs) comprise or consist of the amino acid sequences set forth in SEQ ID NO:11, SEQ ID NO:2, and SEQ ID NO:3; and the light chain CDRs (L-CDRs) comprise or consist of the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the anti-αvβ6 antibody compositions comprises an anti-αvβ6 antibody or αvβ6-binding fragment thereof comprising (i) a VH comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and (ii) a VL comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8. In certain embodiments, the anti-αvβ6 antibody compositions comprises an anti-αvβ6 antibody comprising (i) a heavy chain comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and (ii) a light chain comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the anti-αvβ6 antibodies selectively bind to αvβ6. In some embodiments, these anti-αvβ6 antibodies or αvβ6-binding fragments thereof block the binding of αvβ6 to its ligand, latency associated peptide (LAP), as determined by blocking of ligand binding either to purified hsαvβ6 or to β6-expressing cells. In some embodiments, these anti-αvβ6 antibody or αvβ6-binding fragments thereof have one or more (e.g., one, two, three, four) of these properties: (i) specifically bind with high affinity to αvβ6; (ii) inhibit the binding of αvβ6 to LAP, fibronectin, vitronectin, or tenascin with an IC50 value lower than that of the 10D5 antibody (WO 99/07405); (iii) block or inhibit activation of TGF-0; (iv) specifically bind to the β6 subunit; and (v) recognize αvβ6 in immunostaining procedures such as immunostaining of paraffin-embedded tissues.

[0069] In certain embodiments, these compositions are high concentration anti-αvβ6 antibody compositions. By “high concentration anti-αvβ6 antibody composition” is meant a composition comprising anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of greater than 100 mg/ml and less than 300 mg/ml. In certain instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 50 mg/ml to 250 mg/ml. In certain instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 50 mg/ml to 225 mg/ml. In other instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 75 mg/ml to 225 mg/ml. In certain instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 50 mg/ml to 200 mg/ml. In other instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 75 mg/ml to 165 mg/ml. In other instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 100 mg/ml to 225 mg/ml. In yet other instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 125 mg/ml to 225 mg/ml. In other instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 125 mg/ml to 175 mg/ml. In certain instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 240 mg/ml. In certain instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 225 mg/ml. In certain instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 200 mg/ml. In certain instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 175 mg/ml. In certain instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 150 mg/ml. In other instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 125 mg/ml. In some instances, the anti-αvβ6 antibody composition comprises anti-αvβ6 antibodies or αvβ6-binding fragments thereof at a concentration of 100 mg/ml.

[0070] A composition (e.g., a pharmaceutical composition) comprising an anti-αvβ6 antibody or αvβ6-binding fragment thereof described herein may be in any one of a variety of forms. These include, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions, or suspensions. The preferred form can depend on the intended mode of administration and therapeutic application. In certain embodiments, a pharmaceutical composition described herein is in the form of a sterile injectable or infusible solution.

[0071] Sterile injectable solutions can be prepared by incorporating an antibody described herein in the required amount with one or a combination of ingredients, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an antibody described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, an exemplary method of preparation is vacuum drying and freeze drying that yields a powder of an antibody described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.

[0072] The anti-αvβ6 antibody compositions (e.g., pharmaceutical compositions) may additionally comprise one or more excipients.

[0073] In one embodiment, the excipient lowers/reduces the aggregation and/or viscosity of the antibody in the composition compared to aggregation and/or viscosity of the antibody in the pharmaceutical composition without that excipient. In certain embodiments, such an excipient is arginine. In one instance, the excipient is L-arginine hydrochloride. Arginine (e.g., L-arginine hydrochloride) can be included in the composition at a concentration of 40 mM to 260 mM, 50 mM to 250 mM, 50 mM to 200 mM, 50 mM to 150 mM, 50 mM to 125 mM, 50 mM to 100 mM, 75 mM to 250 mM, 75 mM to 200 mM, 75 mM to 150 mM, or 75 mM to 100 mM. In certain embodiments arginine (e.g., Arg.HCl) is present in the composition at a concentration of 50 mM to 250 mM. In other embodiments, arginine (e.g., Arg.HCl) is present in the composition at a concentration of 50 mM to 200 mM. In certain instances, arginine (e.g., arginine hydrochloride) can be included in the composition at a concentration of 80 mM, 100 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 220 mM, or 260 mM. In a specific instance, arginine (e.g., arginine hydrochloride) can be included in the composition at a concentration of 100 mM. In another specific instance, arginine (e.g., arginine hydrochloride) can be included in the composition at a concentration of 150 mM.

[0074] Sometimes, solutions containing arginine develop visible particles after incubation at room temperature or higher temperatures (e.g., 40° C.). Addition of sucrose can reduce or prevent the formation of visible particles. Furthermore, sucrose can lower the counts of sub visible particulates. In some embodiments, the anti-αvβ6 antibody composition comprises sucrose at a concentration of 0.05% to 5%, 0.05% to 4%, 0.05% to 3%, 1% to 5%, 1% to 4%, 1% to 3%, 2% to 5%, 2% to 4%, or 2% to 3%. In certain embodiments, the anti-αvβ6 antibody composition comprises sucrose at a concentration of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%. In a particular embodiment, the anti-αvβ6 antibody composition comprises sucrose at a concentration of 3%. In another particular embodiment, the anti-αvβ6 antibody composition comprises sucrose at a concentration of 1%.

[0075] In one embodiment, the anti-αvβ6 antibody compositions comprise methionine. In one instance, methionine is included in the composition at a concentration from 0.5 mM to 25 mM. In another instance, methionine is included in the composition at a concentration from 1 mM to 10 mM. In another instance, methionine is included in the composition at a concentration from 3 mM to 8 mM. In one instance, methionine is included in the composition at a concentration of 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM or 25 mM. In a particular instance, methionine is included in the composition at a concentration of 10 mM. In another particular instance, methionine is included in the composition at a concentration of 5 mM.

[0076] Antibody product manufacturing is a complex process that can involve several steps such as, e.g., drug substance and bulk formulation, filtration, shipping, pooling, filling, lyophilization, inspections, packaging, and storage. During these steps, antibodies may be subjected to many different forms of stresses, e.g., agitation, temperature, light exposure, and oxidation. These types of stresses can lead to denaturation and aggregation of the antibody, which compromise the product quality and can even lead to loss of a production batch. Agitation is one of the common physical stresses that antibody therapeutics are subjected to during the course of the manufacturing process. Agitation occurs, e.g., during mixing, ultrafiltration/diafiltration, pumping, shipping, and filling. To protect the antibody composition against agitation-induced stress, the composition may include a polysorbate. In certain embodiments, the composition comprises polysorbate-80 at a concentration of 0.01% to 0.5%, 0.01% to 0.1%, 0.01% to 0.09%, 0.01% to 0.08%, 0.01% to 0.07%, 0.01% to 0.06%, 0.01% to 0.05%, 0.01% to 0.04%, or 0.01% to 0.03%. In certain embodiments, the composition comprises polysorbate-80 at a concentration of 0.02% to 0.08%. In some embodiments, the composition comprises polysorbate-80 at a concentration of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%. In a particular embodiment, the composition comprises polysorbate-80 at a concentration of 0.05%.

[0077] Any antibody composition benefits from a buffer that provides good buffering capacity. In certain embodiments, the antibody composition comprises sodium citrate and citric acid as the buffering agent. In certain embodiments, the composition comprises sodium citrate and citric acid at a concentration of 5 mM to 50 mM, 5 mM to 40 mM, 5 mM to 35 mM, 5 mM to 30 mM, 5 mM to 25 mM, 10 mM to 50 mM, 10 mM to 40 mM, 10 mM to 30 mM, 10 mM to 25 mM, 15 mM to 50 mM, 15 mM to 40 mM, 15 mM to 30 mM, or 15 mM to 25 mM. In certain embodiments, the composition comprises sodium citrate and citric acid at a concentration of 5 mM to 35 mM. In certain embodiments, the composition comprises sodium citrate and citric acid at a concentration of 10 mM to 30 mM. In some embodiments, the composition comprises sodium citrate and citric acid at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 35 mM. In a particular embodiment, the composition comprises sodium citrate and citric acid at a concentration of 20 mM.

[0078] The pH of the antibody composition can be from 5.0 to 6.5. In certain cases, the pH of the antibody composition can be 5.2 to 6.2. In certain instances, the pH of the antibody composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In a particular embodiment, the pH of the antibody composition is 5.5.

[0079] In some instances, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM) and methionine (e.g., 5 mM). In certain cases, these compositions have a pH of 5.5.

[0080] In some instances, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), methionine (e.g., 5 mM) and a buffer (e.g., sodium citrate and citric acid at 20 mM). In certain cases, these compositions have a pH of 5.5.

[0081] In some instances, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), methionine (e.g., 5 mM), and PS80 (e.g., 0.05%). In certain cases, these compositions have a pH of 5.5.

[0082] In certain embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), methionine (e.g., 5 mM), sodium citrate and citric acid (e.g., 20 mM), and PS80 (e.g., 0.05%), and has a pH of 5.2 to 6.2. In some embodiments, the anti-αvβ6 compositions comprise L-arginine hydrochloride (e.g., 150 mM), methionine (e.g., 5 mM), sodium citrate and citric acid (e.g., 20 mM), and PS80 (e.g., 0.05%), and has a pH of 5.5. In certain embodiments, the anti-αvβ6 compositions comprise L-arginine hydrochloride (e.g., 150 mM), methionine (e.g., 5 mM), sodium citrate and citric acid (e.g., 20 mM), PS80 (e.g., 0.05%), and sucrose (up to 3%), and has a pH of 5.2 to 6.2. In some embodiments, the anti-αvβ6 compositions comprise L-arginine hydrochloride, methionine, sodium citrate and citric acid, PS80, and has a pH of 5.5. In all of these embodiments, the anti-αvβ6 antibody is present at a concentration of 100 mg/ml to 165 mg/ml. In one instance, the anti-αvβ6 antibody is present at a concentration of 150 mg/ml. In one instance, the anti-αvβ6 antibody is present at a concentration of 100 mg/ml.

[0083] In some cases, the anti-αvβ6 composition comprises a thiol-containing antioxidant (e.g., reduced glutathione (GSH), oxidized glutathione (GSSG), GSH+GSSG, cysteine, cystine, cysteine+cystine) at a concentration of 0.02 mM to 2 mM (e.g., 0.02, 0.03, 0.05, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mM). Such thiol-containing antioxidants can cleave unfavorable or misbridged disulfide bonds and promote the formation of favorable or properly bridged disulfide bonds. This would result in the stabilization of the native confirmation of the antibody or fragment thereof and slow down aggregation rates. The antioxidant properties of these molecules may slow down oxidative processes that lead to aggregation. In some cases, the composition comprises GSH at a concentration of 0.4 mM. In some cases, the composition comprises GSSG at a concentration of 0.2 mM. In some cases, the composition comprises GSH at a concentration of 0.4 mM and GSSG at a concentration of 0.2 mM. In some cases, the composition comprises cysteine at a concentration of 0.4 mM. In some cases, the composition comprises cystine at a concentration of 0.2 mM. In some cases, the composition comprises cysteine at a concentration of 0.4 mM and cystine at a concentration of 0.2 mM.

[0084] In certain embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), methionine (e.g., 5 mM), sodium citrate and citric acid (e.g., 20 mM), a thiol-containing antioxidant such as GSH, GSSG, GSH and GSSG, cysteine, cystine, or cysteine and cystine (e.g., 0.02 mM to 2 mM), and PS80 (e.g., 0.05%), and has a pH of 5.2 to 6.2. In some embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), methionine (e.g., 5 mM), sodium citrate and citric acid (e.g., 20 mM), a thiol-containing antioxidant such as GSH, GSSG, GSH and GSSG, cysteine, cystine, or cysteine and cystine (e.g., 0.02 mM to 2 mM), and PS80 (e.g., 0.05%), and has a pH of 5.5. In certain embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), methionine (e.g., 5 mM), sodium citrate and citric acid (e.g., 20 mM), PS80 (e.g., 0.05%), a thiol-containing antioxidant such as GSH, GSSG, GSH and GSSG, cysteine, cystine, or cysteine and cystine (e.g., 0.02 mM to 2 mM), and sucrose (up to 3%), and has a pH of 5.2 to 6.2. In some embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride, methionine, histidine, PS80, and a thiol-containing antioxidant such as GSH, GSSG, GSH and GSSG, cysteine, cystine, or cysteine and cystine, and has a pH of 5.5. In all of these embodiments, the anti-αvβ6 antibody is present at a concentration of 100 mg/ml to 165 mg/ml. In one instance, the anti-αvβ6 antibody is present at a concentration of 150 mg/ml. In one instance, the anti-αvβ6 antibody is present at a concentration of 100 mg/ml.

[0085] In certain embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), sodium citrate buffer (sodium citrate and citric acid) (e.g., 20 mM), a thiol-containing antioxidant such as GSH, GSSG, GSH and GSSG, cysteine, cystine, or cysteine and cystine (e.g., 0.02 mM to 2 mM), and PS80 (e.g., 0.05%), and has a pH of 5.2 to 6.2. In some embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), sodium citrate and citric acid (e.g., 20 mM), a thiol-containing antioxidant such as GSH, GSSG, GSH and GSSG, cysteine, cystine, or cysteine and cystine (e.g., 0.02 mM to 2 mM), and PS80 (e.g., 0.05%), and has a pH of 5.5. In certain embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride (e.g., 150 mM), sodium citrate and citric acid (e.g., 20 mM), PS80 (e.g., 0.05%), a thiol-containing antioxidant such as GSH, GSSG, GSH and GSSG, cysteine, cystine, or cysteine and cystine (e.g., 0.02 mM to 2 mM), and sucrose (up to 3%), and has a pH of 5.2 to 6.2. In some embodiments, the anti-αvβ6 antibody compositions comprise L-arginine hydrochloride, histidine, PS80, and a thiol-containing antioxidant such as GSH, GSSG, GSH and GSSG, cysteine, cystine, or cysteine and cystine, and has a pH of 5.5. In all of these embodiments, the anti-αvβ6 antibody is present at a concentration of 100 mg/ml to 165 mg/ml. In one instance, the anti-αvβ6 antibody is present at a concentration of 150 mg/ml. In one instance, the anti-αvβ6 antibody is present at a concentration of 100 mg/ml.

[0086] In certain embodiments, the composition (e.g., a pharmaceutical composition) comprises an anti-αvβ6 antibody or an αvβ6-binding fragment thereof at a concentration of 75 mg/ml to 250 mg/ml, arginine (e.g., L-arginine hydrochloride) at a concentration of 50 mM to 200 mM, methionine at a concentration of 1 mM to 10 mM; polysorbate-80 at a concentration of 0.01% to 0.1%, sodium citrate and citric acid at a concentration of 10 mM to 30 mM, and sucrose at a concentration of 0% to 3%. The composition has a pH of 5.2 to 6.0. In certain embodiments, the anti-αvβ6 antibody or an αvβ6-binding fragment thereof of the composition comprises a VH and a VL comprising the CDRs of STX-100 (e.g., SEQ ID NOs: 1 or 11, 2, 3, 4, 5, and 6). In certain embodiments, the anti-αvβ6 antibody or an αvβ6-binding fragment thereof of the composition comprises a VH and a VL comprising SEQ ID NOs: 7 and 8, respectively. In some embodiments, the anti-αvβ6 antibody or an αvβ6-binding fragment thereof of the composition comprises a heavy chain and a light chain comprising SEQ ID NOs: 9 and 10, respectively. In one embodiment, the composition has a pH of 5.5 and comprises STX-100 or a STX-100-binding fragment thereof at a concentration of 150 mg/ml, L-arginine hydrochloride at a concentration of 150 mM, methionine at a concentration of 5 mM, polysorbate-80 at a concentration of 0.05%, and sodium citrate and citric acid at a concentration of 20 mM. In certain embodiments, the composition further comprises a thiol-containing antioxidant (e.g., GSH, GSSG, GSH+GSSG, cysteine, cystine, cysteine+cystine) at a concentration of 0.02 mM to 2 mM. In some embodiments, the composition further comprises sucrose at a concentration of 0.01% to 3%. In certain embodiments, the anti-αvβ6 antibody or an αvβ6-binding fragment thereof of the composition comprises a VH and a VL comprising the CDRs of STX-100 (e.g., SEQ ID NOs: 1 or 11, 2, 3, 4, 5, and 6). In certain embodiments, the anti-αvβ6 antibody or an αvβ6-binding fragment thereof of the composition comprises a VH and a VL comprising SEQ ID NOs: 7 and 8, respectively. In some embodiments, the anti-αvβ6 antibody or an αvβ6-binding fragment thereof of the composition comprises a heavy chain and a light chain comprising SEQ ID NOs: 9 and 10, respectively.

[0087] In one embodiment, the composition has a pH of 5.5 and comprises STX-100 or a STX-100-binding fragment thereof at a concentration of 150 mg/ml, L-arginine hydrochloride at a concentration of 150 mM, a thiol-containing antioxidant (e.g., GSH, GSSG, GSH+GSSG, cysteine, cystine, cysteine+cystine) at a concentration of 0.02 mM to 2 mM, polysorbate-80 at a concentration of 0.05%, and sodium citrate and citric acid at a concentration of 20 mM. In one embodiment, the thiol-containing antioxidant is GSH at a concentration of 0.4 mM. In one embodiment, the thiol-containing antioxidant is GSH at a concentration of 0.4 mM and GSSG at a concentration of 0.2 mM. In another embodiment, the thiol-containing antioxidant is cysteine at a concentration of 0.4 mM. In another embodiment, the thiol-containing antioxidant is cysteine at a concentration of 0.4 mM and cystine at a concentration of 0.2 mM.

Dosing

[0088] The anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof described above can be administered to a subject, e.g., a human subject, at different doses. The anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof can be administered as a fixed dose (i.e., independent of the weight of the patient), or in a mg/kg dose (i.e., a dose which varies based on the weight of the subject). Dosage unit form or “fixed dose” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in association with the other agent. Single or multiple dosages may be given. The treatment can continue for days, weeks, months or even years.

[0089] In certain embodiments, for treating an indication described herein in an adult human subject, the dosage of the anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof is a fixed dose of 40 mg to 64 mg once weekly. In one embodiment, for treating an indication described herein in an adult human subject, the dosage of the anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof is a fixed dose of 40 mg once weekly. In another embodiment, the dosage of the anti-αvβ6 antibody or αvβ6-binding fragment thereof is a fixed dose of 48 mg once weekly. In another embodiment, the dosage of the anti-αvβ6 antibody or αvβ6-binding fragment thereof is a fixed dose of 56 mg once weekly. In another embodiment, the dosage of the anti-αvβ6 antibody or αvβ6-binding fragment thereof is a fixed dose of 64 mg once weekly.

[0090] In certain embodiments, for treating an indication described herein in an adult human subject, the dosage of the anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof is a mg/kg dose of 0.3 mg/kg to 1.0 mg/kg. In one embodiment, for treating an indication described herein in an adult human subject, the dosage of the anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof is a mg/kg dose of 0.5 mg/kg to 0.8 mg/kg. In one embodiment, for treating an indication described herein in an adult human subject, the dosage of the anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof is a mg/kg dose of 0.5 mg/kg. In another embodiment, for treating an indication described herein in an adult human subject, the dosage of the anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof is a mg/kg dose of 0.6 mg/kg. In another embodiment, for treating an indication described herein in an adult human subject, the dosage of the anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof is a mg/kg dose of 0.7 mg/kg. In yet another embodiment, for treating an indication described herein in an adult human subject, the dosage of the anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof is a mg/kg dose of 0.8 mg/kg.

[0091] In certain instances, the anti-αvβ6 antibody or αvβ6-binding fragment thereof is administered in combination with a therapeutically effective amount of an art recognized treatment for IPF.

[0092] Exemplary art recognized treatment options that can be used in combination with the antibody of the invention include: Corticosteroids (prednisone); Cyclophosphamide (Cytoxan®); Azathioprine (Imuran®); Mycophenolate mofetil (Cellcept®, Myfortic®); N-acetylcysteine (NAC); Nintedanib (Ofev®); Pirfenidone (Esbriet®, Pirfenex®, Pirespa®); Proton pump inhibitors (Prilosec OTC®, Nexium®, others); or Supplemental Oxygen Therapy.

[0093] In one embodiment, an antibody of the invention is combined with prifenidone or nintedanib. In certain cases, the subject is administered prifenidone as follows:

[0094] Treatment Days Dosage

[0095] Days 1 through 7 267 mg three times daily (801 mg/day)

[0096] Days 8 through 14 534 mg three times daily (1602 mg/day)

[0097] Days 15 onward 801 mg three times daily (2403 mg/day)

in combination with the antibody of the invention. In certain cases, the subject is administered a therapeutically effective amount of nintedanib at a fixed dose of 150 mg twice daily in combination with the antibody of the invention.

[0098] In certain instances, the anti-αvβ6 antibody or αvβ6-binding fragment thereof is administered in combination with an antibody that inhibits the activity of connective tissue growth factor (CTGF) such as, but not limited to, the fully-human monoclonal antibody, Pamrevlumab.

[0099] In certain instances, the anti-αvβ6 antibody or αvβ6-binding fragment thereof is administered in combination with a therapeutically effective amount of a selective autotaxin inhibitor (e.g., GLPG1690).

[0100] In certain instances, the anti-αvβ6 antibody or αvβ6-binding fragment thereof is administered in combination with a therapeutically effective amount of GBT-440.

[0101] A pharmaceutical composition may include a “therapeutically effective amount” of an agent described herein. Such effective amounts can be determined based on the effect of the administered agent, or the combinatorial effect of agents if more than one agent is used. A therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic, or detrimental effects, of the composition is outweighed by the therapeutically beneficial effects. In certain embodiment, the therapeutically effective amount of the anti-αvβ6 antibody or αvβ6-binding fragment thereof is 40 mg to 64 mg. In one embodiment, the therapeutically effective amount of the anti-αvβ6 antibody or αvβ6-binding fragment thereof is 40 mg. In another embodiment, the therapeutically effective amount of the anti-αvβ6 antibody or αvβ6-binding fragment thereof is 48 mg. In yet another embodiment, the therapeutically effective amount of the anti-αvβ6 antibody or αvβ6-binding fragment thereof is 56 mg. In yet another embodiment, the therapeutically effective amount of the anti-αvβ6 antibody or αvβ6-binding fragment thereof is 64 mg.

[0102] The route and/or mode of administration of the anti-αvβ6 antibody or αvβ6-binding fragment thereof can be tailored for the individual subject. For many applications, the route of administration is one of: subcutaneous injection (SC), intravenous injection or infusion (IV), intraperitoneal administration (IP), or intramuscular injection. In one embodiment, the route of administration is subcutaneous. In another embodiment, the route of administration is intravenous.

[0103] Pharmaceutical compositions that comprise the anti-αvβ6 antibody or αvβ6-binding fragment thereof alone or in combination with non αvβ6 antibody agent(s) can be administered with a medical device. The device can be designed with features such as portability, room temperature storage, and ease of use so that it can be used in emergency situations, e.g., by an untrained subject or by emergency personnel in the field, removed to medical facilities and other medical equipment. The device can include, e.g., one or more housings for storing pharmaceutical preparations that include the anti-αvβ6 antibody or αvβ6-binding fragment thereof, and can be configured to deliver one or more unit doses of the anti-αvβ6 antibody or other agent.

[0104] For example, the pharmaceutical composition can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other devices, implants, delivery systems, and modules are also known.

[0105] In one embodiment, the anti-αvβ6 antibody or αvβ6-binding fragment thereof is administered to a human subject with a syringe. In another embodiment, the anti-αvβ6 antibody or αvβ6-binding fragment thereof is administered to a human subject with a pump for subcutaneous delivery. In some embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof is administered to a human subject with an autoinjector. In other embodiments, the anti-αvβ6 antibody or αvβ6-binding fragment thereof is administered to a human subject with a subcutaneous large volume injector.

[0106] This disclosure provides a pump or syringe comprising a sterile preparation of an anti-αvβ6 antibody (e.g., STX-100) or αvβ6-binding fragment thereof. The syringe or pump can be adapted for subcutaneous administration of the anti-αvβ6 antibody or αvβ6-binding fragment thereof. In some cases, the syringe or pump delivers a fixed doses(s) (e.g., 40 mg, 48 mg, 56 mg, 64 mg) of the anti-αvβ6 antibody or αvβ6-binding fragment thereof.

[0107] The disclosure also provides a pump, syringe, or injector (e.g., autoinjector, subcutaneous large volume injector) comprising a sterile preparation of the pharmaceutical compositions described above. The syringe or pump can be adapted for subcutaneous administration of the pharmaceutical compositions comprising the anti-αvβ6 antibody or αvβ6-binding fragment thereof. In some instances, the syringe or pump delivers a fixed doses(s) (e.g., 40 mg, 48 mg, 56 mg, 64 mg) of the anti-αvβ6 antibody or αvβ6-binding fragment thereof

Methods of Treatment

[0108] The antibodies of this disclosure are useful in the treatment, including prevention, of αvβ6-mediated diseases. For example, these antibodies can be used to treat fibrosis (e.g., lung fibrosis, kidney fibrosis, liver fibrosis, cardiac fibrosis), acute lung injury, acute kidney injury, Alport's Syndrome, psoriasis, scleroderma, and sclerosis of lung, liver, or kidney, by blocking the activation of TGF-β or blocking the binding of β6 to any other ligands, such as fibronectin, vitronectin, and tenascin. The novelty of this approach includes: (1) it blocks the activation of TGF-β rather than the binding of TGF-β to its receptor, (2) it can inhibit TGF-β locally (i.e., at sites of αvβ6 upregulation) rather than systemically, and (3) it inhibits binding of αvβ6 to a ligand.

[0109] Other than fibrotic diseases or conditions, the antibodies of the disclosure are useful in treating cancer or cancer metastasis (including tumor growth and invasion), particularly epithelial cancers. A subset of epithelial cancers is squamous cell carcinoma, e.g., head and neck, oral, breast, lung, prostate, cervical, pharyngeal, colon, pancreatic and ovarian cancers.

[0110] In additional embodiments of the invention, αvβ6-binding antibodies or fragments thereof, may be used in therapeutic regimens for treating humans having, or at risk of developing carcinomas. Such methods of the invention are useful in treating cancer and associated events, including tumor growth, metastasis and angiogenesis. Particularly amenable to such an approach are those diseases or cancers that are characterized by increased levels of αvβ6 expression in the tissues or cells of a mammal suffering from the disease, and which are responsive to treatments, which target the tissues or cells expressing increased levels of αvβ6 and eliminate those tissues or cells. Diseases that are particularly treatable by these methods include metastatic cancers of epithelial tissues (i.e., metastatic carcinomas and/or adenocarcinomas), including of the breast, ovary, prostate, liver, lung, pancreas, colon, head and neck tissues (e.g., oral, pharyngeal, lingual and laryngeal tissues), endometrium, cervix, stomach and spleen. Particularly suitable for treatment by these methods of the present invention are carcinomas of the endometrium, pancreas, colon (e.g., colorectal carcinomas), cervix, lung and breast (including ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS) of the breast).

[0111] The following are examples of the practice of the invention. They are not to be construed as limiting the scope of the invention in any way.

EXAMPLES

[0112] These Examples relate, in part, to the development of a stable high concentration (e.g., 100 mg/ml or greater) liquid formulation for STX-100.

Example 1: Pre-Formulation Evaluation

[0113] During the initial pre-formulation evaluation, accelerated stability studies were conducted to explore pH, buffer, and excipient components suitable for a high concentration liquid formulation for STX-100. A formulation matrix containing 10 mM Na-citrate/citric acid pH 5.0, 150 mM arginine hydrochloride (Arginine-HCl) was used as a control for comparison. For the excipient screen, amino acids like glycine, lysine, arginine-HCl, and methionine, sugars like sorbitol, trehalose, mannitol, sucrose, and buffer systems such as citrate and acetate were tested. A pH range from 4.4 to 5.7 was also evaluated.

[0114] Accelerated stability evaluation was performed at 40° C. incubation over 4 weeks for the formulations. The following quality attributes were monitored: visible particulates and clarity, % high molecular weight species (via SEC), total sub-visible particulates (via MFI), turbidity (via OD340), pH, fragmentation (via GXII), % total acidic isoforms (via iCIEF), and viscosity at TO. Maximum weightage was assigned to Critical quality attributes (CQA) like aggregate level and particle formation and were utilized in formulation selection. The formulations with least amount of aggregate level and particle formation were selected for further evaluations.

[0115] The data indicated that arginine- and trehalose-containing formulations to be the most stable compared to others (Table 2; FIGS. 1A-1C; and FIG. 2).

TABLE-US-00008 TABLE 2 Results of pre-formulation buffer-excipient screening study Visible appearance Turbidity (OD340) Time at 40° C. Time at 40° C. Excipient/buffer T = 0 1 wk 2 wk 4 wk T = 0 1 wk 2 wk 4 wk 300 mM Glycine 5-10 10-50 <10 <10 1.591549 1.0812 1.193952 NT particles particles particles particles NT cla: 18- cla: 18- cla: 30- 30 NTU 30 NTU 50 NTU 300 mM Lysine 5-10 10-50 Few <10 0.916307 1.1386 1.30471 NT particles particles white particles particles NT cla: 18- cla: 18- cla: 30- 30 NTU 30 NTU 50 NTU 300 mM Sorbitol 50-100 <10 <10 <10 0.85723 1.1036 1.161319 NT particles particles particles particles NT cla: 18- cla: 18- cla: 30- 30 NTU 30 NTU 50 NTU 200 mM Trehalose Few <10 NT <10 1.004 1.005 1.048 NT particles particles particles observed NT cla: 18- NT cla: 30- 30 NTU 50 NTU 100 mM Mannitol 5-10 10-50 <10 <10 0.877126 1.1184 1.181954 NT particles particles particles particles NT cla: 18- cla: 18- cla: 30- 30 NTU 30 NTU 50 NTU 5% Sucrose Few <10 <10 <10 1.902725 1.1286 1.192552 NT particles particles particles particles observed NT cla: 18- cla: 18- cla: 30- 30 NTU 30 NTU 50 NTU 10 mM Acetate <50 <10 <10 <10 0.71461 0.963 1.0212 NT particles particles particles particles NT cla: 18- cla: 18- cla: 30- 30 NTU 30 NTU 50 NTU 10 mM Succinate <50 <10 <10 <10 0.925 0.923 0.929 NT particles particles particles particles NT cla: 18- cla: 18- cla: 30- 30 NTU 30 NTU 50 NTU 10 mM Citrate <50 <10 <10 <10 0.903355 1.1226 1.178035 NT particles particles particles particles NT cla: 18- cla: 18- cla: 30- 30 NTU 30 NTU 50 NTU NT: Not tested

[0116] The presence of additional 25 mM methionine conferred greater aggregation resistance to the arginine-containing formulation (FIG. 1A).

[0117] At pH 4.4, formulations containing 75 mM to 300 mM Arg-HCl displayed gel formation whereas in the pH 5.2 to 5.7 range, no gel formation was observed under accelerated conditions (FIG. 2). As seen in FIGS. 1A and 2, the % total aggregate increase observed in these formulations was also lower in the entire buffer-excipient screen and the pH-arginine screen. At 150 mM ArgHCl, there was no significant difference in aggregation between pH 5.2 and pH 5.7 (FIG. 2) suggesting that this pH range would most likely contain the desired set-point for minimizing aggregation.

Example 2: Medium to Long-term Stability Study & Formulation Selection

[0118] Based on the pre-formulation results in Example 1, the following five liquid formulations and corresponding container-closures (CCs) were selected for pursuing a long-term (24 month) stability study: [0119] 1) Lot #18169-62: 150 mg/mL STX-100 in 20 mM Na-citrate/citric acid, pH 5.3, 150 mM arginine hydrochloride (Arg.HCl), 0.05% PS-80 (1 mL fill in 3 mL Schott vial) [0120] 2) Lot #18169-64: 150 mg/mL STX-100 in 20 mM Na-citrate/citric acid, pH 5.3, 150 mM arginine hydrochloride (Arg.HCl), 0.05% PS-80 (1 mL fill in BD Hypak pre-filled syringe, 27 G needle) [0121] 3) Lot #18169-66: 150 mg/mL STX-100 in 20 mM Na-citrate/citric acid, pH 5.3, 150 mM arginine hydrochloride (Arg.HCl), 25 mM methionine, 0.05% PS-80 (1 mL fill in BD Hypak pre-filled syringe, 27 G needle) [0122] 4) Lot #18169-67: 150 mg/mL STX-100 in 20 mM Na-citrate/citric acid, pH 5.3, 200 mM trehalose, 0.05% PS-80 (1 mL fill in BD Hypak pre-filled syringe, 27 G needle) [0123] 5) Lot #18169-72: 250 mg/mL STX-100 in 20 mM Na-citrate/citric acid, pH 5.3, 150 mM arginine hydrochloride (Arg.HCl), 25 mM methionine, 0.05% PS-80 (1 mL fill in BD Hypak pre-filled syringe, 27 G needle)
Aggregation Data: Stability data indicated that all the five formulations listed above displayed a low aggregation propensity throughout 12 months of storage at 5° C. with only a 0.2-0.3% increase in % total aggregate across all the formulations (Table 3).

TABLE-US-00009 TABLE 3 Long-term % Total aggregate data measured using SEC-UPLC Temperature Lot number (° C.) % Total aggregates (via SEC-UPLC) Time (months) 0 1 2 3 6 9 12 # 18169-62 5 2.5 2.5 2.4 2.5 2.5 2.6 2.6 150 mg/mL, 20 mM 25 2.5 2.6 2.7 2.8 3.2 3.4 3.6 citrate, pH 5.3, 40 2.5 3.7 4.2 5.1 11.6 NT NT 150 mM ArHCl, 0.05% PS-80 1 mL in 3 mL Schott vial) # 18169-64 5 2.5 2.5 2.4 2.5 2.6 2.7 2.7 150 mg/mL, 20 mM 25 2.5 2.7 2.7 2.9 3.2 3.5 3.7 citrate, pH 5.3, 40 2.5 3.6 4.1 5 11.9 NT NT 150 mM ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-66 5 2.5 2.5 2.4 2.5 2.5 2.6 2.6 150 mg/mL, 20 mM 25 2.5 2.6 2.6 2.7 3 3.2 3.2 citrate, pH 5.3, 40 2.5 3.4 3.8 4.7 11 NT NT 150 mM ArHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 5 3 3 2.9 3 3.1 3.3 3.3 150 mg/mL, 20 mM 25 3 3.3 3.5 3.8 4.2 4.6 4.9 citrate, pH 5.3, 40 3 4.5 5.3 6.2 10 NT NT 200 mM trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 5 1.4 1.4 1.3 1.5 1.5 1.7 1.7 250 mg/mL, 20 mM 25 1.4 1.8 1.9 2.2 2.6 3   3.2 citrate, pH 5.3, 40 1.4 3.1 3.9 5.2 14.9 NT NT 150 mM ArHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle)
The data also indicate that there is no significant difference in aggregate level for formulations held in a pre-filled syringe (18169-64) and a vial (18169-62).
Sub-Visible Particulate (SVP) Data: While the aggregation data was promising for a stable liquid formulation, there were some indications of high sub-visible particulate (SVP) counts via micro-flow imaging, MFI (Tables 4, 5 and 6).

TABLE-US-00010 TABLE 4 Total sub-visible particulates/mL (MFI) Temp Lot number (° C.) Total Particulates/mL (MFI) Time (months) 0 1 3 6 9 12 # 18169-62 5 13616 5005 8126 40458 41396 16988 150 mg/mL, 20 mM 25 13616 26252 34202 311593 518112  665292  citrate, pH 5.3, 40 13616 852037 776326 2397797 NT NT 150 mM ArHCl, 0.05% PS-80 1 mL in 3 mL Schott vial) # 18169-64 5 43413 35963 15064 5777  8076  8058 150 mg/mL, 20 mM 25 43413 26300 68631 156553 278577  208307  citrate, pH 5.3, 40 43413 708893 1467637 2111368 NT NT 150 mM ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-66 5 19412 6536 11753 13795 16704 13263 150 mg/mL, 20 mM 25 19412 15750 136088 236386 427144  566722  citrate, pH 5.3, 40 19412 1230328 2023654 2003136 NT NT 150 mM ArHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 5 25444 23998 17266 27329 135928 199303  150 mg/mL, 20 mM 25 25444 113162 701629 527438 1602970  927081  citrate, pH 5.3, 40 25444 178718 1183228 542746 NT NT 200 mM trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 5 13482 17008 7760 10865 10633 21093 250 mg/mL, 20 mM 25 13482 12741 12641 18973 63098 15062 citrate, pH 5.3, 40 13482 25198 10126 21717 NT NT 150 mM ArHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) Ref buffer vial 5 22263 NT 86201 51685 NT 26252 25 22263 NT 57860 40409 NT 27959 40 22263 NT 56260 33898 NT NT Ref buffer PFS 5 38840 NT 76251 64420 NT 15914 25 38840 NT 105080 40830 NT 71146 40 38840 NT 123503 236238 NT NT

TABLE-US-00011 TABLE 5 Sub-visible particulates (>10 um)/mL (MFI) Particulates/mL (size > 10 um) Time (months) Lot number Temp 0 1 3 6 9 12 # 18169-62 5 161 94 59 98 374 78 150 mg/mL, 20 mM 25 161 128 450 6376 3781 10313 citrate, pH 5.3, 150 mM 40 161 4019 50460 57928 NT NT ArHCl, 0.05% PS-80 (1 mL in 3 mL Schott vial) # 18169-64 5 195 274 300 128 1934 52 150 mg/mL, 20 mM 25 195 872 1438 6298 5127 7942 citrate, pH 5.3, 150 mM 40 195 20497 36305 106721 NT NT ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-66 5 331 70 204 74 1014 104 150 mg/mL, 20 mM 25 331 70 71 7838 7696 19841 citrate, pH 5.3, 150 mM 40 331 32528 60842 91013 NT NT ArHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 5 243 698 170 292 4273 2997 150 mg/mL, 20 mM 25 243 3593 15312 41916 52119 67621 citrate, pH 5.3, 200 mM 40 243 5843 35691 27479 NT NT trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 5 995 3121 688 1272 796 870 250 mg/mL, 20 mM 25 995 1112 860 1036 3039 308 citrate, pH 5.3, 150 mM 40 995 2511 786 1816 NT NT ArgHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) Ref buffer vial 5 305 NT 852 20 NT 20 25 305 NT 366 64 NT 54 40 305 NT 108 194 NT NT Ref buffer PFS 5 258 NT 494 370 NT 114 25 258 NT 348 238 NT 420 40 258 NT 822 968 NT NT

TABLE-US-00012 TABLE 6 Sub-visible particulates (>25 um)/mL (MFI) Particulates (>25 um); <=600 Time (months) Lot number Temperature 0 1 3 6 9 12 # 18169-62 5 20 22 6 4 16 0 150 mg/mL, 20 mM 25 20 8 20 94 66 160 citrate, pH 5.3, 150 mM 40 20 3123 4727 679 NT NT ArHCl, 0.05% PS-80 (1 mL in 3 mL Schott vial) # 18169-64 5 27 24 16 2 1644 0 150 mg/mL, 20 mM 25 27 498 132 420 326 640 citrate, pH 5.3, 150 mM 40 27 2395 2735 12288 NT NT ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-66 5 117 2 66 6 560 2 150 mg/mL, 20 mM 25 179 2 162 560 712 1570 citrate, pH 5.3, 150 mM 40 117 3471 6200 9676 NT NT ArHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 5 43 410 42 80 908 428 150 mg/mL, 20 mM 25 43 804 2091 5815 5711 8822 citrate, pH 5.3, 200 mM 40 43 624 5067 2915 NT NT trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 5 563 2307 460 850 450 308 250 mg/mL, 20 mM 25 563 710 402 630 816 124 citrate, pH 5.3, 150 mM 40 563 1512 488 1308 NT NT ArgHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) Ref buffer vial 5 47 NT 88 4 NT 0 25 47 NT 48 11 NT 2 40 47 NT 10 32 NT NT Ref buffer PFS 5 10 NT 10 6 NT 0 25 10 NT 10 2 NT 6 40 10 NT 34 4 NT NT

[0124] However, these SVP are thought to primarily arise from the handling and processing of STX-100 drug substance (DS) during labscale UF/DF process, pre-fill storage, shipping to testing laboratory, and likely issues with the testing method. The growth rate of SVP>10 um (picked up more sensitively by MFI) do not suggest significant instability in any of the arginine-HCl containing formulations except 18169-67 that contains 200 mM trehalose. SVP counts are also observed to be higher in the formulations in pre-filled syringe presentation compared to vial presentation. This indicates that the testing method also identified a significant amount of silicone oil micro-droplets that commonly occur in such syringes. SVP analysis via HIAC method (as per USP-788) that is based on light-obscuration did not indicate instability at the desired storage condition of 5° C. (Tables 7 and 8).

TABLE-US-00013 TABLE 7 Sub-visible particulates (>10 um)/mL (HIAC) Particulates (>10 um); <=6000 Time (months) Lot number Temperature 0 1 3 6 9 12 # 18169-62 5 23 32 22 46 61 63 150 mg/mL, 20 mM 25 23 111 175 55 645 936 citrate, pH 5.3, 150 mM 40 23 1594 3574 83 NT NT ArHCl, 0.05% PS-80 (1 mL in 3 mL Schott vial) # 18169-64 5 74 7 92 69 107 63 150 mg/mL, 20 mM 25 74 185 246 464 732 548 citrate, pH 5.3, 150 mM 40 74 3910 5749 213 NT NT ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-66 5 57 56 104 77 138 40 150 mg/mL, 20 mM 25 57 80 526 260 709 743 citrate, pH 5.3, 150 mM 40 57 4126 10947 267 NT NT ArHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 5 71 83 92 53 215 137 150 mg/mL, 20 mM 25 71 179 4292 289 387 1904 citrate, pH 5.3, 200 mM 40 71 1161 4993 184 NT NT trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 5 51 72 141 96 280 37 250 mg/mL, 20 mM 25 51 132 117 105 442 77 citrate, pH 5.3, 150 mM 40 51 72 105 232 NT NT ArgHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) Ref buffer vial 5 41 NT 92 77 NT 48 25 41 NT 101 53 NT 61 40 41 NT 44 98 NT NT Ref buffer PFS 5 233 NT 251 325 NT 111 25 233 NT 224 416 NT 231 40 233 NT 415 714 NT NT

TABLE-US-00014 TABLE 8 Sub-visible particulates (>25 um)/mL (HIAC) Particulates (>25 um): <=600 Time (months) Lot number Temperature 0 1 3 6 9 12 # 18169-62 5 2 0 1 0 2 0 150 mg/mL, 20 mM 25 2 1 1 0 7 5 citrate, pH 5.3, 150 mM 40 2 47 44 0 NT NT ArHCl, 0.05% PS-80 (1 mL in 3 mL Schott vial) # 18169-64 5 1 0 1 0 2 0 150 mg/mL, 20 mM 25 1 3 5 3 30 5 citrate, pH 5.3, 150 mM 40 1 101 118 8 NT NT ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-66 5 1 2 3 3 2 2 150 mg/mL, 20 mM 25 1 6 10 6 30 3 citrate, pH 5.3, 150 mM 40 1 77 482 18 NT NT ArHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 5 1 4 2 0 10 1 150 mg/mL, 20 mM 25 1 5 102 10 10 16 citrate, pH 5.3, 200 mM 40 1 21 75 12 NT NT trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 5 2 4 5 13 35 2 250 mg/mL, 20 mM 25 2 11 8 2 32 7 citrate, pH 5.3, 150 mM 40 2 7 4 20 NT NT ArgHCl, 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) Ref buffer vial 5 0 NT 1 0 NT 0 25 0 NT 0 0 NT 0 40 0 NT 0 1 NT NT Ref buffer PFS 5 2 NT 0 0 NT 0 25 2 NT 3 7 NT 4 40 2 NT 8 15 NT NT
Oxidation data: Forced oxidation analysis in the past on STX-100 samples had revealed oxidation propensity in Met-55 contained in the second heavy chain CDR along with two other methionines (Met-255 and Met-431) in the Fc region. Structure-activity relationship studies revealed that oxidation in these residues do not lead to any change in binding activity to the antigen. In this study, it was also investigated whether oxidation in these residues over time leads to instability due to the presence of polysorbate-80 as a likely oxidizing agent. The % oxidation was determined using a LCMS method after generating in Met residues contained in corresponding peptides generated (Met-55 in peptide H2, Met-255 in peptide H15, and Met-431 in peptide H30) by LysC cleavage. Overall, there was no major increase in oxidation at each site although the presence of methionine as an excipient in the formulation did suppress this oxidation reaction (Tables 9A and 9B).

TABLE-US-00015 TABLE 9A Oxidation analysis on formulations stored at 5° C. t = 0 t = 6 months at 5° C. t = 12 months at 5° C. Lot# % H2—Ox % H15—Ox % H30—Ox % H2—Ox % H15—Ox % H30—Ox % H2—Ox % H15—Ox % H30—Ox #18169-62 7.9 7.9 5.9 11.2 9.7 6.7 8.40 7.20 4.90 150 mg/mL, 20 mM citrate, pH 5.3, 150 mM ArHCl, 0.05% PS-80 (1 mL in 3 mL Schott vial) #18169-64 7.9 8.1 6.2 10.3 9.4 5.9 8.70 7.40 5.00 150 mg/mL, 20 mM citrate, pH 5.3, 150 mM ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) #18169-66 7.8 8.2 6.3 8.5 8.7 5.3 7.00 7.00 4.90 150 mg/mL, 20 mM citrate, pH 5.3, 150 mM ArHCl 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 8.1 8.3 6.5 9.6 9.5 5.8 7.50 7.10 4.90 150 mg/mL, 20 mM citrate, pH 5.3, 200 mM trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 7 5.7 3.4 9 7.8 4.2 6.60 5.10 2.60 250 mg/mL, 20 mM citrate, pH 5.3, 150 mM ArgHCl, 25 mM methionine, 0 05% PS-80 (1 mL in BD Hypak syringe. 27 G needle

TABLE-US-00016 TABLE 9B Oxidation analysis on formulations stored at 25° C. t = 0 t = 6 months at 25° C. t = 12 months at 25° C. Lot# % H2—Ox % H15—Ox % H30—Ox % H2—Ox % H15—Ox % H30—Ox % H2—Ox % H15—Ox % H30—Ox #18169-62 7.9 7.9 5.9 17.7 10.4 6.8 18.40 8.50 5.80 150 mg/mL, 20 mM citrate, pH 5.3, 150 mM ArHCl, 0.05% PS-80 (1 mL in 3 mL Schott vial) #18169-64 7.9 8.1 6.2 17 10.2 6.2 18.40% 8.40 5.60 150 mg/mL, 20 mM citrate, pH 5.3, 150 mM ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) #18169-66 7.8 8.2 6.3 10.2 8.8 5.8 9.00% 7.20 5.20 150 mg/mL, 20 mM citrate, pH 5.3, 150 mM ArHCl 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 8.1 8.3 6.5 13.6 9.8 6.2 12.90% 8.10 5.50 150 mg/mL, 20 mM citrate, pH 5.3, 200 mM trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 7 5.7 3.4 10.8 7.8 4 9.10% 5.70 2.90 250 mg/mL, 20 mM citrate, pH 5.3, 150 mM ArgHCl, 25 mM methionine, 0 05% PS-80 (1 mL in BD Hypak syringe. 27 G needle
Visible particulate data: Appearance (particulate) observations did not reveal any significant increase in visible particulates for any formulation throughout the 12-month storage period at 5° C. (Table 10A). Visible particulates do appear at 25° C. over long-term storage (Table 10B) and are probably linked to the increase in large SVP (>25 um) at this temperature.

TABLE-US-00017 TABLE 10A Long-term assessment of visible particulates at 5° C. Months at 5° C. Lot# 0 1 2 3 6 9 12 #18169-62 No visible No visible No visible No visible No visible No visible *No visible 150 mg/mL, particulates particulates particulates particulates particulates particulates particulates 20 mM citrate, observed observed observed observed observed observed observed pH 5.3, 150 mM ArHCl, 0.05% PS-80 (1 mL in 3 mL Schott vial) #18169-64 No visible No visible No visible No visible No visible No visible No visible 150 mg/mL, particles particulates particulates particulates particulates particulates particulates 20 mM citrate, observed observed observed observed observed observed observed pH 5.3, 150 mM ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) #18169-66 No visible No visible No visible No visible No visible No visible *No visible 150 mg/mL, particles particulates particulates particulates particulates particulates particulates 20 mM citrate, observed observed observed observed observed observed observed pH 5.3, 150 mM ArHCl 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 No visible No visible No visible No visible No visible No visible No visible 150 mg/mL, particles particulates particulates particulates particulates particulates particulates 20 mM citrate, observed observed observed observed observed observed observed pH 5.3, 200 mM trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 No visible No visible No visible No visible No visible No visible *No visible 250 mg/mL, particles particles particles particles particulates particulates particulates 20 mM citrate, observed observed observed observed observed observed observed pH 5.3, 150 mM ArgHCl, 25 mM methionine, 0 05% PS-80 (1 mL in BD Hypak syringe. 27 G needle Ref Buffer vial No visible NT NT No visible White flake NT No visible particles particles particle particles observed observed observed Ref Buffer PFS No visible NT NT No visible No visible NT No visible particles particles particles particles observed observed observed observed *Samples were re-examined after initial report revealed some particulates. The internal examinations on triplicate vials/syringes failed to show any visible particulates. The initial observations are therefore thought to arise from error in handling or human error.

TABLE-US-00018 TABLE 10B Long-term assessment of visible particulates at 25° C. Months at 25° C. Lot# 0 1 2 3 6 9 12 #18169-62 No visible No visible No visible No visible No visible No visible No visible 150 mg/mL, particulates particulates particulates particulates particulates particulates particulates 20 mM citrate, observed observed observed observed observed observed observed pH 5.3, 150 mM ArHCl, 0.05% PS-80 (1 mL in 3 mL Schott vial) #18169-64 No visible No visible No visible No visible No visible No visible No visible 150 mg/mL, particles particulates particulates particulates particulates particulates particulates 20 mM citrate, observed observed observed observed observed observed observed pH 5.3, 150 mM ArHCl, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) #18169-66 No visible No visible No visible No visible Two other White fiber Small rounc 150 mg/mL, particles particulates particulates particulates white like particulate 20 mM citrate, observed observed observed observed particles particulates pH 5.3, 150 observed mM ArHCl 25 mM methionine, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-67 No visible No visible No visible No visible No visible No visible Small fiber 150 mg/mL, particles particulates particulates particulates particulates particulates like 20 mM citrate, observed observed observed observed observed observed particulate pH 5.3, 200 mM trehalose, 0.05% PS-80 (1 mL in BD Hypak syringe, 27 G needle) # 18169-72 No visible No visible No visible No visible No visible No visible No visible 250 mg/mL, particles particles particles particles particulates particulates particulates 20 mM citrate, observed observed observed observed observed observed observed pH 5.3, 150 mM ArgHCl, 25 mM methionine, 0 05% PS-80 (1 mL in BD Hypak syringe. 27 G needle Ref Buffer vial No visible NT NT White fiber No visible NT No visible particles and 10 other particulates particulates observed particles observed observed Ref Buffer PFS No visible NT NT No visible No visible NT No visible particles particles particles particles observed observed observed observed
Milestone assays: Other stability assays such as CE-SDS (non-reduced), icIEF, osmolality, viscosity and potency tested at t=0, 6, 12 months are grouped herein as milestone assays. This data does not reveal any significant degradation in the samples at 5° C. over 12 months (Tables 11A through 11E).

TABLE-US-00019 TABLE 11A Milestone assays performed at t = 0 Formulation Assay (t = 0) 18169-62 18169-64 18169-66 18169-67 18169-72 CE-SDS % Purity 95.68 95.315 95.58 95.435 93.735 (non- % Single Largest 1.75 1.825 1.78 1.8 3.245 reduced) Impurity iCIEF % Main Peak 53 53 53.55 52.85 55.2 % Acidic 41.75 42.3 41.55 42.35 40.15 Isoform % Basic Isoform 5.2 4.75 4.9 4.75 4.65 Osmolality mOsm/kg 368 4330 417 350 417 (Freezing pt) Viscosity (cP) at 25° C. 6.14 6.49 6.73 9.64 46.7 Potency % Relative 103 106 95 96 96 potency (109, 97 (120, 95) (102, 88) (102, 91) (107, 86) (95% UCL, 95% LCL)

TABLE-US-00020 TABLE 11B Milestone assays performed at t = 6 months on formulations stored at 5° C. Formulation Assay (t = 6 M) 5° C. 18169-62 18169-64 18169-66 18169-67 18169-72 CE-SDS % Purity 94.49 94.76 94.82 94.62 92.9 (non- % Single Largest 1.86 1.83 1.8 1.77 3.08 reduced) Impurity iCIEF % Main Peak 51.6 52.1 52.5 49.6 54.3 % Acidic 42.5 41.7 41.6 44.2 40.5 Isoform % Basic Isoform 5.9 6.2 5.9 6.1 5.3 Osmolality mOsm/kg NT NT NT NT NT (Freezing pt) Viscosity (cP) at 5° C. 14 13.9 14.8 24 146.5 Potency % 98 105 96 114 107 (88, 108) (89, 125) (77, 120) (108, 119) (92, 123)

TABLE-US-00021 TABLE 11C Milestone assays performed at t = 6 months on formulations stored at 25° C. Formulation Assay (t = 6 M) 25° C. 18169-62 18169-64 18169-66 18169-67 18169-72 CE-SDS % Purity 90.5 90.4 90.43 91.55 90.04 (non- % Single Largest 2.13 2.11 2.09 2.02 3.48 reduced) Impurity iCIEF % Main Peak 41.6 41.8 42.2 39.6 43.9 % Acidic 48.6 48.3 48 51.8 46.2 Isoform % Basic Isoform 9.8 9.9 9.8 8.6 9.9 Osmolality mOsm/kg NT NT NT NT NT (Freezing pt) Viscosity (cP) at 25° C. 6.3 6.4 6.4 9.3 46.2 Potency % 87 89 92 93 95 (85, 89) (85, 92) (91, 94) (86, 102) (93, 97)

TABLE-US-00022 TABLE 11D Milestone assays performed at t = 12 months on formulations stored at 5° C. Formulation Assay (t = 12 M) 5° C. 18169-62 18169-64 18169-66 18169-67 18169-72 CE-SDS % Purity 95.21 95.03 94.95 94.69 92.88 (non- % Single Largest 1.61 1.7 1.75 1.88 3.44 reduced) Impurity iCIEF % Main Peak 51.6 51.5 51 51.1 53.4 % Acidic 42.5 42.5 42.9 43.2 41.2 Isoform % Basic Isoform 5.9 6 6.1 5.7 5.4 Osmolality mOsm/kg 360 170 181 146 182 (Freezing pt) Viscosity (cP) at 5° C. 15.2 14.4 15 24.5 152.6

TABLE-US-00023 TABLE 11E Milestone assays performed at t = 12 months on formulations stored at 25° C. Formulation Assay (t = 12 M) 25° C. 18169-62 18169-64 18169-66 18169-67 18169-72 CE-SDS % Purity 87.32 87.25 87.24 88.72 87.35 (non- % Single Largest 2.85 2.94 2.92 2.46 4.24 reduced) Impurity iCIEF % Main Peak 33.5 34 34.1 30.3 34.9 % Acidic 55.3 54.9 54.8 60.6 53.3 Isoform % Basic Isoform 11.3 11 11.1 9.1 11.8 Osmolality mOsm/kg 370 169 182 130 174 (Freezing pt) Viscosity (cP) at 25° C. 15.2 6.5 6.71 6.69 9.79
Color, Clarity and pH Data: The visually observed color in all formulations except 18169-72 remained below BY3 (BY4-BY5 or BY3-BY4) throughout the period of 1 year at 5° C. The color of 18169-72 was between BY3-BY4 up to 9 months and was observed to be BY3-BY2 at 12 months. The clarity of all formulations remained below 30 NTU (6-18 NTU or 18-30 NTU) throughout the storage at 5° C.
Conclusion: Formulations 18169-62, 18169-64, 18169-66 and 18169-72 were found to be stable within acceptable limits over 1 year. The trends in the most critical attributes: % total aggregates and Sub-visible particulates (HIAC) over this time period suggest that formulation 18169-64 and 18169-66 are both suitable to be pursued for a pre-filled syringe (PFS) drug product (DP).

Example 3: Characterization of the Viscosity of the Formulation

[0125] The impact of pH and methionine concentration on the viscosity of STX-100 formulation at high concentration was evaluated using a full-factorial design of experiment (DOE) study. The following formulation parameters were varied: [0126] 1) pH: 5.0, 5.5, 6.0 [0127] 2) Methionine: 0, 10, 25 mM [0128] 3) Protein concentration: 150, 220, 240 and 260 mg/mL
The core formulation buffer was: 20 mM Citric acid/Na-citrate, 150 mM arginine HCl, 0.05% PS80.

[0129] The data indicated that the viscosity of STX-100 formulation was not significantly impacted by either the pH or the methionine concentration around a core formulation containing 20 mM Na-citrate/citric acid, 150 mM Arginine-HCl, 0.05% PS-80. Both the 5′C and 25° C. data did not reveal p-values lower than 0.05 for each of the two formulation parameters. The only solution parameter with a significant impact on viscosity was the protein concentration which was expected in the range examined. These results show that the pH and excipient levels can be varied within this design space without negatively impacting the viscosity of the formulation.

Example 4: Drug Product Bracketing Study

[0130] This study was done to examine the effect of increasing the methionine content from 5 mM to 10 mM as well as lowering the polysorbate-80 level from 0.05% to 0.03% on long-term stability attributes.

[0131] The following two formulations were prepared and filled into representative pre-filled syringes (0.8 mL fill in BD Hypak STW 27G PFS).

[0132] Formulation A: 150 mg/mL STX-100, 20 mM Na-citrate/citric acid, pH 5.5, 150 mM Arginine-HCl, 10 mM Methionine, 0.05% polysorbate-80.

[0133] Formulation B: 150 mg/mL STX-100, 20 mM Na-citrate/citric acid, pH 5.5, 150 mM Arginine-HCl, 5 mM Methionine, 0.03% polysorbate-80.

[0134] The results from long-term stability at 2-8° C. displayed equivalent stability based on the trends in % HMW and sub-visible particulates. Stability data was also collected at 25° C. and 40° C. for information purposes. The formulations did not appear significantly different in their oxidized species content.

[0135] Thus, the data shows flexibility in polysorbate-80 and methionine concentration for the formulation.

Example 5: Process Stability Study

[0136] This study assessed the impact of different polysorbate-80 surfactant levels on stability of STX-100 in small-scale DS containers (PC bottles or bags), and representative DP in pre-filled syringes (PFS). The formulation was subjected to two different stresses:

[0137] a) Multiple freeze-thaw cycles (1, 3 and 5 freeze-thaw cycles),

[0138] b) Shaking-induced agitation stress (orbital shaking at 650 rpm for 72 hours at ambient conditions) and

[0139] c) Representative ambient hold-times (selected PS-80 level only).

[0140] The different PS-80 levels selected for evaluations were 0, 0.01, 0.02, 0.05, 0.08, 0.1% w/v in 150 mg/mL STX-100 formulation containing 20 mM Na-citrate/citric acid, pH 5.5, 150 mM Arginine-HCl, 5 mM Methionine. The container closure system used for the evaluations were Polycarbonate bottles (1 mL fill in 5 mL bottle), Small DS bag (30 mL capacity, 5 or 15 mL fill), PFS syringes (BD Hypak 47368319 with plungers (47165919) filled with either 0.8 mL or 0.3 mL at 150 mg/mL or 0.3 mL at 40 mg/mL).

[0141] The product quality attributes examined were: Visible appearance (particulates), Turbidity (A340), % Total aggregates (SEC), Protein concentration (SoloVPE method), and Sub-visible particulates (MFI)

[0142] The results from target drug product fill volume of 0.8 mL at 150 mg/mL STX-100 showed that agitating the STX-100 syringes at 650 rpm for 72 hours at ambient temperature protected from light has minimal impact on the visible particulates as long as there is 0.01% PS-80 present in the formulation. One dust-like particle was observed in the 0.02% PS80 sample but this appears to be environmental. There was only a 0.05-0.1% increase in soluble aggregate after agitation in the formulations containing 0-0.01% PS80 while no observable increase in soluble aggregate in any other formulations. The turbidity data indicated no substantial increase in OD340 for all formulations except the one with 0.1% PS80 indicating some contribution from a relatively high level of PS80. However, the SVP data indicates no substantial particle formation tendency as long as PS80 is present. The process study results suggested that 0.05% w/v was an optimal level of polysorbate-80 to protect the formulation against freeze-thaw stress, agitation stress, and process hold times. A suggested specification for PS80 level for product development purposes is 0.05+/−0.025% w/v.

Example 6: Selection of Formulation

[0143] Based on all the above studies the following STX-100 formulation displayed acceptable stability over long-term storage (1 year at 2-8° C.), worst-case agitation stress (650 rpm for 72 h) and worst-case freeze-thaw stress (5 freeze-thaw cycles): 150 mg/mL STX-100, 20 mM Na-citrate/citric acid, 150 mM Arginine-HCl, 5 mM Methionine, 0.05% w/v polysorbate-80, pH 5.5.

[0144] Based on trends in stability attributes, this formulation guarantees greater than 24 month stability at 2-8° C. in a representative pre-filled syringe product.

Example 7: Stability of STX-100 Formulations Comprising Thiol Group Containing Excipients

[0145] The addition of thiol group containing excipients to an STX-100 formulation reduces aggregation as determined by the development of high molecular weight species during storage.

[0146] The control STX-100 formulation had 150 mg/mL STX-100, 20 mM citrate/citric acid, 150 mM L-Arginine HCl, 5 mM Methionine, 0.05% Polysorbate-80, pH 5.5. The control formulation was spiked with a thiol group containing excipient: GSH. The formulations were stored at 25° C. and 40° C. As shown in FIG. 3, the addition of GSH reduced aggregation during storage.

[0147] Addition of glutathione negatively impacted another antibody, STX200, where an increase in aggregation was observed (FIG. 5). STX 200 is an aglycosylated molecule, demonstrating poor conformational stability at higher temperatures. Hence, unfolding of the molecule exposes the thiol group making it more susceptible to crosslinking with the thiol in glutathione and promoting further aggregation. Glutathione did not have any effect on the aggregation kinetics of SB4 (BENEPALI®, an etanercept biosimilar referencing Enbrel®) at 25° C., but facilitated faster aggregation at 40° C. (FIG. 4).

Example 8: Stability Data for STX-100 Formulations

[0148] Stability study data for 50 and 100 mg/mL STX-100 formulations in 20 mM sodium citrate buffer containing 150 mM Arg.HCL, 5 mM methionine, 0.05% PS80, at pH 5.5 filled into syringes (0.8 mL/syringe) supports stability for 36 months when stored at 2-8° C. This is based on stability data at the long term storage condition of 2-8° C. See Tables 12 and 13 below. Based upon this drug product data, a stability for 36 months can be assigned to a formulation at 70 mg/mL (0.8 mL/syringe) selected to deliver a dose of 56 mg.

TABLE-US-00024 TABLE 12 Stability Data for STX-100 Drug Product at 100 mg/mL in 1 mL Syringe, Stored at 2-8° C. Test/Attribute Acceptance Criteria 0 mo 01 mo 03 mo.sup.2 06 mo.sup.2 09 mo.sup.2 12 mo.sup.2 18 mo Appearance - Clarity (NTU) Report Results 6 NTU < 18 NTU < N/A N/A N/A N/A 6 NTU < Sample < Sample < Sample < 18 NTU 30 NTU 18 NTU Appearance - Clarity: LT 50 Conforms Conforms Conforms N/A N/A N/A N/A Conforms NTU Appearance - Color (BY Scale) Report Results 4 <= 5 <= N/A N/A N/A N/A 5 <= Sample < 3 Sample < 4 Sample < 4 Appearance - Color (BY Scale): Conforms Conforms Conforms N/A N/A N/A N/A Conforms LT BY2 Appearance - Essentially free of Conforms Conforms Conforms N/A N/A N/A N/A Conforms visible particles pH 5.0-6.0 5.6 5.5 N/A N/A N/A N/A 5.4 Protein Concentration (RI) 90-110 mg/ml 100 100 N/A N/A N/A N/A 99 DELFIA Blocking - Binding 75-133% 101 100 N/A N/A N/A N/A 96 relative to Reference Standard icIEF - Lower pl Isoforms (%) Report Results 43.3 39.8 N/A N/A N/A N/A 43.9 icIEF - Main pl Isoform (%) Report Results 53.3 55.1 N/A N/A N/A N/A 48.3 Size Exclusion NMT 5.0% 1.3 1.4 N/A N/A N/A N/A 2.0 Chromatography (SEC) - Aggregates Non-Reducing CE-SDS - Report Results 1.4 1.7 N/A N/A N/A N/A 1.8 Highest Single Impurity (%) Non-Reducing CE-SDS - Total NLT 90.0% 96.5 95.2 N/A N/A N/A N/A 95.6 Purity Endotoxin (USP, EP) - NMT 130.00 EU/ml <8.00 N/S N/S N/A N/A N/A <8.00 Endotoxin Particulates - NLT 10 um NMT 6000 Counts/container 165 N/S N/S N/A N/A N/A 76.59 Particulates - NLT 25 um NMT 600 Counts/container 4 N/S N/S N/A N/A N/A 1.07 Container Closure Integrity - Conforms Conforms.sup.1 N/S N/S N/A N/A N/A Conforms Seal Integrity

TABLE-US-00025 TABLE 13 Stability Data for STX-100 Drug Product at 50 mg/mL in 1 mL Syringe, Stored at 2-8° C. Description: PS TAB-14-10-033 (Cycle 2 drug product} stability data Lot No.: TR-PPD-009928 Stability Protocol: PSTAB-14-10-033 Study Start Date: 22 Oct. 1914 Manufacturing Date 16 Oct. 1914 my CIMS Portocol: TR-PPD-010008 Concentration: 50 mg/mL Manufacturing Site PPD, Cambridge Storage Conditions: 2-8° C. Sample Container PES 1 mL (0.8 mL fill) Time Point (Months and TD Labware LIMS submission # PPD- PPD- 38228 38229 38230 38231 38232 38233 15-6943 15-6944 Test Method Acceptance Criteria.sup.1 0 1 3 6 9 12 18 24 Appearance Color: Report Relults BY6-BY5 BY6-BY5 BY6-BY5 BY6-BY5 BY5-BY4 BY5-BY4 N/A BY7-BY6 Clarity. Report Results 18-30 18-30 18-30 18-30 18-30 18-30 N/A 6-18 Essentialy free of visible N/O N/O N/O N/O N/O N/O N/A N/O particles pH 5.0-6.0 5.5 5.5 5.5 5.8 5.5 5.5 N/A 5.5 Osmolality Report Results 332 NT NT NT NT NT NT NT Protein Conc. 45-55 mg/mL 52 52 51 52 52 51 N/A 52 SEC ≤5.0% Aggregates 1.0 1.1 1. 1.3 1.4 1.4 N/A 1.7 ieIEF Report % Lower pI 43.5 42.8 42.3 42.4 43.8 41.0 N/A 43.0 Isoforms (xx.x %) Report % Main Peak 49.8 50.5 51.2 49.6 48.8 53.1 N/A 50.4 (xx.x %) Report % Higher pI 6.8 6.7 6.5 8.0 7.4 5.9 N/A 6.7 Informs (xx.x %) Non-Redeucing ≥90.0 % purity reuslts 97.1 97.1 96.5 96.6 96.5 96.4 N/A 96.3 CE-SDS Report Highest Single 1.7 1.7 1.7 1.7 1.6 1.8 N/A 1.7 Impurity (x.x %) Reducing Report % purity results 97.1 97.3 96.5 96.7 96.8 96.0 N/A 96.6 CE-SDS Report Highest Single 1.4 1.3 1.3 1.4 1.4 1.4 N/A 1.5 Impurity (x.x %) Potency 75 in  % Binding relative N/A.sup.2 102 98 95 100 N/A 106 to R  Standard PS80 Report % 0.05 NT NT NT NT 0.057 NT 0.05 Oxidation % H2—Ox 5.3 NT 5.8 6.8 NT N/A.sup.2 N/A 7 % H15—Ox 4.9 NT 5.6 7.0 NT N/A.sup.2 N/A 6.6 % H30—Ox 2.9 NT 3.6 4.1 NT N/A.sup.2 N/A 2.5 Subvisible ≥10 μm Particles: Report 77 98 122 41 39 47 N/A 42 Particles Results, Particles/mL (HIAC) ≥25 μm Particles: Report 37 0 5 0 0 0 N/A 0 Results, Particles/mL Subvisible ≥10 μm Particles: Report 197 NT 21 400 150 235 N/A 43 Particles Results, Particles/mL.sup.3 (MFI) ≥25 μm Particles: Report 13 NT 7 59 11 14 N/A 1 Results, Particles/mL.sup.3 Subvisible ≥10 μm Particles ≤ 6000 62 78 98 33 24 38 N/A 34 Particles Particles/Container (HIAC).sup.4 ≥25 μm Particles ≤ 6000 30 0 4 0 0 0 N/A 0 Particles/Container Subvisible ≥10 μm Particles ≤ 6000 158 NT 17 320 120 188 N/A 34 Particles Particles/Container (MFI).sup.4 ≥25 μm Particles ≤ 6000 10 NT 8 47 9 11 N/A 1 Particles/Container .sup.1Based on platform specification and esperimental data. This Acceptance Criteria has not been approved and may be used For Information Only. .sup.2Data is not available due to sample mishandling. .sup.3T0 (0 month) data is from ELE: EXP-8 January 2015-0065; .sup.4Calculated by using particles/mL × fill volume (0.8 mL) N/O: No particulates observed; NT: Not tested according to protocol; N/A: Study was not performed due to program changes indicates data missing or illegible when filed

Other Embodiments

[0149] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.