Modulating antibody effector functions

Abstract

To provide modulation of antibody effector functions.SOLUTION: There are provided methods of modulating Fc gamma Receptor (FcγR)-mediated cytotoxicity of an antibody composition. In exemplary embodiments, the method comprises (1) increasing or decreasing an amount of terminal β-galactose at an N-297 glycosylation site of panitumumab, or increasing or decreasing an amount of panitumumab molecules that comprise G1, G1a, G1b and / or G2 galactosylated glycan at the N-297 site, (2) increasing or decreasing the amount of panitumumab molecules that comprise fucosylated glycan at the N-297 site, or increasing or decreasing the amount of panitumumab molecules that comprise afucosylated glycan at the N-297 site, and (3) increasing or decreasing the amount of panitumumab molecules that comprise high-mannose glycan at the N-297 site.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to modulating the effector functions of therapeutic antibodies. [Background technology]

[0002] Monoclonal antibodies have become widely used as therapeutics to treat a wide range of metabolic, inflammatory, and oncological disease conditions. The most common human antibody subclasses used as biotherapeutics, IgG1 and IgG2, have widely different immunological properties and are usually selected as drug candidates based on the desired mechanism of action. Target cell killing, which may be desirable for cancer indications, seeks to exploit IgG1-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC). While these may be easily mediated by IgG1 antibodies, IgG2 is thought to be inherently incapable of producing such effects (RAVETCH, JV, AND S. BOLLAND. 2001, ANNU. REV. IMMUNOL. 19:275-290). However, it has recently been found that panitumumab, a human IgG2 EGFR antagonist indicated for the treatment of metastatic colorectal cancer, can mediate cytotoxicity (SCHNEIDER-MERCK, J IMMUNOL 2010; 184:512-520). This was shown to be mediated primarily through myeloid cells (monocytes and neutrophils), IgG1 through lymphocyte-derived natural killer (NK) cells, and FcγRIIa, in contrast to conventional ADCC associated with FcγRIIIa.

[0003] In the manufacture of therapeutic monoclonal antibodies, to ensure the required product quality, it is necessary to define and monitor the key quality attributes that affect the safety and efficacy of the product. It is well established that specific glycan structures of IgG1 associated with conserved glycans in the Fc CH2 domain can strongly influence the interaction with FcγRs that mediate C1q binding, which initiates ADCC, ADCP, and CDC (Reusch D, Tejada ML.,GLYCOBIOLOGY 2015;25:1325-34). However, there are no studies investigating the influence of product quality characteristics of IgG2 molecules that affect immune-mediated cytotoxic activity. Fc receptors are key immunoregulatory receptors that connect the antibody-mediated (humoral) immune response to cellular effector functions. Fc gamma receptors on the surface of effector cells (such as natural killer cells, macrophages, or monocytes) bind the Fc region of IgG, which itself binds to the target cell. Upon Fc binding, signaling pathways are triggered that result in the secretion of various substances that mediate the destruction of the target cell. The level of cytotoxic effector function varies with the human IgG subtype. Human IgG1 and IgG3 bind better to FcγR and therefore mediate higher effector functions compared to IgG2 or IgG4 (JEFFERIS, R. 2007, EXPERT OPIN. BIOL. THER. 7:1401-1413; DAERON M, FC RECEPTOR BIOLOGY; ANNU REV IMMUNOL. 1997; 15:203-234). The contribution of IgG2-mediated cytotoxicity in therapeutic efficacy is not fully understood, nor are the quality attributes that affect IgG2-mediated cytotoxicity. Quality attributes that affect IgG2-mediated cytotoxicity and are predictive and therefore suitable for monitoring during the manufacture of IgG2 antibodies are not well established. Therefore, there is a need to understand how certain quality attributes affect IgG2-mediated cytotoxicity and adjust such quality attributes accordingly. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] RAVETCH,JV,AND S.BOLLAND.2001,ANNU.REV.IMMUNOL.19:275-290 [Non-Patent Document 2] SCHNEIDER-MERCK,J IMMUNOL 2010; 184:512-520 [Non-Patent Document 3] REUSCH D,TEJADA ML.,GLYCOBIOLOGY 2015;25:1325-34 Summary of the Invention [Means for solving the problem]

[0005] Given the description herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein, which equivalents are intended to be encompassed by embodiment (E) below.

[0006] E1. A method of modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab comprising increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or increasing or decreasing the amount of panitumumab molecules containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0007] E2. A method of increasing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab comprising increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab or increasing the amount of panitumumab molecules containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0008] E3. The method of E1 or E2, wherein about a 1 percent increase in β-galactose increases FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, e.g., about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent.

[0009] E4. A method of reducing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab comprising decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab or decreasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0010] E5. The method of E1 or E4, wherein about a 1 percent reduction in β-galactose reduces FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, e.g., about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent.

[0011] E6. The method according to any one of E1 to E5, wherein the FcγR is FcγRIIa.

[0012] E7. The FcγR-mediated cytotoxicity is measured using an in vivo assay, such as the KILR™ cytotoxicity assay. The method according to any one of E1 to E6, wherein the cytotoxicity is measured by an in vitro cytotoxicity assay.

[0013] E8. The method according to any one of E1 to E7, wherein the FcγR-mediated cellular cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0014] E9. A method for matching Fc gamma receptor (FcγR) mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab samples; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or by increasing or decreasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is about 35% or less.

[0015] E10. The method of E9, wherein the difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 5% or less.

[0016] E11. The method of E9 or E10, wherein FcγR-mediated cytotoxicity of the panitumumab sample is increased by increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or by increasing the amount of panitumumab molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0017] E12. The method of any one of E9 to E11, wherein about a 1 percent increase in β-galactose increases FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, for example, about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent.

[0018] E13. The method of E9 or E10, wherein FcγR-mediated cytotoxicity of the panitumumab sample is reduced by decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or decreasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0019] E14. The method of any one of E9, E10, or E13, wherein about a 1 percent reduction in β-galactose reduces FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, e.g., about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent.

[0020] E15. The method according to any one of E9 to E14, wherein the FcγR is FcγRIIa.

[0021] E16. The method of any one of E9 to E15, wherein said FcγR mediated cytotoxicity is measured by an in vitro cytotoxicity assay, such as the KILR™ cytotoxicity assay.

[0022] E17. The method according to any one of E9 to E16, wherein the FcγR-mediated cellular cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0023] E18. A method for modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing or decreasing the amount of panitumumab molecules containing a fucosylated glycan at the N-297 site.

[0024] E19. A method for modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing or decreasing the amount of panitumumab molecules containing a non-fucosylated glycan at the N-297 site.

[0025] E20. A method for increasing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing the amount of panitumumab molecules containing a fucosylated glycan at the N-297 site.

[0026] E21. A method for increasing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing the amount of panitumumab molecules comprising a non-fucosylated glycan at the N-297 site.

[0027] E22. The method of any one of E18 to E21, wherein an increase of about 1 percent in fucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent.

[0028] E23. The method of any one of E18-E21, wherein about a 1 percent reduction in nonfucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent.

[0029] E24. A method for reducing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising reducing the amount of panitumumab molecules containing fucosylated glycans at the N-297 site.

[0030] E25. A method for reducing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing the amount of panitumumab molecules comprising a non-fucosylated glycan at the N-297 site.

[0031] E26. The method of any one of E18-E19 and E24-E25, wherein about a 1 percent reduction in fucosylated panitumumab molecules reduces FcγR-mediated cytotoxicity by about 2.70 percent to about 3 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent.

[0032] E27. The method of any one of E18-E19 and E24-E25, wherein an increase of about 1 percent of nonfucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent.

[0033] E28. The method according to any one of E18 to E27, wherein the FcγR is FcγRIIa.

[0034] E29. The method of any one of E18 to E28, wherein said FcγR mediated cytotoxicity is measured by an in vitro cytotoxicity assay, such as a KILR™ cytotoxicity assay.

[0035] E30. The method according to any one of E18 to E29, wherein the FcγR-mediated cellular cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0036] E31. A method for matching Fc gamma receptor (FcγR) mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab samples; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and a reference value is about 35% or less.

[0037] E32. A method for matching Fc gamma receptor (FcγR) mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab samples; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of panitumumab molecules that contain a nonfucosylated glycan at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and a reference value is about 35% or less.

[0038] E33. The method of E31 or E32, wherein the difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 5% or less.

[0039] E34. The method of any one of E31-E33, wherein FcγR-mediated cytotoxicity of the panitumumab sample is increased by increasing the amount of panitumumab molecules comprising a fucosylated glycan at the N-297 site.

[0040] E35. The method of any one of E31-E34, wherein an increase of about 1 percent in fucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent.

[0041] E36. The method of any one of E31-E33, wherein FcγR-mediated cytotoxicity of the panitumumab sample is increased by decreasing the amount of panitumumab molecules comprising a non-fucosylated glycan at the N-297 site.

[0042] E37. The method of any one of E31-E34 and E36, wherein about a 1 percent reduction in nonfucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent.

[0043] E38. The method of any one of E31-E33, wherein FcγR-mediated cytotoxicity of the panitumumab sample is reduced by decreasing the amount of panitumumab molecules comprising a fucosylated glycan at the N-297 site.

[0044] E39. The method of any one of E31-E33 and E38, wherein about a 1 percent reduction in fucosylated panitumumab molecules reduces FcγR-mediated cytotoxicity by about 2.70 percent to about 3 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent.

[0045] E40. The method of any one of E31-E33, wherein FcγR-mediated cytotoxicity of the panitumumab sample is reduced by increasing the amount of panitumumab molecules comprising a non-fucosylated glycan at the N-297 site.

[0046] E41. The method of any one of E31-E33 and E40, wherein an increase of about 1 percent of nonfucosylated panitumumab molecules reduces FcγR-mediated cytotoxicity by about 2.70 percent to about 3 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent.

[0047] E42. The method according to any one of E31 to E41, wherein said FcγR is FcγRIIa.

[0048] E43. The method of any one of E31-E42, wherein said FcγR mediated cytotoxicity is measured by an in vitro cytotoxicity assay, such as a KILR™ cytotoxicity assay.

[0049] E44. The method according to any one of E31 to E43, wherein said FcγR-mediated cellular cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0050] E45. A method of modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab comprising increasing or decreasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0051] E46. A method of increasing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab comprising decreasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0052] E47. The method of E45 or E46, wherein about a 1 percent reduction in high mannose glycans increases FcγR-mediated cytotoxicity by about 1.20 percent to about 1.40 percent, e.g., about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent.

[0053] E48. A method for reducing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site.

[0054] E49. The method of E45 or E48, wherein about a 1 percent reduction in high mannose glycans reduces FcγR-mediated cytotoxicity by about 1.20 percent to about 1.40 percent, e.g., about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent.

[0055] E50. The method according to any one of E45 to E49, wherein the high mannose is mannose-5 (Man-5).

[0056] E51. The method according to any one of E45 to E50, wherein said FcγR is FcγRIIa.

[0057] E52. The method of any one of E45-E51, wherein said FcγR mediated cytotoxicity is measured by an in vitro cytotoxicity assay, such as a KILR™ cytotoxicity assay.

[0058] E53. The method according to any one of E45 to E52, wherein the FcγR-mediated cellular cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0059] E54. A method for matching Fc gamma receptor (FcγR) mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab samples; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and a reference value is about 35% or less.

[0060] E55. The method of E54, wherein the difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 5% or less.

[0061] E56. The method of E54 or E55, wherein FcγR-mediated cytotoxicity of a panitumumab sample is increased by decreasing the amount of panitumumab molecules comprising high mannose glycans at the N-297 site.

[0062] E57. The method of any one of E54-E56, wherein about a 1 percent reduction in high mannose glycans increases FcγR-mediated cytotoxicity by about 1.2 percent to about 1.40 percent, e.g., about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent.

[0063] E58. The method of E54 or E55, wherein FcγR-mediated cytotoxicity of a panitumumab sample is reduced by increasing the amount of panitumumab molecules comprising high mannose glycans at the N-297 site.

[0064] E59. The method of any one of E54, E55, or E58, wherein about a 1 percent increase in high mannose glycans reduces FcγR-mediated cytotoxicity by about 1.2 percent to about 1.40 percent, e.g., about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent.

[0065] E60. The method according to any one of E54 to E59, wherein the high mannose is mannose-5 (Man-5).

[0066] E61. The method according to any one of E54 to E60, wherein said FcγR is FcγRIIa.

[0067] E62. The method of any one of E54-E61, wherein said FcγR mediated cytotoxicity is measured by an in vitro cytotoxicity assay, such as a KILR™ cytotoxicity assay.

[0068] E63. The method according to any one of E54 to E62, wherein the FcγR-mediated cellular cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0069] E64. A method of modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising: (i) increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or increasing or decreasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site; (ii) increasing or decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or increasing or decreasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site, and / or (iii) increasing or decreasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0070] E65. A method for increasing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising: (i) increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or increasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site; (ii) increasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or decreasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site, and / or (iii) reducing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0071] E66. A method of reducing Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising: (i) decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or decreasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site; (ii) decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or increasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site, and / or (iii) increasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0072] E67. An antibody composition produced by the method according to any one of E1 to E66.

[0073] E68. A pharmaceutical composition comprising the antibody composition of E67 and a pharma- ceutically acceptable carrier, diluent or excipient. [Brief description of the drawings]

[0074] [Figure 1A]FIG. 1A is an illustration of the three types of N-glycans (oligomannose, complex and hybrid) and the symbols commonly used for such saccharides. [Figure 1B] Figure 1B shows the predominant N-linked glycan found in human IgG at N-glycosylation site asparagine (Asn) 297, with representative linkages of oligosaccharide structures. These glycans typically contain a heptasaccharide core and outer arms constructed by the variable addition of fucose, N-acetylglucosamine (GlcNAc), galactose, sialic acid (SA), and bisected N-GlcNAc. [Figure 1C] FIG. 1C summarizes the structures of the three major glycan species evaluated in the cytotoxicity reporter gene assay, including nonfucosylated species (i.e., species lacking core fucose, including G0 or G1), high mannose species (including M5 species), or terminal β-galactose species (i.e., terminal β-galactose, including G1F or G2F). [Figure 1D] FIG. 1D provides a basic schematic flow chart summarizing the glycan enrichment and preparation of engineered samples. [Figure 1E] FIG. 1E is a diagram of the salvage and de novo pathways of fucose metabolism. In the salvage pathway, free L-fucose is converted to GDP-fucose, whereas in the de novo pathway, GDP-fucose is synthesized through three reactions catalyzed by GMD and FX. GDP-fucose is then transported from the cytosol to the lumen of the Golgi by GDP-Fuc transferase and transferred to acceptor oligosaccharides and proteins. The other reaction product, GDP, is converted to guanosine 5-monophosphate (GMP) and inorganic phosphate (Pi) by luminal nucleotide diphosphatase. The former is exported to the cytosol (via an antiport system coupled to the transport of GDP-fucose), whereas the latter is assumed to leave the Golgi lumen via the Golgi anion channel, GOLAC. See, for example, Nordeen et al. 2000; Hirschberg et al. 2001. [Figure 2A-2B]Figure 2A shows FcγRIIa signaling activity as a function of β-galactosylation level. Figure 2B shows a representative dose-response curve overlay. A linear regression line (equation shown) was fitted to the plot of measured activity (Figure 2A) and representative dose-response curves were shown for various activity levels in a correlated line graph (Figure 2B). This data demonstrated the quantitative nature and range of the ADCC reporter gene assay and its suitability for the assessment of quality attributes that affect ADCC activity. Higher levels of β-galactose generally result in higher cytotoxicity. [Figure 3A] FIG. 3A shows FcγRIIa signaling activity as a function of defucosylation level. [Figure 3B] Figure 3B shows a representative dose-response curve overlay. A linear regression line (equation shown) was fitted to the plot of measured activity (Figure 3A) and representative dose-response curves were shown for various activity levels in a correlated line graph (Figure 3B). Higher levels of defucosylation generally result in lower cytotoxicity. [Figure 4A-4B] Figure 4A shows FcγRIIa signaling activity as a function of high mannose levels. Figure 4B shows a representative dose-response curve overlay. A linear regression line (equation shown) was fitted to the plot of measured activity (Figure 4A) and representative dose-response curves were shown for various activity levels in a correlated line graph (Figure 4B). Higher levels of high mannose generally result in lower cytotoxicity. [Diagram 5] FIG. 5 shows representative dose curves of PBMC-mediated cellular cytotoxicity by enriched glycan samples using the KILR assay. [Figure 6A-6B] Figures 6A-6D are graphs showing PBMC-mediated cytotoxicity as a function of panitumumab nonfucosylation levels from donors with (A) HHVV, (B) HHFF, (C) RRFV, and (D) HHFV polymorphisms of the FcγRIIa and FcγRIIIa receptors, respectively. [Figure 6C-6D] Same as above. [Figure 7A-7B]Figures 7A-7D are graphs showing PBMC-mediated cytotoxicity as a function of panitumumab high mannose levels from donors with (A) HHVV, (B) HHFF, (C) RRFV, and (D) HHFV polymorphisms of the FcγRIIa and FcγRIIIa receptors, respectively. [Figure 7C-7D] Same as above. [Figure 8A-8B] Figures 8A-8D are graphs showing PBMC-mediated cytotoxicity as a function of panitumumab β-galactose levels from donors with (A) HHVF, (B) RRVF, (C) HHVV, and (D) RRFF polymorphisms of the FcγRIIa and FcγRIIIa receptors, respectively. [Fig. 8C-8D] Same as above. [Figure 9A-9B] Figure 9A is a representative panitumumab dose curve of PBMC cytotoxicity. Figure 9B is a plot showing FcγR blocking using antibodies to the indicated receptors or controls. [Figure 10A-10B] 10A-10C are SPR sensograms showing that panitumumab or control binds to (A) FcγRI at up to 10 μM antibody; (B) FcγRIIIa-158F at up to 10 μM antibody; and (C) FcγRIIa-131H at up to 10 μM antibody. [Figure 10C] Same as above. [Figure 11A-11B] 11A-11B are SPR equilibrium binding curves of panitumumab binding to huFcgRIIa-131H with apparent KDs of approximately 7.9 μM and 8 μM for (A) the fucose-enriched sample and (B) the non-fucose-enriched sample, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] 1. Overview IgG1 and IgG2, the most common human antibody subclasses used as biotherapeutics, have very different immunological properties. General effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) may be an important mechanism of action for IgG1 antibodies. Previously, IgG2 antibodies were not known to exhibit effector functions. However, it has been shown recently that panitumumab can mediate cytotoxic effects similar to ADCC by binding to FcγRIIa. This is in contrast to conventional ADCC, which is mediated by IgG1 binding to FcγRIIIa.

[0076] Panitumumab is a human IgG2 monoclonal antibody that binds to the human epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1, and HER1). Panitumumab has a molecular weight of approximately 147 kD. The sequences of the heavy and light chains are shown in Table 1 as SEQ ID NOs: 1 and 2, respectively. Panitumumab has two N-glycosylation sites located in the second constant domain of each heavy chain. The N-glycosylation site is commonly referred to as residue N-297 according to Kabat EU numbering. The actual residue number is residue 295 of SEQ ID NO: 1.

[0077] As described and exemplified herein, the inventors have conducted extensive research into the mechanisms by which panitumumab cytotoxicity is mediated and the product quality attributes that influence the FcγR-mediated cytotoxicity level of panitumumab. The inventors discovered that cytotoxicity is primarily mediated by myeloid cells (monocytes, macrophages and neutrophils). The inventors then investigated the impact of different glycans in this IgG2 molecule on effector function. Using a highly sensitive cytotoxicity assay combined with glycoengineered forms of panitumumab, the inventors discovered that the impact of different glycans on FcγRIIa-mediated cytotoxicity is substantial and variable depending on the glycoform.

[0078] For example, galactosylation at the N-297 site was positively correlated with reporter genes, whereas non-fucosylation and high mannose levels at the N-297 site were inversely correlated with cell killing. Therefore, FcγR-mediated cytotoxicity of panitumumab can be increased by (1) increasing galactosylation at the N-297 site; (2) decreasing non-fucosylation levels at the N-297 site; and / or (3) decreasing high mannose levels at the N-297 site. Conversely, FcγR-mediated cytotoxicity of panitumumab can be decreased by (1) decreasing galactosylation at the N-297 site; (2) increasing non-fucosylation levels at the N-297 site; and / or (3) increasing high mannose levels at the N-297 site.

[0079] Panitumumab is currently produced in genetically engineered mammalian (Chinese Hamster Ovary) cells. During the recombinant production process, glycan moieties are attached to the antibody by post-translational modification. The discovery made by the inventors herein provides a quantifiable relationship between the glycoform profile of panitumumab and its cytotoxicity. This discovery can be used to adjust the glycosylation pattern during the CHO cell production process so that the cytotoxicity level meets the desired reference level.

[0080] 2.Definition "Panitumumab" (trade name Vectibix®) refers to a human monoclonal antibody comprising a heavy chain comprising SEQ ID NO: 1 and a light chain comprising SEQ ID NO: 2. The amino acid sequences of the heavy and light chains of panitumumab are shown in Table 1. The nucleic acid sequences encoding SEQ ID NOs: 1 and 2 are shown as sequences 3 and 4, respectively. As shown in the Examples, the glycan profile of panitumumab can vary.

[0081] [Table 1]

[0082] [Table 2]

[0083]

Table 3

[0084] The terms "glycan", "glycans", "glycoform" or "glycoforms" refer to oligomers of monosaccharide species linked by various glycosidic bonds. Examples of monosaccharides commonly found in mammalian N-linked glycans include hexose (Hex), glucose (Glc), galactose (Gal), mannose (Man) and N-acetylglucosamine (GlcNAc). The major N-glycan species found on recombinant IgG2 antibodies include fucose, galactose, mannose, sialic acid and GlcNAc, as shown in Figures 1B, 1C and Table 2. In the case of panitumumab, the glycan oligosaccharide structure is attached to the N-glycosylation site at Asn-297 (Kabat EU numbering) and generally consists of a seven-saccharide core with outer arms constructed by variable addition of fucose, N-acetylglucosamine (GlcNAc), galactose, sialic acid (SA), and bisected N-GlcNAc. Each of the potential oligosaccharide structures can be abbreviated as follows: G0, G1, or G2 refer to the core GlcNAc and mannose oligosaccharide structure with 0, 1, or 2 terminal galactose molecules, respectively. Within G1, there can be two additional structures, abbreviated G1a and G1b, where G1a or G1b indicates whether the terminal galactose group is attached to the 6-arm or 3-arm of the core structure. See Figures 1B and 1C. If fucosylated (i.e., when a fucose group is attached to the core glycan structure), the G0, G1 (G1a / G1b) or G2 types can be abbreviated as G0F, G1F (G1aF / G1bF) or G2F. If sialic acid is present, these abbreviations include "S", e.g., G2FS2 refers to a glycan having two galactose, fucose and two sialic acid groups. There may also be additional glycans linked to the antibody, including high mannose (HM) structures formed by the incorporation of additional mannose groups (e.g., "Man5" or "M5" as shown in FIG. 1C and Table 2).As used herein, the term "glycan" or "glycans" refers to any of the oligomers of monosaccharide species described herein, or any other oligomers of monosaccharide species linked to an antibody.

[0085] The N-glycosylation site of IgG2 (located in the second constant domain of the heavy chain) is typically referred to as N-297 based on the EU numbering system. A complete chart comparing the different numbering systems is provided by the International Immunogenetics Information System ("IMGT Scientific Chart"). The IMGT Scientific Chart refers to IgG1, and the corresponding number for IgG2 can be easily obtained by aligning the respective sequences.

[0086] The terms "terminal β-galactose", "galactosylated glycan" or "G1, G1a, G1b and / or G2 galactosylated glycan" refer to a glycan that comprises one or two galactose molecules linked to an IgG antibody at the N-glycosylation site (Asn-297) via an N-acetylglucosamine moiety that is linked to a core mannose structure. Exemplary glycans that comprise "terminal β-galactose", "galactosylated glycan" or "G1, G1a, G1b and / or G2 galactosylated glycan" are shown in Figures 1B and 1C. In some embodiments, the G1, G1a, G1b and / or G2 galactosylated glycans may or may not comprise a core fucose.

[0087] The term "core fucose" or "fucosylated species" refers to a glycan comprising a fucose molecule (alpha 1-6) linked to an IgG antibody at the N-glycosylation site (Asn-297) via an N-acetylglucosamine moiety linked to a core mannose structure. Exemplary glycans comprising "core fucose" or "fucosylated glycans" are shown in Figures 1B and 1C. In some embodiments, antibodies containing core fucose and / or fucosylated glycans may or may not contain other glycans (including terminal β-galactose and / or high mannose glycans).

[0088] The terms "nonfucosylated," "nonfucosylated glycan," or "nonfucosylated" refer to the removal or absence of core fucose on an antibody. Exemplary nonfucosylated glycans are shown in Figures 1B and 1C. In some embodiments, antibodies lacking core fucose may or may not include other glycans (including terminal β-galactose and / or high mannose glycans). Nonfucosylated glycoforms include, but are not limited to, A1G0, A1G1a, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. See, e.g., Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015).

[0089] The term "high mannose", "high mannose glycan" or "HM" refers to a glycan containing more than three mannose molecules linked to an IgG antibody at the N-glycosylation site (Asn-297). Exemplary high mannose antibodies are shown in FIG. 1C and Table 2, including the "Man-5 high mannose glycan" that contains two additional mannose molecules. High mannose glycans encompass glycans containing 5, 6, 7, 8 or 9 mannose residues, abbreviated as Man5 or M5, Man6 or M6, Man7 or M7, Man8 or M8, and Man9 or M9, respectively. Exemplary structures of Man6, Man7, and Man8 are shown below.

[0090] [Table 4]

[0091] The term "FcγR" or "Fc-gamma receptor" refers to a protein belonging to the IgG superfamily that is involved in inducing phagocytosis of opsonized cells or microorganisms. See, e.g., Fridman WH. Fc receptors and immunoglobulin binding factors. FASEB Journal. 5(12):2684-90 (1991). Members of the Fc gamma receptor family include FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). The sequences of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcγRIIIB can be found in many sequence databases, for example the Uniprot database (www.uniprot.org) under accession numbers P12314 (FCGR1_HUMAN), P12318 (FCG2A_HUMAN), P31994 (FCG2B_HUMAN), P08637 (FCG3A_HUMAN), and P08637 (FCG3A_HUMAN), respectively.

[0092] The terms "a", "an" and "the" and similar referents with respect to the description of this disclosure (especially with respect to the claims below) as used herein should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to" and allowing for the presence of one or more features or components) unless otherwise specified. The terms "a" (or "an"), as well as "one or more" and "at least one" can be used interchangeably herein. Furthermore, "and / or" as used herein should be construed as a specific disclosure of each of two specific features or components (with or without the other feature or component). As such, the term "and / or," when used herein in phrases such as "A and / or B," is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0093] The term "about" as used in connection with numerical values ​​throughout the specification and claims indicates an interval of accuracy well known and accepted by those of skill in the art. Typically, such an interval of accuracy is ±10%.

[0094] 3. Post-translational Glycosylation and FcγR-Mediated Cytotoxicity 3.1 Post-translational glycosylation Many secreted proteins are post-translationally glycosylated, a process by which sugar moieties (e.g., glycans, sugars) are covalently attached to specific amino acids of proteins. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which the glycan is linked to an asparagine at the recognition sequence Asn-X-Thr / Ser (where "X" is any amino acid except proline), and (2) O-linked glycosylation, in which the glycan is linked to a serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), there is a wide range of glycan structures attached to each site (O or N), so there is microheterogeneity in protein glycoforms. For IgG2 antibodies, N-linked glycosylation occurs at the asparagine-297 (N-297) site (Eu numbering system). For panitumumab, the actual position of this asparagine occurs at residue number 295, but nevertheless the N-glycosylation site is commonly referred to as N-297 to be consistent with the EU numbering system.

[0095] All N-glycans have a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr (Man3GlcNAc2Asn) and are classified into one of three types: (A) high mannose (HM) or oligomannose (OM) types, consisting of two N-acetylglucosamine (GalNAc) moieties and multiple (e.g., 5, 6, 7, 8, or 9) mannose (Man) residues, (B) complex types, containing three or more GlcNAc moieties and any number of other sugar types, or (C) hybrid types, containing a Man residue on one branch and a GlcNAc at the base of the complex branch. Figure 1A (Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2 nd ed., Cold Spring Harbor Laboratory Press; 2009) shows three types of N-glycans:

[0096] N-linked glycans typically contain one or more monosaccharides of galactose (Gal), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), mannose (Man), N-acetylneuraminic acid (Neu5Ac), and fucose (Fuc). Commonly used symbols for such sugars are shown in Figure 1A.

[0097] The sugar composition and structural configuration of the glycan structures vary depending, inter alia, on the glycosylation machinery in the ER and Golgi apparatus, the accessibility of the glycan structures to the enzymes of the machinery, the order of action of each enzyme, and the stage at which the protein is released from the glycosylation machinery. Controlling the glycan structure is important in the recombinant production of therapeutic monoclonal antibodies, since the glycan structure attached to the Fc domain influences the interaction with FcγR, which mediates cytotoxicity.

[0098] 3.2 Glycans that influence FcγR-mediated cytotoxicity The present disclosure elucidates the effect of various glycans (e.g., containing β-galactose, core-fucose and / or high mannose) on FcγR-mediated cytotoxicity of IgG2 antibodies such as panitumumab. Accordingly, the present disclosure provides a method for modulating Fc gamma receptor (FcγR)-mediated cytotoxicity of an IgG2 antibody (such as panitumumab) or a composition comprising the antibody (antibody composition). In an exemplary embodiment, the method comprises modulating the amount of (a) a galactosylated glycan of the antibody, (b) a non-fucosylated glycan of the antibody, (c) a high mannose glycan of the antibody, or (d) a combination thereof. Without being bound to a particular theory, it is believed that the methods disclosed herein provide a means to tailor-made compositions containing specific amounts of specific glycoforms of a given antibody that exhibit a target level of FcγR-mediated cytotoxicity. Particularly relevant glycan structures are shown in FIG. 1C.

[0099] In an exemplary aspect, a method provided by the present disclosure relates to modulating an IgG2 antibody (such as panitumumab), or a composition comprising the antibody (antibody composition), where a step is taken to achieve a desired or predetermined reference level of glycoforms of the IgG2 antibody, such that the antibody or antibody composition exhibits a desired or predetermined reference level of FcγR-mediated cytotoxicity. In an exemplary embodiment, the method comprises modulating (increasing or decreasing) the amount of (a) galactosylated glycans, (b) non-fucosylated glycans, (c) high mannose glycans, or (d) a combination thereof, of the IgG2 antibody (such as panitumumab) to modulate (increase or decrease) antibody-induced or stimulated FcγR-mediated cytotoxicity. In an exemplary embodiment, the method comprises modulating (increasing or decreasing) the amount of a glycoform, e.g., (a) a galactosylated glycoform, (b) a non-fucosylated glycoform, (c) a high mannose glycoform; or (d) a combination thereof, to modulate (increase or decrease) antibody-induced or stimulated FcγR-mediated cytotoxicity.

[0100] The term "amount", particularly when referring to the amount of glycan (e.g., including (1) the amount of terminal β-galactose, (2) the amount of G1, G1a, G1b and / or G2 galactosylated glycans, (3) the amount of core fucose, (4) the amount of fucosylated glycans, (5) the amount of non-fucosylated glycans, (6) the amount of high mannose glycans, and / or (7) the amount of Man-5 glycans), refers to the relative percentage of a particular glycan at N-297 compared to the total amount of glycans at N-297. Since it is impractical / impossible to count glycan species at the individual molecule level, the amount of glycan content described herein is generally calculated based on the relative percentage by commonly used analytical methods. For example, as illustrated in Example 2.2, an enzyme is used to release all N-glycans from a protein, and the glycans are then separated by hydrophobic interaction liquid chromatography (HILIC). HILIC results in various peaks, each peak representing a glycan species. The amount of a particular glycan is calculated as a relative percentage based on the area of ​​that peak out of the total area of ​​all peaks, and therefore, unless otherwise stated, the amount of a glycan refers to the relative percentage of that particular glycan species out of all N-glycans at the N-297 site using any of the commonly used analytical methods (e.g., HPAEC, CE-SDS, HILIC, or LC-MS).

[0101] Methods for measuring and determining the amount or relative percentage of glycans (e.g., including terminal β-galactose, G1, G1a, G1b and / or G2 galactosylated glycans, core fucose, fucosylated glycans, non-fucosylated glycans, high mannose glycans, and / or Man-5 glycans) are well known in the art and include, for example, hydrophilic interaction liquid chromatography (HILIC) as described in the Examples. See also Pace et al., Characterizing the Effect of Multiple Fc Glycan Attributes on the Effector Functions and FcγRIIIa Receptor Binding Activity of an IgG1 Antibody, Biotechnol. Prog., 2016, Vol. 32, No. 5 pages 1181-1192, and Shah, B. et al. LC-MS / MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins J. Am. Soc. Mass Spectrom. (2014) 25:999, each of which is incorporated by reference herein for all purposes. In some embodiments, the amount can be determined or calculated as a mole percent incorporation.

[0102] In some aspects, the methods disclosed herein comprise modulating the amount of terminal β-galactose, core fucose, or high mannose, or a combination thereof, attached to a particular IgG2 molecule (such as panitumumab).

[0103] For example, the method may include increasing the amount of terminal β-galactose on IgG2 (such as panitumumab) (e.g., by effectively changing the glycan from G0 to G1 or G2, or from G1 to G2) to increase FcγR-mediated cytotoxicity. Alternatively, FcγR-mediated cytotoxicity may be increased by increasing the amount of antibody molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. Also, for example, the method may include decreasing the amount of terminal β-galactose on IgG2 (such as panitumumab) (e.g., by effectively changing the glycan from G0 to G1 or G2, or from G1 to G2) to decrease FcγR-mediated cytotoxicity. Alternatively, FcγR-mediated cytotoxicity may be decreased by decreasing the amount of antibody molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0104] In other exemplary aspects, the method may include increasing the amount of core fucose on IgG2 (such as panitumumab) to increase FcγR-mediated cytotoxicity. FcγR-mediated cytotoxicity may be increased by increasing the amount of antibody molecules that contain fucosylated glycans at the N-297 site or decreasing the amount of antibody molecules that contain non-fucosylated glycans at the N-297 site. The method may also include decreasing the amount of core fucose on IgG2 (such as panitumumab) to decrease FcγR-mediated cytotoxicity. FcγR-mediated cytotoxicity may be decreased by decreasing the amount of antibody molecules that contain fucosylated glycans at the N-297 site or increasing the amount of antibody molecules that contain non-fucosylated glycans at the N-297 site.

[0105] In other exemplary embodiments, the method may include decreasing the amount of high mannose (e.g., Man-5) on IgG2 (such as panitumumab) to increase FcγR-mediated cytotoxicity. FcγR-mediated cytotoxicity may be increased by decreasing the amount of antibody molecules that contain high mannose glycans at the N-297 site. The method may also include increasing the amount of mannose (e.g., Man-5) on IgG2 (such as panitumumab) to decrease FcγR-mediated cytotoxicity. FcγR-mediated cytotoxicity may be decreased by increasing the amount of antibody molecules that contain mannose (e.g., Man-5) at the N-297 site.

[0106] 3.3 Regulation of FcγR-mediated cytotoxicity Fc gamma receptors exist in two distinct classes, those that activate cells upon cross-linking ("activating FcR") and those that inhibit activation upon co-binding ("inhibitory FcR"). In humans, there are two low affinity activating FcRs for IgG, FcγRIIa and FcγRIIIa. FcγRIIa (or FcγRIIA) is a single-chain low affinity receptor for IgG with an ITAM sequence in the cytoplasmic tail. It is expressed on macrophages, mast cells, monocytes, neutrophils, and some B cells. It has an ITIM sequence in the cytoplasmic domain and is 90% homologous in the extracellular domain to the human inhibitory FcRIIb molecule, which is expressed on B cells, macrophages, mast cells, neutrophils, and monocytes, but not on NK cells or T cells. FcγRIIIa (or FcγRIIIA) is an oligomeric activating receptor consisting of a ligand-binding subunit and ITAM containing gamma or zeta subunits. It is expressed on NK cells, macrophages, and mast cells. It is not expressed on neutrophils, B cells, or T cells. In addition, a receptor with over 95% sequence identity in the extracellular domain, called FcRIIIb, is found on human neutrophils as a GPI-anchored protein. In the absence of binding to ITAM-containing receptors such as FcRIIa, it can bind immune complexes but cannot activate cells. FcRII and FcRIII are approximately 70% identical in their ligand-binding extracellular domains.

[0107] Thus, in humans, IgG cytotoxic antibodies interact with four different low affinity receptors - two of which are FcRIIa and FcRIIIa that can activate cellular responses, one of which is inhibitory FcRIIb, and one of which is FcRIIIb that binds to IgG complexes but does not trigger cellular responses. Macrophages express FcRIIa, FcRIIb, and FcRIIIa, neutrophils express FcRIIa, FcRIIb, and FcRIIIb, while NK cells express only FcRIIIa. Thus, the efficacy of therapeutic anti-tumor antibodies depends on their specific interactions with activating, inhibitory, and inactive low affinity FcRs that are differentially expressed on different cell types.

[0108] Well-defined tumor models are known for studying the cytotoxicity of therapeutic antitumor antibodies. For example, Matui et al. described an in vitro system using A431 cells and an in vivo system using A431 cell xenografts in athymic mice to study the cytotoxicity of IgG1 and IgG2 antibodies that bind to EGFR.

[0109] In certain aspects, the FcγR-mediated cytotoxicity described herein is mediated by FcγRIIa.

[0110] In a specific embodiment, the FcγR-mediated cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0111] In certain embodiments, FcγR-mediated cytotoxicity as described herein is measured or determined using an FcγR reporter gene assay. In certain embodiments, the reporter gene assay comprises Jurkat cells. In certain embodiments, the reporter gene assay comprises Jurkat cells expressing FcγR receptors, NFAT response elements, and / or reporter genes. The reporter gene can be any gene whose expression provides a measurable signal. Exemplary reporter genes include green fluorescent protein (GFP), antibiotic resistance proteins (e.g., chloramphenicol transferase), virulence proteins (e.g., GATA-1 DNA binding domain, colicin lysis protein), β-galactosidase, E. coli β-galactosidase (LacZ), Halobacterium β-galactosidase, Neuropsora tyrosinase, human placental alkaline phosphatase, chloramphenicol acetyltransferase (CAT), aequorin (jellyfish bioluminescence), firefly luciferase (EC 1.13.12.7) from the American firefly, Photinus pyralis, Renilla luciferase (EC 1.13.12.7) from the sea pansy, Renilla reniformis, Examples of reporter genes include genes encoding luciferase from Photobacterium fischeri (EC 1.13.12.5), and bacterial luciferase from Photobacterium fischeri (EC 1.14.14.3). A variety of other reporter genes are known to those of skill in the art. In an exemplary embodiment, the reporter gene encodes luciferase.

[0112] In certain embodiments, FcγR-mediated cytotoxicity as described herein is measured using an ADCC assay kit, such as that commercially available from Promega (catalog number G7010 or G7018) under the name "ADCC Reporter Bioassay."

[0113] In certain aspects, the disclosure provides methods of increasing FcγR-mediated cytotoxicity of an IgG2 antibody (such as panitumumab) or a composition comprising the antibody, compared to a control or reference value. In exemplary embodiments, the increase is an increase of at least or about 0.1% to about 100% (e.g., an increase of at least or about 0.1%, an increase of at least or about 0.2%, an increase of at least or about 0.3%, an increase of at least or about 0.4%, an increase of at least or about 0.5%, an increase of at least or about 0.55%, an increase of at least or about 0.6%, an increase of at least or about 0.65%, an increase of at least or about 0.7%, an increase of at least or about 0.75%, an increase of at least or about 0.8%, an increase of at least or about 0.9%, an increase of at least or about 1%, an increase of at least or about 1.2%, an increase of at least or about 1.25%, an increase of at least or about 1.2% or ... or about a 1.3% increase, at least or about a 1.35% increase, at least or about a 1.4% increase, at least or about a 1.5% increase, at least or about a 2% increase, at least or about a 2.5% increase, at least or about a 2.7% increase, at least or about a 2.75% increase, at least or about a 2.8% increase, at least or about a 2.85% increase, at least or about a 2.9% increase, at least or about a 2.95% increase, at least or about a 3% increase, at least or about a 4% increase, at least or about a 5% increase, at least or about a 6% increase, at least or about a 7% increase, at least or about a 8% increase, at least or about a 9% increase, at least or about a 10 ...5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about an 80% increase, at least or about an 85% increase, at least or about a 90% increase, at least or about a 95% increase, or at least or about a 100% increase). In exemplary embodiments, the increase is greater than 100%, e.g., at least or about 125%, at least or about 150%, at least or about 175%, at least or about 200%, at least or about 300%, at least or about 400%, at least or about 500%, at least or about 600%, at least or about 700%, at least or about 800%, at least or about 900%, or at least or about 1000% compared to a control or reference value. In exemplary embodiments, FcγR-mediated cytotoxicity of the antibody or composition comprising the antibody is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, or at least about 1.9-fold compared to a control or reference value. In exemplary embodiments, the FcγR-mediated cytotoxicity of the antibody or composition comprising the antibody is increased by at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold compared to a control or reference value. In exemplary embodiments, the FcγR-mediated cytotoxicity of the antibody or composition comprising the antibody is increased by at least about 1.The increase is from 1 to about 10 times, from about 1.2 to about 10 times, from about 1.3 to about 10 times, from about 1.4 to about 10 times, from about 1.5 to about 10 times, from about 1.1 to about 5 times, from about 1.2 to about 5 times, from about 1.3 to about 5 times, from about 1.4 to about 5 times, or from about 1.5 to about 5 times.

[0114] In certain aspects, the disclosure provides methods of reducing FcγR-mediated cytotoxicity of an IgG2 antibody (such as panitumumab) or a composition comprising the antibody, compared to a control or reference value. In exemplary embodiments, the reduction is a reduction of at least or about 0.1% to about 100% (e.g., at least or about 0.1% reduction, at least or about 0.2% reduction, at least or about 0.3% reduction, at least or about 0.4% reduction, at least or about 0.5% reduction, at least or about 0.55% reduction, at least or about 0.6% reduction, at least or about 0.65% reduction, at least or about 0.7% reduction, at least or about 0.75% reduction, at least or about 0.8% reduction, at least or about 0.9% reduction, at least or about 1% reduction, at least or about 1.2% reduction, at least or about 1.25% reduction, at least or about 1.2 ... or about a 1.3% decrease, at least or about a 1.35% decrease, at least or about a 1.4% decrease, at least or about a 1.5% decrease, at least or about a 2% decrease, at least or about a 2.5% decrease, at least or about a 2.7% decrease, at least or about a 2.75% decrease, at least or about a 2.8% decrease, at least or about a 2.85% decrease, at least or about a 2.9% decrease, at least or about a 2.95% decrease, at least or about a 3% decrease, at least or about a 4% decrease, at least or about a 5% decrease, at least or about a 6% decrease, at least or about a 7% decrease, at least or about a 8% decrease, at least or about a 9% decrease, at least or about a 10 ...a 5% decrease, at least or about a 10% decrease, at least or about a 15% decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about a 80% decrease, at least or about a 85% decrease, at least or about a 90% decrease, at least or about a 95% decrease, or at least or about a 100% decrease). In exemplary embodiments, the decrease is greater than 100%, e.g., at least or about 125%, at least or about 150%, at least or about 175%, at least or about 200%, at least or about 300%, at least or about 400%, at least or about 500%, at least or about 600%, at least or about 700%, at least or about 800%, at least or about 900%, or at least or about 1000%, as compared to a control or reference value. In exemplary embodiments, FcγR-mediated cytotoxicity of the antibody or composition comprising the antibody is decreased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, or at least about 1.9-fold as compared to a control or reference value. In exemplary embodiments, FcγR-mediated cytotoxicity of the antibody or composition comprising the antibody is reduced by at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold compared to a control or reference value. In exemplary embodiments, FcγR-mediated cytotoxicity of the antibody or composition comprising the antibody is reduced by at least about 1.It decreases by about 1 to about 10 times, about 1.2 to about 10 times, about 1.3 to about 10 times, about 1.4 to about 10 times, about 1.5 to about 10 times, about 1.1 to about 5 times, about 1.2 to about 5 times, about 1.3 to about 5 times, about 1.4 to about 5 times, or about 1.5 to about 5 times.

[0115] As used herein, a "control" or "reference value" herein refers to the level of FcγR-mediated cytotoxicity of an antibody or a composition comprising an antibody before experimental intervention aimed at modulating the glycan profile (such as the level of cytotoxicity when first measured). If an antibody or a composition comprising an antibody has undergone experimental intervention aimed at modulating the glycan profile, but additional modulation is desired, the "control" or "reference value" may be the level of FcγR-mediated cytotoxicity before any additional experimental intervention aimed at further modulating the glycan profile.

[0116] In certain embodiments, the reference value is the level of FcγR-mediated cytotoxicity exhibited by a commercially available panitumumab sample at the same dose (e.g., the same amount of antibody molecule). In certain embodiments, the reference value is a predetermined level that provides a therapeutic effect.

[0117] In certain aspects, the present disclosure provides methods for increasing the amount of a particular glycan (e.g., galactosylated glycan, G1, G1a, G1b and / or G2 galactosylated glycan, fucosylated glycan, non-fucosylated glycan, core fucose, high mannose glycan, Man-5 glycan, or a combination thereof) of an antibody to at least or about 0.5%, at least or about 1%, at least or about 2%, at least or about 3%, at least or about 5%, at least or about 7%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45% of the total amount. , at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, about 0.5% to about 98%, about 0.5% to about 98%, about 0.5% to about 98%, about 0.1% to about 99%, about 0.5% to about 98%, about 0.5% to about 95%, about 1% to about 90%, about 1% to about 85%, about 5% to about 85%, about 10% to about 85%, or about 10% to about 80%. As mentioned above, when describing a particular glycan species, the percentage generally refers to the relative percentage of the particular glycan species in the total glycan content at the N-297 site, calculated according to any of the art-recognized analytical methods (HILIC, LC-MS, etc.). In one exemplary embodiment, the relative percentage is calculated according to the area of ​​the chromatographic peak.

[0118] In certain aspects, the disclosure provides methods of modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab comprising increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or increasing or decreasing the amount of panitumumab molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0119] In certain embodiments, the disclosure provides a method of increasing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab comprising increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab or increasing the amount of panitumumab molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. In certain embodiments, an increase in β-galactose of about 1 percent increases FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, e.g., about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent. As stated above, the percentage of β-galactose, or G1, G1a, G1b, G2 galactosylated glycans refers to the relative percentage of the relative glycan species within the total glycan content at the N-297 site.

[0120] When quantifying the relationship between FcγR-mediated cytotoxicity and various glycans (e.g., the change in the percentage level of a particular glycan and the corresponding change in the level of cytotoxicity), FcγR-mediated cytotoxicity is often expressed as a relative value quantified against a standard. For example, "percent relative activity" (with respect to a standard) can be used to express the level of FcγR-mediated cytotoxicity. "Percent relative activity" can be calculated as: (i) cytotoxic activity of the sample / cytotoxic activity of the standard (" / " means to divide); or (ii) cytotoxic activity of the standard / cytotoxic activity of the sample (" / " means to divide). For example, if sample A shows a cytotoxicity level of 50% compared to the standard and sample B shows a cytotoxicity level of 51% compared to the same standard, then it can be said that FcγR-mediated cytotoxicity increases by 1% from sample A to sample B.

[0121] In certain embodiments, the standard is the level of FcγR-mediated cytotoxicity exhibited by commercially available panitumumab samples at the same dose (e.g., the same amount of antibody molecules). Thus, in certain embodiments, a quantitative relationship is established using the relative cytotoxicity level. For example, referring to FIG. 2A, when the terminal β-galactose is about 0%, the relative cytotoxicity level (calculated for commercially available panitumumab samples at the same dose) is about 88%. When the terminal β-galactose is increased to about 10%, the relative cytotoxicity level (calculated for commercially available panitumumab samples at the same dose) is about 95%. Thus, terminal β-galactose correlates with about a 0.67 percent increase in FcγR-mediated cytotoxicity. This means that for every 1% increase in terminal β-galactose, the relative cytotoxicity level of the panitumumab sample increases by 0.67%.

[0122] In certain embodiments, the relative cytotoxicity level can be calculated based on the EC50 value measured in a bioassay. For example, if a reporter gene is used to determine the EC50 of the cytotoxicity of a sample antibody, the relative cytotoxicity level can be calculated as EC50 sample / EC50 standard or EC50 standard / EC50 sample (" / " means to divide).

[0123] If desired, the relative cytotoxicity value of a sample can be determined multiple times (eg, two, three, four times) and the results can be reported as the average of these multiple values.

[0124] In certain embodiments, the disclosure provides a method of reducing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising reducing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab or reducing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. In certain embodiments, a reduction in β-galactose of about 1 percent reduces FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, e.g., about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent. Additionally, the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0125] In certain aspects, the disclosure provides a method of modulating Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab comprising increasing or decreasing the amount of panitumumab molecules that comprise a fucosylated glycan at the N-297 site, or increasing or decreasing the amount of panitumumab molecules that comprise a non-fucosylated glycan at the N-297 site.

[0126] In certain embodiments, the present disclosure provides a method of increasing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising increasing the amount of panitumumab molecules comprising a fucosylated glycan at the N-297 site. In certain embodiments, an increase of about 1 percent of fucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3.0 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent. In certain embodiments, the present disclosure provides a method of increasing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising decreasing the amount of panitumumab molecules comprising a non-fucosylated glycan at the N-297 site. In certain embodiments, a decrease of about 1 percent of nonfucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3.0 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent, and the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0127] In certain embodiments, the disclosure provides a method of reducing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising decreasing the amount of panitumumab molecules comprising a fucosylated glycan at the N-297 site. In certain embodiments, a decrease of about 1 percent of fucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3.0 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent. In certain embodiments, the disclosure provides a method of reducing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising increasing the amount of panitumumab molecules comprising a non-fucosylated glycan at the N-297 site. In certain embodiments, an increase of about 1 percent of nonfucosylated panitumumab molecules reduces FcγR-mediated cytotoxicity by about 2.70 percent to about 3.0 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent, and the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0128] In an exemplary embodiment, the fucosylated glycans on the antibody that are modulated (increased or decreased) include one or more fucosylated glycans selected from the group consisting of: A1G0, A1G1, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. In an exemplary embodiment, the non-fucosylated glycans on the antibody that are modulated (increased or decreased) include one or more non-fucosylated glycans selected from the group consisting of: A1G0, A1G1, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5.

[0129] In certain aspects, the disclosure provides a method of modulating Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising increasing or decreasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site. In exemplary embodiments, the high mannose can be Man-5, Man-6, Man-7, Man-8, or Man-9. In exemplary embodiments, the high mannose is Man-5.

[0130] In certain aspects, the present disclosure provides a method of increasing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising decreasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site. In certain embodiments, a decrease of about 1 percent of high mannose glycans increases FcγR-mediated cytotoxicity by about 1.2 percent to about 1.4 percent, e.g., about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent. Additionally, the change in cytotoxicity level is generally calculated based on the relative cytotoxicity value as described above. In certain embodiments, the high mannose is mannose-5 (Man-5).

[0131] In certain aspects, the disclosure provides a method of decreasing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising increasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site. In certain embodiments, an increase in high mannose glycans of about 1 percent decreases FcγR-mediated cytotoxicity by about 1.2 percent to about 1.4 percent, e.g., about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent. Additionally, the change in cytotoxicity level is generally calculated based on the relative cytotoxicity value as described above. In certain embodiments, the high mannose is mannose-5 (Man-5).

[0132] Methods provided herein also include methods of matching FcγR-mediated cytotoxicity of an IgG2 antibody sample (such as a panitumumab sample) to a reference value by adjusting the amount of glycans (e.g., galactosylated glycans, terminal β-galactose, G1, G1a, G1b and / or G2 galactosylated glycans, fucosylated glycans, non-fucosylated glycans, core fucose, high mannose glycans, Man-5 glycans, or combinations thereof) in the sample antibody to match the reference value. In certain aspects, the reference value is the level of FcγR-mediated cytotoxicity exhibited by a commercially available panitumumab sample at the same dose (e.g., the same amount of antibody molecules). In certain aspects, the reference value is a predetermined level that provides a therapeutic effect. In exemplary embodiments, the method includes measuring the cytotoxic activity of the antibody sample and / or the reference sample using the methods described herein. In exemplary embodiments, the cytotoxic activity of the antibody sample and / or reference sample is determined or measured (i) before adjusting the amount of glycans in the antibody, (ii) after adjusting the amount of glycans in the antibody, or (iii) before and after adjusting the amount of glycans in the antibody.

[0133] In certain aspects, the disclosure provides a method of matching Fc gamma receptor (FcγR) mediated cytotoxicity of an IgG2 antibody sample (such as a panitumumab sample) with a reference value, comprising: (1) obtaining a reference value for FcγR mediated cytotoxicity; (2) determining the FcγR mediated cytotoxicity of said IgG2 antibody sample (such as a panitumumab sample); and (3) modifying the FcγR mediated cytotoxicity of said IgG2 antibody sample (such as a panitumumab sample) by increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of the antibody, or increasing or decreasing the amount of IgG2 molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site, such that the difference in FcγR mediated cytotoxicity between the antibody sample and the reference value is about 35% or less. In certain embodiments, the difference in FcγR-mediated cytotoxicity between an IgG2 antibody sample (such as a panitumumab sample) and a reference value is about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 5% or less. In some examples, step (1) ("obtaining a reference value of FcγR-mediated cytotoxicity") is performed before, after, or simultaneously with step (2) ("determining FcγR-mediated cytotoxicity of said IgG2 or panitumumab sample") and / or step (3) ("modifying FcγR-mediated cytotoxicity of said IgG2 or panitumumab sample"), while in other examples, step (2) is performed before, after, or simultaneously with step (1) and / or step (3).

[0134] In certain aspects, the FcγR-mediated cytotoxicity of an IgG2 sample or a panitumumab sample is increased by increasing the amount of terminal β-galactose at the N-297 glycosylation site of the antibody, or by increasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. In certain embodiments, an increase of about 1 percent in β-galactose increases FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, e.g., about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent. Calculations of glycan levels and cytotoxicity levels are as described above, and generally the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0135] In certain aspects, FcγR-mediated cytotoxicity of an IgG2 sample or a panitumumab sample is reduced by decreasing the amount of terminal β-galactose at the N-297 glycosylation site of the antibody or by decreasing the amount of antibody molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. In certain embodiments, a reduction of about 1 percent of β-galactose increases FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, e.g., about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent. Additionally, the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0136] In certain aspects, the disclosure provides a method of matching Fc gamma receptor (FcγR)-mediated cytotoxicity of an IgG2 antibody sample (such as a panitumumab sample) with a reference value, comprising: (1) obtaining a reference value of FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of said IgG2 antibody sample (such as a panitumumab sample); and (3) modifying the FcγR-mediated cytotoxicity of said IgG2 antibody sample (such as a panitumumab sample) by increasing or decreasing the amount of IgG2 molecules comprising a fucosylated glycan at the N-297 site, or increasing or decreasing the amount of IgG2 molecules comprising a non-fucosylated glycan at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the antibody sample and the reference value is about 35% or less. In certain embodiments, the difference in FcγR-mediated cytotoxicity between an IgG2 antibody sample (such as a panitumumab sample) and a reference value is about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 5% or less. In some examples, step (1) ("obtaining a reference value of FcγR-mediated cytotoxicity") is performed before, after, or simultaneously with step (2) ("determining FcγR-mediated cytotoxicity of said IgG2 or panitumumab sample") and / or step (3) ("modifying FcγR-mediated cytotoxicity of said IgG2 or panitumumab sample"), while in other examples, step (2) is performed before, after, or simultaneously with step (1) and / or step (3).

[0137] In certain aspects, FcγR-mediated cytotoxicity of an IgG2 sample or a panitumumab sample is increased by increasing the amount of panitumumab molecules that comprise a fucosylated glycan at the N-297 site or decreasing the amount of panitumumab molecules that comprise a non-fucosylated glycan at the N-297 site. In certain embodiments, an increase of about 1 percent of fucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.7 percent to about 3.0 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent. In certain embodiments, a decrease of about 1 percent of nonfucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.7 percent to about 3.0 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent, and the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0138] In certain aspects, FcγR-mediated cytotoxicity of an IgG2 sample or a panitumumab sample is reduced by decreasing the amount of panitumumab molecules that comprise a fucosylated glycan at the N-297 site or by increasing the amount of panitumumab molecules that comprise a non-fucosylated glycan at the N-297 site. In certain embodiments, a reduction of about 1 percent of fucosylated panitumumab molecules reduces FcγR-mediated cytotoxicity by about 2.7 percent to about 3.0 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent. In certain embodiments, an increase of about 1 percent of nonfucosylated panitumumab molecules reduces FcγR-mediated cytotoxicity by about 2.7 percent to about 3.0 percent, e.g., about 3.0 percent, about 2.95 percent, about 2.90 percent, about 2.85 percent, or about 2.70 percent, and the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0139] In certain aspects, the disclosure provides a method of matching Fc gamma receptor (FcγR)-mediated cytotoxicity of an IgG2 antibody sample (such as a panitumumab sample) with a reference value, comprising: (1) obtaining a reference value of FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of said IgG2 antibody sample (such as a panitumumab sample); and (3) modifying the FcγR-mediated cytotoxicity of said IgG2 antibody sample (such as a panitumumab sample) by increasing or decreasing the amount of IgG2 molecules comprising high mannose glycans at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the antibody sample and the reference value is about 35% or less. In certain embodiments, the difference in FcγR-mediated cytotoxicity between an IgG2 antibody sample (such as a panitumumab sample) and a reference value is about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 5% or less. In some examples, step (1) ("obtaining a reference value of FcγR-mediated cytotoxicity") is performed before, after, or simultaneously with step (2) ("determining FcγR-mediated cytotoxicity of said IgG2 or panitumumab sample") and / or step (3) ("modifying FcγR-mediated cytotoxicity of said IgG2 or panitumumab sample"), while in other examples, step (2) is performed before, after, or simultaneously with step (1) and / or step (3).

[0140] In certain aspects, FcγR-mediated cytotoxicity of an IgG2 sample or a panitumumab sample is increased by decreasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site. In certain embodiments, about a 1 percent decrease in high mannose glycans increases FcγR-mediated cytotoxicity by about 1.2 percent to about 2.4 percent, e.g., about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent. Additionally, the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0141] In certain aspects, FcγR-mediated cytotoxicity of an IgG2 sample or a panitumumab sample is decreased by increasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site. In certain embodiments, an increase in high mannose glycans of about 1 percent decreases FcγR-mediated cytotoxicity by about 1.2 percent to about 2.4 percent, e.g., about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent. Additionally, the change in cytotoxicity level is generally calculated based on the relative cytotoxicity values ​​as described above.

[0142] 3.4 Methods for Modulating Glycans Suitable methods for modulating the amount of glycans, such as galactosylated glycans (e.g., including terminal β-galactose, G1, G1a, G1b, and / or G2 galactosylated glycans), non-fucosylated glycans, fucosylated glycans, or glycans containing core fucose, and / or high mannose glycans (e.g., including Man-5 glycans), on glycoproteins, such as antibodies, are known in the art. See, for example, Zhang et al. See, al., Drug Discovery Today 21(5):2016. Thus, in some embodiments, glycosylation-competent cells (which can be used to recombinantly produce glycoproteins, e.g., antibodies) are cultured under specific conditions to achieve a desired level of glycans.

[0143] For example, WO 2013 / 114164, WO 2013 / 114245, WO 2013 / 114167, WO 2015128793, and WO 2016 / 089919 each describe recombinant cell culture techniques useful for modulating glycans, e.g., galactosylated glycans (e.g., including terminal β-galactose, or G1, G1a, G1b and / or G2 galactosylated glycans), non-fucosylated glycans, fucosylated glycans, or glycans containing core fucose, and / or high mannose glycans (e.g., including Man-5 glycans), e.g., glycotans having an increased proportion of total non-fucosylated glycans. These publications teach methods for obtaining glycoproteins with increased proportions of Man5 glycans and / or nonfucosylated glycans (WO 2013 / 114164), glycoproteins with increased proportions of Man5 glycans and / or nonfucosylated glycans (WO 2013 / 114245), glycoproteins with specific amounts of high mannose glycans, nonfucosylated glycans and G0F glycans (WO 2013 / 114167); glycoproteins with high mannose glycans and reduced galactosylated and / or highly galactosylated glycans (WO 2015128793); and methods for manipulating the fucosylated glycan content on recombinant proteins (WO 2016 / 089919). Cell culture methods described by WO 2013 / 114164, WO 2013 / 114245, WO 2013 / 114167, WO 2015128793, and WO 2016 / 089919 include modifying one or more cell culture parameters, such as temperature, pH, culturing the cells with manganese ions or a salt thereof (e.g., 0.35 μM to about 20 μM manganese), and / or culturing the cells with copper (e.g., 10-100 ppb) and manganese (e.g., 50-1000 nM) to modulate specific glycans.

[0144] Furthermore, WO 2015 / 140700 describes culturing cells in the presence of betaine to increase nonfucosylated glycans, or culturing cells with manganese, galactose and betaine to obtain target values ​​of mannosylated, galactosylated and nonfucosylated glycans. US 2014 / 0356910 teaches how to increase high mannose glycoforms by manipulating the mannose to total hexose ratio in cell culture medium formulations. Pacis et al., Biotechnology and Bioengineering 108(10):2348-2358 (2011) teaches obtaining high levels of Man5 glycans by increasing the osmolality level of the cell culture medium and extending the culture period. Similarly, Konno et al., Cytotechnology 64:249-3+6 (2012) describes a method to control antibody fucose content by the osmolality of the culture medium. Wong et al., Biotechnology and Bioengineering 89(2):164-177 (2004) teaches a method to reduce recombinant protein sialylation and increase high mannose glycans by using low glutamine fed-batch culture. WO 2017 / 079165 describes a method to increase or decrease the non-fucosylated or fucosylated forms of recombinant proteins by using host cells genetically engineered to have no GMD or FX and culturing the host cells with fucose. WO 2017 / 134667 describes culturing cells with nicotinamide and fucose to produce antibodies with reduced levels of non-fucosylation. Sha et al., TIBs 34(10):835-846 (2016) also outline several methods for modulating glycans, including increasing the level of galactosylation on antibodies by incubation with uridine, manganese, and galactose, and using mannose as a carbon source to increase high mannose glycoforms.

[0145] Thus, the methods of the present disclosure, in exemplary embodiments, include employing one or more of the procedures, cell culture media, and / or cell culture conditions taught in any one or more of the above references or other references described herein to modulate the amount of galactosylated glycans (e.g., including terminal β-galactose, or G1, G1a, G1b, and / or G2 galactosylated glycans), non-fucosylated glycans, fucosylated glycans, or glycans containing core fucose, and / or high mannose glycans (e.g., including Man-5 glycans). In exemplary embodiments, the methods include culturing glycosylation-competent cells expressing an antibody in a cell culture medium under conditions that modulate the levels of galactosylated glycans (e.g., including terminal β-galactose, or G1, G1a, G1b, and / or G2 galactosylated glycans), non-fucosylated glycans, fucosylated glycans, or glycans containing core fucose, and / or high mannose glycans (e.g., including M5 high mannose species). For example, the methods, in some embodiments, include culturing glycosylation-competent cells expressing the antibody in a cell culture medium under conditions that modulate glycan levels, the cell culture medium comprising fucose, or fucose and glucose.

[0146] In the method comprising maintaining or culturing cells in cell culture, the cell culture can be maintained according to any set of conditions suitable for recombinant glycosylated protein or antibody production. For example, in some embodiments, the cell culture is maintained at a specific pH, temperature, cell density, culture volume, dissolved oxygen level, pressure, osmolality, etc. In a representative embodiment, the pre-inoculated cell culture is shaken (e.g., 70 rpm) in a CO2 incubator under standard humidified conditions with 5% CO2. In an exemplary embodiment, the method comprises culturing glycosylation-competent cells expressing an antibody in a cell culture medium under conditions that regulate the level of glycans, e.g., increasing the osmolality of the cell culture medium to reduce the level of non-fucosylated glycans of the antibody, as taught by Konno et al. (supra). In an exemplary embodiment, the method includes culturing glycosylation-competent cells expressing the antibody in a cell culture medium under conditions that modulate the level of glycans, e.g., modulating the pH and temperature of the cell culture, as taught by WO 2013 / 114164, WO 2013 / 114245, WO 2013 / 114167, or WO 2015 / 128793 (each of which is incorporated by reference herein).

[0147] In an exemplary embodiment, the method of the disclosure comprises maintaining glycosylation-competent cells in a cell culture medium at a suitable pH, temperature, osmolality, and dissolved oxygen level for the production of a recombinant glycosylated protein or antibody, as is well known in the art. In an exemplary embodiment, the cell culture is maintained in a medium suitable for cell growth and / or is provided with one or more feed media according to any suitable feeding schedule, as is well known in the art.

[0148] In an exemplary embodiment, the glycosylation competent cell is a eukaryotic cell, including but not limited to a yeast cell, a filamentous fungal cell, a protist cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In an exemplary embodiment, the eukaryotic cell is a mammalian cell. In an exemplary embodiment, the mammalian cell is a non-human mammalian cell. In some embodiments, the cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonal carcinoma cells P19, mouse embryonic fibroblast cells NIH 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatoma Hep G2, mouse B myeloma J558L, or baby hamster kidney (BHK) cells (Gaillet et al. al.2007;Khan,Adv Pharm Bull 3(2):257-263(2013)).

[0149] Cells that are not competent for glycosylation can also be transformed into glycosylation-competent cells, for example, by introducing into them genes encoding the relevant enzymes required for glycosylation. Exemplary enzymes include, but are not limited to, oligosaccharyltransferase, glycosidase, glucosidase I, glucosidase II, calnexin / calreticulin, glycosyltransferase, mannosidase, GlcNAc transferase, galactosyltransferase, and sialyltransferase.

[0150] In further or alternative aspects, glycosylation competent cells recombinantly producing antibodies are engineered to modulate the glycans of the antibodies, such as galactosylated glycans (e.g., including terminal β-galactose, or G1, G1a, G1b and / or G2 galactosylated species), non-fucosylated glycans or glycans containing core fucose, and / or high mannose glycans (e.g., including M5 high mannose species). In exemplary aspects, glycosylation competent cells are engineered to alter the activity of an enzyme in the de novo or salvage pathway. Optionally, glycosylation competent cells are engineered to knock out a gene encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase. In exemplary embodiments, glycosylation competent cells are engineered to alter the activity of an enzyme in the de novo or salvage pathway. These two pathways of fucose metabolism are well known in the art and are shown in Figure IE. In an exemplary embodiment, the glycosylation competent cells are engineered to alter the activity of any one or more of fucosyl-transferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, the glycosylation competent cells are engineered to knock out the gene encoding FX. In an exemplary embodiment, the glycosylation competent cells are engineered to alter the activity of β(1,4)-N-acetylglucosaminyltransferase III (GNTIII) and / or GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). In an exemplary aspect, the glycosylation competent cells are engineered to overexpress GNTIII and / or RMD. In an exemplary embodiment, the glycosylation competent cells are engineered to have altered beta-galactosyltransferase activity.In some embodiments, glycosylation competent cells are genetically engineered to modulate the expression levels of genes encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase, β1-4 galactosyltransferase, and / or β1-4 N-acetylgalactosaminyltransferase.

[0151] Several methods are known in the art to reduce or eliminate fucosylation of Fc-containing molecules, such as antibodies. These include recombinant expression in certain mammalian cell lines, including FUT8 knockout cell lines, mutant CHO line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNA specific for the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Alternatively, Fc-containing molecules can be expressed in non-mammalian cells, such as plant cells, yeast, or prokaryotic cells (e.g., E. coli).

[0152] In exemplary embodiments, the target glycan amount is achieved by chemical or enzymatic treatment of the antibody after production. In exemplary embodiments, the methods of the present disclosure include treating the antibody chemically or enzymatically after the antibody is recombinantly produced. In exemplary embodiments, the chemical or enzyme is selected from the group consisting of Endo-S, Endo-S2, Endo-D, Endo-M, Endo-LL, α-fucosidase, β-(1-4)-galactosidase, Endo-H, Endo-F1, Endo-F2, Endo-F3, β-1,4-galactosyltransferase, kifunensine, and PNGase-F. In exemplary embodiments, the chemical or enzyme is incubated with the antibody for various times to generate antibodies with various amounts of glycans. In some embodiments, the antibody is incubated with β-1,4-galactosyltransferase, as described in the Examples. In some further embodiments, antibodies with varying levels of galactose can be generated by incubating the antibodies with β-1,4-galactosyltransferase for a period of time, such as, but not limited to, about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 9 hours, or a period of time ranging from about 10 minutes to about 9 hours.

[0153] 3.5 Glycan Measurement Methods A variety of methods are known in the art for assessing glycoforms present in a glycoprotein-containing composition, e.g., an antibody, or for determining, detecting, or measuring the glycoform profile of a particular sample containing a glycoprotein. Suitable methods include, but are not limited to, hydrophilic interaction liquid chromatography (HILIC), liquid chromatography-tandem mass spectrometry (LC-MS), positive ion MALDI-TOF analysis, negative ion MALDI-TOF analysis, HPLC, weak anion exchange (WAX) chromatography, normal phase chromatography (NP-HPLC), exoglycosidase digestion, Biogel P-4 chromatography, anion exchange chromatography, and one-dimensional NMR spectroscopy, and combinations thereof. For example, Pace et al.,Biotechnol.Prog.,2016,Vol.32,No.5 pages 1181-1192, Shah,B.et al.J.Am.Soc.Mass Spectrom.(2014)25:999, Mattu et al.,JBC 273:2260-2272(1998), Field et al.,Biochem J 299(Pt 1):261-275(1994), Yoo et al.,MAbs 2(3):320-334(2010),Wuhrer M.et al.,Journal of Chromatography B,2005,Vol.825,Issue 2,pages 124-133,Ruhaak LR,Anal Bioanal Chem,2010,Vol.397:3457-3481, Kurogochi et al.,PLOS One 10(7):e0132848;doi:10.1371 / journal.pone.0132848, Thomann et al.,PLOS See, for example, Pace et al., Biotechnol. Prog. 32(5):1181-1192 (2016), and Geoffrey, RGet. al. Analytical Biochemistry 1996, Vol. 240, pages 210-226. The examples provided herein also describe suitable methods for assessing glycoforms present in a glycoprotein-containing composition, such as an antibody.

[0154] For example, glycan content can be measured by high pH anion exchange chromatography (HPAEC) as described by Wuhrer et al. (Journal of Chromatography B Vol.825:124-133,2005) and Dell et al. (Science Vol.291:2351-2356). In summary, N-glycans are enzymatically removed from recombinant glycoproteins, such as recombinant monoclonal antibodies, and labeled with a fluorescent tag (e.g., 2-aminobenzamide or 2-aminobenzoic acid) at the reducing end. Fluorescent N-glycans are separated by HPAEC and detected by fluorescence detection. In general, separation of neutral N-glycans is based on the increasing complexity of N-glycan structures. Separation of charged N-glycans is based on the number and type of sialic acid, sulfate, or other modifications present that can derive a charge number. These glycan profiles of the test samples are visually compared to appropriate standards.

[0155] Example 2.2 uses hydrophilic interaction liquid chromatography (HILIC). In summary, glycan species can be analyzed based on the following steps: (i) release of N-glycans (e.g., by an enzyme such as PNGase F), (ii) labeling (e.g., by 2-aminobenzoic acid or 2-aminobenzamide), (iii) removal of free label (e.g., by gel filtration or solid phase extraction), (iv) separation of glycan species by HILIC, and (v) detection (e.g., by fluorescence spectroscopy). Further details of HILIC can be found in Melmer et al. et al., Analytical and Bioanalytical Chemistry, September 2010, Volume 398, Issue 2, pp905-914.

[0156] Another commonly used method is liquid chromatography-tandem mass spectrometry (LC-MS). After release of N-glycans, labeling, and removal of free labels, the sample can be analyzed by a technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). See, for example, Wang et al., Biotech Method, 17 January 2018, doi.org / 10.1002 / biot.201700185.

[0157] 3.6 Antibody composition Compositions comprising recombinant glycosylated proteins and antibodies produced by the methods described herein are also provided herein. In an exemplary embodiment, the compositions are prepared by a method that modulates the amount of glycans (e.g., galactosylated glycans, terminal β-galactose, G1, G1a, G1b and / or G2 galactosylated glycans, non-fucosylated glycans, fucosylated glycans, core fucose, high mannose glycans, Man-5 glycans, or combinations thereof) in the antibody. In an exemplary aspect, the recombinant glycosylated protein is an IgG2 antibody, such as panitumumab. Thus, antibody compositions are provided herein that include an IgG2 antibody (e.g., panitumumab) with increased or decreased FcγR-mediated cytotoxicity, where the IgG2 antibody (e.g., panitumumab) has been engineered to increase or decrease FcγR-mediated cytotoxicity compared to a control or reference value by modulating the glycan profile as described above.

[0158] In an exemplary embodiment, the antibody compositions provided herein are combined with a pharma- ceutically acceptable carrier, diluent or excipient. Thus, provided herein is a pharmaceutical composition comprising a recombinant glycosylated protein composition (e.g., an antibody composition) described herein and a pharma- ceutically acceptable carrier, diluent or excipient. As used herein, the term "pharma- ceutically acceptable carrier" includes some of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents.

[0159] The following examples are offered merely to illustrate the present disclosure and are not intended to limit its scope in any way. EXAMPLES

[0160] 1. Introduction To improve our understanding of IgG2-mediated cytotoxicity, we developed sensitive cytotoxicity assays using panitumumab as a model IgG2, using specific responder cell types. We deployed FcγRIIa signaling assays using engineered cell lines and reporter genes, as well as primary cells derived from PBMCs isolated from whole blood of genotyped donors to study cytotoxic activity. Donors expressing common FcγRIIa and FcγRIIIa receptor allotypes were used. To understand the impact of quality attributes that may vary as a function of the manufacturing process, we generated panitumumab species that contained a broad range of major glycan species, such as galactosylated, nonfucosylated, and mannosylated, and assessed their impact on activity against panitumumab in various assays.

[0161] 2. Materials and Methods Panitumumab was produced in CHO cells by standard manufacturing processes at Amgen (Thousand Oaks, Calif.).

[0162] 2.1 Glycan species enrichment and enzymatic remodeling High mannose-containing species were enriched from the mAb using a ProSwift ConA-1S affinity column (5 x 50 mm, ThermoFisher, PN 074148) on an Agilent 1100 series HPLC system with a flow rate of 0.5 mL / min. The column was first kept at initial conditions with 100% buffer A (50 mM sodium acetate, 0.2 M NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 5.3) for 10.5 min and then eluted with 100% buffer B (50 mM sodium acetate, 0.2 M NaCl, 1 mM CaCl2, 1 mM MgCl2, 100 mM α-methyl-mannopyranoside pH 5.3) for 17.5 min. Both the flow-through and elution fractions were collected and treated with β-(1-4)-galactosidase (QA Bio, PN E-BG07) to remove terminal galactose. Specifically, the mAb fraction was incubated with β-(1-4)-galactosidase at a ratio of 1 / 50 (μg / μg) in the presence of reaction buffer (pH 6.0) containing 50 mM sodium phosphate for 1 h at 37° C. The reaction was stopped by flash freezing.

[0163] Nonfucosylated species were prepared from mAbs by enzymatic treatment with Endo-H (QA-Bio, PN E-EH02). Specifically, mAbs were incubated with Endo-H for 24 h at 37 °C in a reaction buffer of 50 mM sodium phosphate (pH 5.5). The final mAb concentration was 4 mg / mL. Nonfucosylated mAbs were subsequently separated by affinity chromatography on an Agilent 1100 series HPLC using a customized glycap-3A column (low density FcγIIIa, 3 × 150 mm, Zepteon, PN R3AVD1P1ML). Mobile phase A contained 20 mM Tris, 150 mM NaCl (pH 7.5), and mobile phase B was 50 mM sodium citrate (pH 4.2). Both non-fucose-depleted (flow-through) and concentrated (eluted) mAbs were separated using a gradient (hold at 0% B for 8 min, 0% to 18% B for 22 min) at a flow rate of 0.5 mL / min, and enzymatic treatment with β-(1-4)-galactosidase was performed (as above) to remove any potential contribution of terminal galactose.

[0164] Galactose remodeling samples were generated by in vitro activity of β-1,4-galactosyltransferase (Sigma / Roche). First, fucosylated mAb (mainly G0F) was prepared by collecting the flow-through fraction from an FcγIIIa column and treated with galactosidase to remove terminal galactose. G0F enriched mAb was then incubated with β-1,4-galactosyltransferase at 37°C in a reaction buffer containing 10 mM UDP-galactose, 100 mM MES (pH 6.5), 20 mM MnCl2, and 0.02% sodium azide. The final enzyme to mAb ratio was 6 (μL / mg) and the mAb concentration was 2 mg / mL. MAbs with different levels of galactose were obtained by taking samples from the reaction mixture at various time points (10 min, 20 min, 30 min, 1 h, 2 h, 4 h, and 9 h) and then flash freezing to stop the reaction.

[0165] Protein A chromatographic purification was performed on all concentrated and remodeled samples to remove enzymes and other components. Purification was performed on an Agilent 1100 series HPLC system using a pre-packed Protein A column (Poros A / 20, 4.6 x 100 mm, Applied Biosystems, PN 1-5022-26) at a flow rate of 3 mL / min. After loading an appropriate amount of each sample, the column was first maintained at the initial conditions with 100% Buffer A (20 mM Tris-HCl / 150 mM NaCl, pH 7.0) for 1.4 min, and then eluted with 100% Buffer B (0.1% acetic acid) for 2.9 min. All eluted mAbs were diafiltered into formulation buffer using Amicon Ultra centrifugal filters with a 3 kDa cut-off membrane. Protein concentrations were typically around 1 mg / mL for all concentrated / remodeled mAb samples.

[0166] 2.2 Characterization of glycan species enrichment and remodeling All enriched and remodeled samples were characterized by hydrophilic interaction liquid chromatography (HILIC) and size-exclusion chromatography to ensure the desired glycan properties and minimal levels of high molecular weight species. Glycans from mAbs were released using PNGase F (New England BioLabs) at an E / S ratio of 1 / 25 (μL / μg) and labeled with 12 mg / mL 2-aminobenzoic acid (2-AA, Sigma-Aldrich) by incubating the reaction mixture at 80°C for 75 min. 2-AA labeled glycans were separated using a BEH glycan column (1.7 μm, 2.1 × 100 mm, Waters) on a Waters Acuity or H-Class UPLC system equipped with a fluorescence detector. The column temperature was maintained at 55°C. Mobile phase A contained 100 mM ammonium formate (pH 3.0) and mobile phase B was 100% acetonitrile. Glycans were bound to the column in high organic solvent and then eluted with an increasing gradient of aqueous ammonium formate buffer (76% B held for 5 min, followed by elution with a gradient from 76% B to 65.5% B over 14 min). Verification that the required procedures did not result in the formation of high molecular weight species was assessed using a size exclusion column (SEC) TSK-Gel G3000SWLXL (7.8 x 300 mm, Tosoh Bioscience) on an Agilent 1100 HPLC system at a flow rate of 0.5 mL / min. Sample loads of 20-40 μg of sample were typically separated isocratically using a mobile phase containing 100 mM sodium phosphate (pH 6.8) and 250 mM NaCl.

[0167] 2.3 FcγRIIa reporter gene assay The FcγRIIa reporter luciferase reporter gene assay uses genetically engineered Jurkat T cells as effector cells. Jurkat reporter cells express the IgG Fc receptor FcγRIIa (H131 variant) on the cell surface as well as a luciferase reporter gene with response elements for factor for T cell activator (NFAT). Simultaneous binding between an antibody on a target cell and an antibody bound to an antibody Fc domain with stably expressed FcγRIIa on Jurkat effector cells activates the transcription factor NFAT. Activated NFAT translocates into the nucleus of Jurkat cells and induces luciferase reporter gene expression. Addition of a luciferase substrate containing luciferin and detergent generates a luminescent signal, allowing detection of FcγRIIa reporter activity. For panitumumab, reference standards, assay controls, and test samples were serially diluted across 8 concentration levels in RPMI1640 assay medium with low IgG FBS to final plate well concentrations ranging from (0.004 μg / mL to 2 μg / mL). Effector Jurkat reporter cells and target (A431) cells are mixed at an effector to target (E to T) cell ratio of 3:2 to prepare a cell suspension. The plate is then incubated for 5.5 hours in a humidified incubator at 5% CO2 and 37°C. At the end of the incubation, the cells are lysed by detergent in luciferase assay buffer. The luciferase reaction in luciferase assay buffer produces a luciferin signal of its substrate and is detected by an EnVision plate reader. The data was fitted to the mean luminescence values ​​using a 4-parameter fit with SoftMaxPro, and the EC50 standard / EC50 sample was calculated and reported as percent activity. Each sample was tested in three independent assays and the final sample result is reported as the average of the triplicate determinations.

[0168] 2.4 Donor allotyping and cell-mediated cytotoxicity assay using PBMCs Allotyping of PBMC donors. PBMCs were isolated from healthy donor blood using Becton Dickinson cell preparation tubes (BD-CPT). 8 mL of blood was collected from each donor using venipuncture into BD CPT tubes. The tubes were then centrifuged at 1500 RPM for 30 minutes to separate the blood into different layers. The plasma layer was aspirated and lymphocytes were collected in a 15 mL centrifuge tube. The lymphocytes were then washed twice with PBS to remove the plasma and the cells were counted before DNA was isolated. DNA was extracted from the cells using a QIAGEN Blood and Cell Culture DNA kit. The DNA was then subjected to Taqman single nucleotide polymorphism (SNP) genotyping analysis using a qualified set specific for each receptor (FcγRIIa and FcγRIIIa) on a 7900HT real-time PCR system. The qPCR assay was set for 40 cycles using the master mix, DNA, and assay oligomix (fluorescently labeled probes). Each probe anneals specifically to a complementary sequence, if present. The exonuclease activity of the DNA polymerase cleaves the probe hybridized to the target, releasing the reporter dye and increasing fluorescence. If the specific sequence is not present, the probe will not attach during amplification, thereby releasing the dye, so the presence of the dye indicates a specific polymorphism. The SDS software reads each well and the genotype is determined by calling. The software also provides an allele discrimination plot where clustering indicates individual genotypes. The TaqMan® 5'-nuclease assay chemistry provided a method to obtain single nucleotide polymorphism (SNP) genotyping results. Each pre-designed TaqMan® SNP genotyping assay contained two allele-specific TaqMan® MGB probes with different fluorescent dyes, and a PCR primer pair to detect the specific SNP target. These TaqMan® probe and primer sets (assays) uniquely align with the genome, providing unparalleled specificity for the allele of interest. The SNP assay for FcγRIIA 131 histidine or arginine polymorphism (H / R) is C_9077561_20.The SNP assay for FcγRIIIA 158 phenylalanine or valine (F / V) is C_25815666_10.

[0169] KILR™ Cellular Cytotoxicity Assay. This assay utilized U2OS target cells overexpressing EGFR and a proprietary housekeeping protein fused to an inactive fragment of the β-galactosidase (β-gal) reporter, a component of the Eurofins DiscoverX KILR™ Cytotoxicity Assay. The engineered target cells were mixed with PBMCs at a ratio of 1:200 in the presence of various concentrations of panitumumab or glycoengineered samples, respectively. Upon lysis of the target cells, the tagged housekeeping protein was released into the medium. The tagged housekeeping protein is detected in the medium by the addition of a reagent containing another fragment of the β-gal reporter that leads to the formation of active β-gal enzyme. Light emission occurs in a dose-dependent manner when the chemiluminescent reagent is hydrolyzed in response to β-gal. The luminescence response data was directly proportional to the amount of cytotoxicity. In this assay, fresh PBMCs from healthy donors were used as effector cells. Luminescence signals were detected with a plate reader. Luminescence responses were plotted with respect to the test concentrations to generate dose-response curves.

[0170] PBMCs isolated from healthy volunteers with known FcγRIIa and FcγRIIIa genotypes were generated by Amgen (Thousand Oaks, CA). The KILR™ ADCC assay was performed by isolating PBMCs using BD-CPT tubes. PBMCs were harvested, washed with D-PBS, and plated at 1.2 × 10 per well of a 96-well plate. 6Cells were dispensed. KILR™ U2OS target cells (6,000 / well) were added to wells containing PBMCs and incubated for 12 h with increasing concentrations of panitumumab or glycoengineered samples (0.148-200 ng / mL). A dose-dependent increase in luminescence signal is detected by reading the assay plates on a Perkin Elmer Envision plate reader. Data analysis is performed using SoftMax Pro v5.4.1 and dose-response curves are reported.

[0171] 2.5 FcγR blocking assays to demonstrate specificity Receptor antibody blocking studies were performed by individually blocking CD16 (FcγRIIIa), CD32 (FcγRIIa), and CD64 (FcγRI) with antibodies that specifically bind and block these receptors, and the resulting cytotoxic activity was measured. Panitumumab was used at a constant concentration of 200 mg / mL and different blocking mAbs (anti-FcγRI [mouse monoclonal, BioLegend catalog no. 360701], anti-FcγRIIa [goat polyclonal, R&D Systems catalog no. AF1330], and anti-FcγRIIIa [goat polyclonal, R&D Systems catalog no. AF1257]) at various concentrations (2000 ng / mL to 1 ng / mL). Goat isotype control: polyclonal goat, R&D Systems catalog number AB-108-C; Mouse IgG1 / k isotype control: mouse IgG1 / k, BD Biosciences catalog number 550979.

[0172] U2OS target cells engineered with Eurofins DiscoverX's KILR® housekeeping gene were harvested and seeded in 96-well plates at a density of 6000 cells / well. A fixed concentration of panitumumab (200ng / mL) was mixed with blocking reagent at concentrations ranging from 2000ng / mL to 1ng / mL and added to the target cells. Healthy donor PBMCs were used as effector cells by drawing whole blood and isolating PBMCs in BD-CPT tubes. These PBMCs were then added to the mixture of target cells and antibody mixture at a density of 1.2e6 cells / well to achieve an effector to target ratio of 200:1. The assay plates were co-incubated at 37°C for approximately 18 hours, after which KILR® detection reagent was added and the luminescence signal was read on an Envision plate reader.

[0173] 2.6 FcγR binding by SPR Surface plasmon resonance experiments were performed using an SPR T-200 instrument. His-tagged human FcγRs were expressed in CHO cells and purified in mouse anti-his capture antibodies immobilized at approximately 5000 RU on a series S sensor chip CM5 (GE Healthcare) using Instrument buffer (0.005% P20 in PBS). FcγRs were diluted to 3.3-10 nM in running buffer (0.005% P20, 0.1 mg / mL BSA in PBS) and injected for 1.5 min at 10 μL / min for the capture step. Panitumumab samples were diluted in running buffer (PBS + 0.005% P20 + 0.1 mg / mL BSA) in a concentration range of 0.4 nM-20000 nM and injected over the captured FcγR at 50 μL / min with 3 min binding and dissociation times. The chip surface was regenerated by injecting 10 mM glycine, pH 1.7 at 30 μL / min for 30 s.

[0174] 3.Results 3.1 Effect of glycan species on panitumumab-mediated cytotoxicity As previously described, panitumumab can mediate cell-mediated cytotoxicity activity that has not been previously described for a human IgG2 therapeutic antibody. To ascertain which product quality attributes may affect its activity, a series of enrichment and enzymatic treatments (see Materials and Methods) were used to modify the glycan profile of panitumumab, generating a wide range of each of the major glycan species: terminal galactose, core fucose, and high mannose. Because this activity was previously attributed to FcγRIIa, a sensitive FcγRIIa reporter gene assay was also devised to read out the impact of quality attributes on activity.

[0175] The first glycan species evaluated for impact was terminal galactose. Panitumumab samples were enzymatically treated as described in the methods section and displayed a wide range of terminal galactose from 0.4% to 88.3%. As shown in Figures 2A-2B, across that range of galactose levels, activity measured by reporter gene assays ranged nearly 60%, with activity levels exhibiting a supralinear response to galactose levels. We quantified the relative impact of β-galactose on FcγRIIa signaling activity by expressing it in terms of the slope of an activity / property correlation plot, which can be considered to represent a response factor. Using this approach in panitumumab, the impact of FcγRIIa signaling activity was measured with an R of 0.98. 2 Using this value, it can be calculated that for β-galactose it is 0.6681.

[0176] Next, we investigated the effect of the level of core fucose species on the FcγRIIa reporter gene assay. Panitumumab also showed a linear response to various fucose (non-fucosylated) levels. The dose-response curve of FcγRIIa signaling activity for non-fucosylation is shown in Figure 3A-3B. The calculated activity yielded a supra-linear negative response to the amount of non-fucosylation. The data show that panitumumab mediated higher cytotoxicity at lower non-fucosylated levels and lower cytotoxicity at higher non-fucosylated levels, thereby inversely correlating with the non-fucosylated percent of the mAb. We note that this is an interesting reversal of the effect of non-fucose levels on FcγRIIIa-mediated ADCC activity by IgG1.

[0177] The final component of this glycan study involved testing the effect of high mannose on the ability of panitumumab to affect the FcγRIIa reporter gene assay. The FcγRIIa signaling activity response as a function of high mannose levels is shown in Figures 4A-4B. Again, panitumumab shows a linear, but inverse response to high mannose.

[0178] A summary of the effects of different glycan types is shown in Table 3.

[0179] [Table 5]

[0180] 3.2 Allotyping of PBMCs To extend these observations to additional assay formats that better reflect the physiological situation, we developed a primary PBMC assay to assess the impact of panitumumab-mediated cytotoxicity (see Materials and Methods). To assess the impact of FcγR allotypes in this method, DNA from several donors was genotyped to determine the alleles at amino acid position 131 of FcγRIIA (H / R) and at amino acid position 158 of the FcγRIIIA receptor (V / F). The allelic clusters of the FcγRIIIa receptor polymorphism were 52% homozygous for the FF genotype, 36% heterozygous for FV, and 12% homozygous for VV at amino acid position 158, while the allelic discrimination plot for FcγRIIa showed 26% homozygous for the RR genotype, 58% heterozygous for HR, and 16% homozygous for HH at amino acid position 131. Primary PBMC from these donors were used in subsequent cytotoxicity assays.

[0181] 3.3 PBMC cytotoxicity data A wide range of nonfucosylated, mannosylated, and galactosylated panitumumab samples were tested in the cytotoxicity assay described in Materials and Methods using PBMCs from donors with HHVV, HHFF, RRFV, and HHFV allotypes of FcγRIIa and FcγRIIIa, respectively. Representative dose-response curve overlays of methods varying specific glycan levels are shown in Figure 5. Panitumumab also showed a linear inverse relationship response to a range of 0.4% to 27.4% fucose (nonfucosylated). Calculated activity yielded a supralinear negative response to the amount of nonfucosylation when tested in donors with different allotypes (HHVV, HHFF, RRFV, and HHFV, respectively) (Figures 6A-6D). All donors except HHVV showed a linear negative correlation between cell killing and percent nonfucosylation.

[0182] Panitumumab also showed a linear response to a high mannose range of 2.9% to 75.6%. Calculated activity yielded a supralinear negative response to amounts of high mannose when tested in donors with different allotypes (HHVV, HHFF, RRFV, and HHFV, respectively) (Figures 7A-7D). In this assay, all four donors showed a strong negative correlation between cell killing and high mannose levels.

[0183] Panitumumab samples with a wide range of terminal galactose from 0.4% to 88.3% were also tested in a PBMC-mediated cytotoxicity assay. In this case, the results showed that there was no substantial correlation between cytotoxic activity and various levels of galactosylation, unlike that seen in the FcγRIIa reporter gene assay. Using this approach for panitumumab, it was not possible to quantify the relative impact of β-galactose on cell-mediated cytotoxicity. The assay was performed with PBMCs from four donors with different allotypes, HHFV, RRFV, HHVV, and RRFF, respectively, as shown in Figures 8A-8D.

[0184] 3.4 FcγRIIa is the sole receptor involved in IgG2-mediated cell killing. Receptor antibody blocking studies were performed by individually blocking CD16 (FcγRIIIa), CD32 (FcγRIIa), and CD64 (FcγRI) with antibodies that specifically bind and block these receptors, and the resulting cytotoxic activity was measured. Blocking experiments were set up using KILR assays with PBMCs and panitumumab. Panitumumab was used at a constant concentration of 200 mg / mL, and by varying the concentrations of different blocking mAbs (2000 ng / mL to 1 ng / mL), it was shown that only cells incubated with anti-FcγRIIa showed a decrease in cell death with increasing concentrations of blocking mAb. Cells treated with panitumumab (0.1 to 200 ng / mL) without blocking antibodies mediated cell-mediated cytotoxicity as expected in a dose-dependent manner (Figure 9A). Cells incubated with anti-FcγRI or anti-FcγRIIIa, as well as isotype control mAb, showed no difference in percent cell death at any concentration of blocking mAb (FIG. 9B), indicating that IgG2 (panitumumab)-mediated cytotoxicity was primarily due to engagement of the FcγRIIa receptor and not FcγRI or FcγRIIIa.

[0185] 3.5 Binding of panitumumab to FcγR To assess the affinity of panitumumab and various enriched glycan species to FcγR, binding of panitumumab samples to all three human Fcγ receptors was measured by surface plasmon resonance (SPR). Panitumumab at 10 μM showed no detectable binding to human FcγRI or FcγRIIIa-158F, but did show binding to huFcγRIIa-131H (Figures 10A-10C). Panitumumab and huIgG2 controls bound to huFcγRIIa-131H with apparent KDs of approximately 20 μM and 25 μM, respectively (Figure 10C). Binding was further assessed for fucose and non-fucose enriched panitumumab samples by equilibrium binding (Figures 11A-11B). Two glycan enriched samples (fucose enriched and non-fucose enriched KDs of approximately 7.9 μM and 8 μM, respectively) showed no detectable binding to human FcγRI or FcγRIIIa-158F, but did show binding to huFcγRIIa-131H (Figures 10A-10C). DThe differences in binding by SPR between the 1990 and 1995 (values) were not as pronounced as those seen in the FcγRIIa signaling activity or PBMC cytotoxicity assays. This is likely due to signal amplification provided by the cell-based assays via receptor clustering, signaling, and gene expression aspects of the binding response (see, e.g., Unkeless et al, Semin Immunol. 1995;7(1):37-44; Amigorena et al., Science. 1992;256(5065):1808-1812; Amigorena et al., Nature. 1992;358(6384):337-341; Regnault et al., J Exp Med. 1999;189(2):371-380).

[0186] To summarize the effect of different glycans on the cytotoxic activity of panitumumab, Table 3 shows the slope values ​​and R for each donor when tested with either the nonfucosylated, high mannose, or β-galactose samples for their ability to mediate cell-mediated cytotoxicity. 2 Shows.

[0187] 4. Discussion The goal of this study is to understand the mechanisms and product quality attributes that influence therapeutic human IgG2 monoclonal antibody-mediated cytotoxicity. Sensitive and reproducible quantitative functional assays are needed to assess the impact of quality attributes. To understand the impact of quality attributes that may vary as a function of the manufacturing process, we generated panitumumab species that contained a wide range of major glycan species, such as galactosylated, nonfucosylated, and mannosylated, and evaluated the impact of each on activity for panitumumab in various assays. Through the manipulation process, we were able to achieve a substantially wider range than would be possible through process modifications to more precisely find the relationship between properties and activity. Gal levels ranged from 0.4% to 88.3%, nonfucose levels ranged from 0.4% to 27.4%, and high mannose levels ranged from 2.9% to 75.6%.

[0188] Many assays that use primary cells to determine phagocytic activity are prone to inconsistencies in effector types present in the donor-derived population at the time of the assay, as well as receptor allotypes and other background genetic diversity that may affect assay activity. Also, as described elsewhere, it is generally more difficult to identify the relevant receptors on phagocytes due to the diversity of receptors expressed (Parren et al., J. Clin. Invest. 90:1537-1546, 1992; Salmon et al, 1992, J. Clin. Invest. 89(4):1274-81; Ackerman et al., J Immunol Methods. 2011;366:8-19). From an operational perspective, assay throughput is also limited by the number of cells that can be harvested. Thus, these types of assays are not suitable for drug development and are characterized in a quality control setting. Recognizing these challenges, Tada et al. (PLOS ONE, April 2, 2014, Vol. 9, No. 4, e95787) developed a reporter gene assay for FcγRIIa signaling activity that overcomes many of the limitations mentioned above.

[0189] Effector function also depends on the receptor polymorphism (158V or F for FcγRIIIa, 131H or R for FcγRIIa). To further study the impact of quality attributes in a more physiologically relevant setting, PBMC donors expressing common FcγRIIa and FcγRIIIa receptor allotypes were used to evaluate whether there is an impact of receptor allotype on panitumumab-mediated cytotoxicity and conclusions regarding the impact of glycans. As the kinetics of IgG2 is much slower than IgG1, reliable functional assays could be developed using PBMCs that could tolerate long read assays, which leads to longer incubation times for these assays. KILR (Killing Immunolytic Reaction) by DisCoverX is a non-radioactive assay of kinetically slow cytotoxicity. The KILR assay was set up using panitumumab-coated U2OS target cells overexpressing EGFR transduced with the KILR housekeeping gene and PBMCs as effector cells.

[0190] These studies show for the first time that the glycans of IgG2 panitumumab have substantial and different effects on the cellular cytotoxic activity. We observed that non-fucosylation and mannosylation negatively impact cellular cytotoxicity in both FcγRIIa signaling activity and PBMC-mediated cell killing assays. This is the exact opposite phenomenon to IgG1. Increased non-fucosylation significantly reduces the cell killing ability of the antibody, and similarly, increasing the high mannose content of the antibody reduces the cellular cytotoxicity mediated by panitumumab. For these two glycans, a supralinear negative response is observed in both primary cell and reporter gene assays. Galactosylation, on the other hand, appears to have a more modest but positive correlation between β-galactose content and cell killing. This was more pronounced in the FcγRIIa signaling activity assay than in the PBMC-mediated cell killing assay. It must be mentioned that there is still no clear mechanistic explanation available for the observed association of FcγRIIa with IgG2, since the affinity of fucosylated panitumumab for FcγRIIa-H131 (KD ∼7.9 μM) is only slightly higher than that of nonfucosylated panitumumab for FcγRIIa-H131 (KD ∼8.0 μM) as measured by surface plasmon resonance.

[0191] It is acknowledged that PMBCs are a complex and variable population. To confirm that FcγRIIa is mediating activity, receptor blocking studies were performed to confirm the specificity showing that panitumumab mediates cytotoxicity through FcγRIIa and not FcγRIIIa. This was achieved by using blocking antibodies against FcγRI, FcγRIIa, and FcγRIIIa. Cytotoxicity was only inhibited by increasing concentrations of anti-FcγRIIa, and cytotoxicity was not affected when blocked with other antibodies against FcγRI or FcγRIIIa. The difference in cytotoxicity levels between different donors with the same FcγRIIa allele could be due to a variety of reasons, including receptor density, membrane mobility, or interactions / cooperation with other molecules that may affect intracellular signaling and the resulting cellular cytotoxicity. It remains to be seen whether and how the role of inhibitory receptors will affect the overall cellular cytotoxicity of PBMCs.

[0192] The involvement of FcγR was further investigated by SPR binding assay, where panitumumab samples up to 10 μM were tested for binding to FcγRI, FcγRIIa 131H, and FcγRIIIa 158V. Binding was detected only to FcγRIIa 131H with an apparent KD of 20 μM. The IgG2 control mAb used in this study also showed similar binding activity to only FcγRIIa with a KD of 25 μM. Furthermore, fucose-enriched and non-fucose-enriched samples were generated to detect differences in binding activity. In the context of the SPR binding assay, the significant difference in binding between the two glycan-enriched samples and the fucose- and non-fucose-enriched samples was not as dramatic as seen in the functional assay, with the samples binding with KDs of 7.9 μM and 8 μM, respectively. Engagement of FcγRs with immunoreceptor tyrosine-based activation motifs (ITAMs) by IgG complexes initiates a number of signaling cascades that lead to cell activation and subsequent induction of effector functions. The cellular response to Fc-FcγR interactions varies depending on the myeloid cell type; however, aggregation of FcγRs typically leads to rapid internalization of FcγRs and activation of various signaling pathways that affect cell activation (Unkekess et al.,Semin Immunol.1995;7(1):37-44.PubMed PMID:7612894;Amigorena et al.,Science.1992;256(5065):1808-1812;Amigorena et al.,Nature.1992;358(6384):337-341;Regnault et al.,J Exp Med.1999;189(2):371-380). The difference between non-fucosylated and fucosylated panitumumab may be less evident in the Biacore binding assay due to these missing elements such as clustering and signal amplification that the cell-based assay may introduce.

[0193] This study, with extensive characterization combined with responsive functional assays, revealed significant impact of protected Fc glycans on novel IgG2-mediated cytotoxic activity, an understanding that needs to be taken into account when designing and characterizing therapeutic IgG2 drug candidates.

[0194] All publications, patents, and patent applications cited herein are incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present disclosure that certain changes and modifications can be made thereto without departing from the spirit or scope of the disclosed embodiments. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0195] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each of the separate values ​​falling within the range, and each of the endpoints, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0196] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended only to clarify the disclosure and does not impose limitations on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0197] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the present disclosure. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will adopt such variations as necessary, and the inventors intend for the present disclosure to be practiced in other forms than those specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed in the present disclosure unless otherwise indicated herein or clearly contradicted by context.

[0198] The present invention relates in particular to the following embodiments: 1. A method of modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab comprising increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or increasing or decreasing the amount of panitumumab molecules containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0199] 2. The method of embodiment 1, wherein FcγR-mediated cytotoxicity of panitumumab is increased by increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab or by increasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0200] 3. The method of embodiment 1, wherein FcγR-mediated cytotoxicity of panitumumab is reduced by decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab or by increasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0201] 4. The method of embodiment 1, wherein said FcγR is FcγRIIa.

[0202] 5. The method of embodiment 1, wherein said FcγR-mediated cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0203] 6. A method for matching Fc gamma receptor (FcγR)-mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab samples; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or by increasing or decreasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is about 35% or less.

[0204] 7. The method of embodiment 6, wherein FcγR-mediated cytotoxicity of the panitumumab sample is increased by increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or by increasing the amount of panitumumab molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0205] 8. The method of embodiment 6, wherein FcγR-mediated cytotoxicity of the panitumumab sample is reduced by decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or by decreasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0206] 9. The method of embodiment 6, wherein the FcγR is FcγRIIa.

[0207] 10. The method of embodiment 6, wherein said FcγR-mediated cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0208] 11. A method for modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing or decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or increasing or decreasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site.

[0209] 12. The method of embodiment 11, wherein the FcγR-mediated cytotoxicity of panitumumab is increased by increasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site or decreasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site.

[0210] 13. The method of embodiment 11, wherein the FcγR-mediated cytotoxicity of panitumumab is reduced by decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site or by increasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site.

[0211] 14. The method of embodiment 11, wherein said FcγR is FcγRIIa.

[0212] 15. The method of embodiment 11, wherein said FcγR-mediated cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0213] 16. A method for matching Fc gamma receptor (FcγR)-mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab samples; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or by increasing or decreasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and a reference value is about 35% or less.

[0214] 17. The method of embodiment 16, wherein FcγR-mediated cytotoxicity of the panitumumab sample is increased by increasing the amount of panitumumab molecules comprising a fucosylated glycan at the N-297 site or decreasing the amount of panitumumab molecules comprising a non-fucosylated glycan at the N-297 site.

[0215] 18. The method of embodiment 16, wherein FcγR-mediated cytotoxicity of the panitumumab sample is reduced by decreasing the amount of panitumumab molecules that comprise a fucosylated glycan at the N-297 site or by increasing the amount of panitumumab molecules that comprise a non-fucosylated glycan at the N-297 site.

[0216] 19. The method of embodiment 16, wherein said FcγR is FcγRIIa.

[0217] 20. The method of embodiment 16, wherein said FcγR-mediated cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0218] 21. A method for modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing or decreasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0219] 22. The method of embodiment 21, wherein the FcγR-mediated cytotoxicity of panitumumab is increased by decreasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0220] 23. The method of embodiment 21, wherein the FcγR-mediated cytotoxicity of panitumumab is reduced by increasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0221] 24. The method of embodiment 21, wherein the FcγR is FcγRIIa.

[0222] 25. The method of embodiment 21, wherein said FcγR-mediated cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0223] 26. A method for matching Fc gamma receptor (FcγR)-mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab samples; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and a reference value is about 35% or less.

[0224] 27. The method of embodiment 26, wherein FcγR-mediated cytotoxicity of the panitumumab sample is increased by decreasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0225] 28. The method of embodiment 26, wherein FcγR-mediated cytotoxicity of the panitumumab sample is reduced by increasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0226] 29. The method of embodiment 26, wherein the FcγR is FcγRIIa.

[0227] 30. The method of embodiment 26, wherein said FcγR-mediated cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

[0228] 31. A method for increasing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising: (i) increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or increasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site; (ii) increasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or decreasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site, and / or (iii) reducing the amount of panitumumab molecules containing high mannose glycans at the N-297 site.

[0229] 32. A method for reducing Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab, comprising: (i) decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or decreasing the amount of panitumumab molecules that contain G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site; (ii) decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or increasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site, and / or (iii) increasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site. The present invention provides, for example, the following items. (Item 1) A method of modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or increasing or decreasing the amount of panitumumab molecules containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. (Item 2) A method for modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing or decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or increasing or decreasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site. (Item 3) A method for modulating Fc gamma receptor (FcγR) mediated cytotoxicity of panitumumab, comprising increasing or decreasing the amount of panitumumab molecules containing high mannose glycans at the N-297 site. (Item 4) 1. A method for matching Fc gamma receptor (FcγR) mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab sample; (3) altering the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or by increasing or decreasing the amount of panitumumab molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is about 35% or less. (Item 5) 1. A method for matching Fc gamma receptor (FcγR) mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab sample; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of panitumumab molecules that contain a fucosylated glycan at the N-297 site, or by increasing or decreasing the amount of panitumumab molecules that contain a non-fucosylated glycan at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is about 35% or less. (Item 6) 1. A method for matching Fc gamma receptor (FcγR) mediated cytotoxicity of a panitumumab sample with a reference value, comprising: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the panitumumab sample; (3) modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of panitumumab molecules that contain high mannose glycans at the N-297 site, such that the difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is about 35% or less. (Item 7) 7. The method of any one of items 1 to 6, wherein the FcγR-mediated cytotoxicity of panitumumab is increased by increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab or by increasing the amount of panitumumab molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. (Item 8) 7. The method of any one of items 1 to 6, wherein the FcγR-mediated cytotoxicity of panitumumab is reduced by decreasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab or by decreasing the amount of panitumumab molecules comprising G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. (Item 9) The method according to any one of items 1 to 6, wherein the FcγR is FcγRIIa. (Item 10) The method of item 1, wherein the FcγR-mediated cytotoxicity is FcγRIIa-mediated cellular cytotoxicity.

Claims

[Claim 1] The invention described in the specification.

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