Adjustment of antibody effector function

Abstract

Provided herein are methods for modulating Fc gamma receptor (FcγR)-mediated cytotoxicity of an antibody composition. In exemplary embodiments, the method comprises: (1) 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; (2) increasing or decreasing the amount of panitumumab molecules containing fucosylated glycans at the N-297 site, or increasing or decreasing the amount of panitumumab molecules containing non-fucosylated glycans at the N-297 site; and (3) increasing or decreasing the amount of panitumumab molecules containing high-mannose glycans at the N-297 site.

Description

Technical Field

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

Background Art

[0002] Monoclonal antibodies have become widely used as therapeutic agents for treating the pathophysiology of a wide range of metabolic, inflammatory, and oncology diseases. IgG1 and IgG2, which are the most common human antibody subclasses used as biotherapeutics, have significantly different immunological properties and are usually selected as drug candidates based on the desired mechanism of action. The killing of target cells that may be desirable as cancer indicators attempts to utilize IgG1-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). These can potentially be easily mediated by IgG1 antibodies, but IgG2 is originally thought to be unable to bring about such effects (RAVETCH, J.V., AND S. BOLLAND. 2001, ANNU. REV. IMMUNOL. 19:275-290). However, in recent years, it has been found that panitumumab, a human IgG2 EGFR antagonist adapted for the treatment of metastatic colorectal cancer, can mediate cytotoxicity (SCHNEIDER-MERCK, J IMMUNOL 2010; 184:512-520). This has been shown to be mediated mainly through myeloid cells (monocytes and neutrophils) and is mediated by FcγRIIa, in contrast to the conventional ADCC mediated by IgG1 through lymphocyte-derived natural killer (NK) cells and associated with FcγRIIIa).

[0003] In the manufacture of therapeutic monoclonal antibodies, it is necessary to define and monitor key quality characteristics that affect product safety and efficacy in order to ensure the required product quality. It is well established that specific glycan structures of IgG1 related to the conserved glycans of the Fc CH2 domain can strongly influence interactions with ADCC, ADCP, and FcγR, which mediates C1q binding that initiates CDC (REUSCH D, TEJADA ML., GLYCOBIOLOGY 2015;25:1325-34). However, there are no studies investigating the impact of product quality characteristics of the IgG2 molecule on immune-mediated cytotoxic activity. The Fc receptor is a crucial immunomodulatory receptor that connects antibody-mediated (humoral) immune responses to cellular effector function. The Fc gamma receptor, located on the surface of effector cells (such as natural killer cells, macrophages, or monocytes), binds to the Fc region of IgG, causing IgG itself to bind to target cells. Fc binding triggers signaling pathways that lead to the secretion of various substances that mediate the destruction of target cells. The level of cytotoxic effector function varies depending on the human IgG subtype. Human IgG1 and IgG3 mediate higher effector function compared to IgG2 or IgG4 due to their good binding to FcγR (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 to therapeutic efficacy is not fully understood, nor are the quality characteristics that affect IgG2-mediated cytotoxicity well understood. Quality characteristics that influence and predict IgG2-mediated cytotoxicity, and are therefore suitable for monitoring during the production of IgG2 antibodies, have not been well established. Therefore, it is necessary to understand how specific quality characteristics affect IgG2-mediated cytotoxicity and to adjust such characteristics 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 [Overview of the Initiative] [Means for solving the problem]

[0005] Based on the description herein, those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments of the invention described herein through simple routine experiments. Such equivalents shall be included in the following embodiment (E).

[0006] E1. A method for modulating panitumumab-mediated Fc gamma receptor (FcγR)-mediated cytotoxicity, 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 a panitumumab molecule containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0007] E2. A method for increasing panitumumab-mediated Fc-gamma receptor (FcγR) mediated cytotoxicity, 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 according to E1 or E2, wherein an increase of approximately 1 percent in β-galactose increases FcγR-mediated cytotoxicity by approximately 0.55 percent to approximately 0.75 percent, for example, approximately 0.55 percent, approximately 0.6 percent, approximately 0.65 percent, approximately 0.7 percent, or approximately 0.75 percent.

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

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

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

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

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

[0014] E9. A method for matching a panitumumab sample with a reference value for Fc-gamma receptor (FcγR)-mediated cytotoxicity, wherein: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) To determine FcγR-mediated cytotoxicity in the panitumumab sample; (3) A method for modifying the FcγR-mediated cytotoxicity of a 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 a panitumumab molecule containing 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 a reference value is approximately 35% or less.

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

[0016] E11. The method according to E9 or E10, wherein FcγR-mediated cytotoxicity of a 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 containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

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

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

[0019] A method according to any one of E9, E10, or E13, wherein a decrease of about 1 percent of β-galactose decreases FcγR-mediated cytotoxicity by about 0.55 percent to about 0.75 percent, such as about 0.55 percent, about 0.6 percent, about 0.65 percent, about 0.7 percent, or about 0.75 percent.

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

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

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

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

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

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

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

[0027] E22. The method according to any one of E18-E21, wherein an increase of approximately 1 percent in the fucosylated panitumumab molecule increases FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent.

[0028] E23. The method according to any one of E18-E21, wherein a reduction of approximately 1 percent in the non-fucosylated panitumumab molecule increases FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent.

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

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

[0031] E26. The method according to any one of E18-E19 and E24-E25, wherein a reduction of approximately 1 percent in the fucosylated panitumumab molecule reduces FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent.

[0032] E27. The method according to any one of E18-E19 and E24-E25, wherein an increase of approximately 1 percent in the non-fucosylated panitumumab molecule increases FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent.

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

[0034] E29. The method according to any one of E18 to E28, wherein the above-mentioned FcγR-mediated cytotoxicity is measured by an in vitro cytotoxic assay such as the KILR® cytotoxicity assay.

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

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

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

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

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

[0040] E35. The method according to any one of E31-E34, wherein an increase of approximately 1 percent in the fucosylated panitumumab molecule increases FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent.

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

[0042] E37. The method according to any one of E31-E34 and E36, wherein a decrease of approximately 1 percent in the non-fucosylated panitumumab molecule increases FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent.

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

[0044] E39. The method according to any one of E31-E33 and E38, wherein a reduction of approximately 1 percent in the fucosylated panitumumab molecule reduces FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent.

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

[0046] E41. The method according to any one of E31-E33 and E40, wherein an increase of approximately 1 percent in the non-fucosylated panitumumab molecule reduces FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent.

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

[0048] E43. The method according to any one of E31 to E42, wherein the above-mentioned FcγR-mediated cytotoxicity is measured by an in vitro cytotoxic assay such as the KILR® cytotoxicity assay.

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

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

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

[0052] E47. The method according to E45 or E46, wherein a decrease of approximately 1 percent in high mannose glycan increases FcγR-mediated cytotoxicity by approximately 1.20 percent to approximately 1.40 percent, for example, approximately 1.2 percent, approximately 1.25 percent, approximately 1.3 percent, approximately 1.35 percent, or approximately 1.40 percent.

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

[0054] E49. The method according to E45 or E48, wherein a reduction of approximately 1 percent in high-mannose glycan reduces FcγR-mediated cytotoxicity by approximately 1.20 percent to approximately 1.40 percent, for example, approximately 1.2 percent, approximately 1.25 percent, approximately 1.3 percent, approximately 1.35 percent, or approximately 1.40 percent.

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

[0056] E51. The method described in any one of E45 to E50, wherein the above FcγR is FcγRIIa.

[0057] E52. The method according to any one of E45 to E51, wherein the above-mentioned FcγR-mediated cytotoxicity is measured by an in vitro cytotoxic assay such as the KILR® cytotoxicity assay.

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

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

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

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

[0062] E57. The method according to any one of E54-E56, wherein a decrease of approximately 1 percent in high mannose glycan increases FcγR-mediated cytotoxicity by approximately 1.2 percent to approximately 1.40 percent, for example, approximately 1.2 percent, approximately 1.25 percent, approximately 1.3 percent, approximately 1.35 percent, or approximately 1.40 percent.

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

[0064] E59. The method according to any one of E54, E55, or E58, wherein an increase of approximately 1 percent in high mannose glycan reduces FcγR-mediated cytotoxicity by approximately 1.2 percent to approximately 1.40 percent, for example, approximately 1.2 percent, approximately 1.25 percent, approximately 1.3 percent, approximately 1.35 percent, or approximately 1.40 percent.

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

[0066] E61. The method described in any one of E54-E60, wherein the above FcγR is FcγRIIa.

[0067] E62. The method according to any one of E54 to E61, wherein the above-mentioned FcγR-mediated cytotoxicity is measured by an in vitro cytotoxic assay such as the KILR® cytotoxicity assay.

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

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

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

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

[0072] An antibody composition produced by any one of the methods described in E67.E1 to E66.

[0073] A pharmaceutical composition comprising the antibody composition described in E68.E67 and a pharmaceutically acceptable carrier, diluent, or excipient. [Brief explanation of the drawing]

[0074] [Figure 1A]Figure 1A illustrates three types of N-glycans (oligomannose type, complex type, and hybrid type) and the symbols commonly used for such sugars. [Figure 1B] Figure 1B shows the major N-linked glycans found at the N-glycosylation site asparagine (Asn) 297 in human IgG, which have typical oligosaccharide linkages. These glycans typically contain a heptasaccharide core and outer arms constructed by variable additions of fucose, N-acetylglucosamine (GlcNAc), galactose, sialic acid (SA), and bisected N-GlcNAc. [Figure 1C] Figure 1C summarizes the structures of three major glycan species evaluated in a cytotoxicity reporter gene assay, including non-fucosylated species (i.e., core-fucose-deficient species including G0 or G1), high-mannose species (including M5), or terminal β-galactose species (i.e., terminal β-galactose species including G1F or G2F). [Figure 1D] Figure 1D provides a basic schematic flowchart summarizing the preparation of glycan concentration and manipulated samples. [Figure 1E] Figure 1E shows the salvage and de novo pathways of fucose metabolism. In the salvage pathway, free L-fucose is converted to GDP-fucose, while in the de novo pathway, GDP-fucose is synthesized via three reactions catalyzed by GMD and FX. Next, GDP-fucose is transported from the cytoplasm to the Golgi lumen by GDP-Fuctransferase and transferred to acceptor oligosaccharides and proteins. The other reaction product, GDP, is converted to guanosine 5-monophosphate (GMP) and inorganic phosphate (Pi) by nucleotide diphosphatases in the lumen. The former is effluxed into the cytoplasm (via a countertransport system coupled with GDP-fucose transport), while the latter is presumed 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 levels. Figure 2B shows an overlay of representative dose-response curves. 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 the correlated line graph (Figure 2B). This data demonstrated the quantitative nature and range of the ADCC reporter gene assay and its suitability for evaluating quality characteristics affecting ADCC activity. Higher levels of β-galactose generally result in greater cytotoxicity. [Figure 3A] Figure 3A shows FcγRIIa signaling activity as a function of the non-fucosylation level. [Figure 3B] Figure 3B shows an overlay of representative dose-response curves. A linear regression line (equation shown) was fitted to the plot of measured activity (Figure 3A), and representative dose-response curves are shown for various activity levels in the correlated line graph (Figure 3B). Higher levels of non-fucosylation generally result in less cytotoxicity. [Figure 4A-4B] Figure 4A shows FcγRIIa signaling activity as a function of high mannose levels. Figure 4B shows an overlay of representative dose-response curves. 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 the correlated line graph (Figure 4B). Higher levels of high mannose generally result in less cytotoxicity. [Figure 5] Figure 5 shows a typical dose curve of PBMC-mediated cytotoxicity using concentrated glycan samples with the KILR assay. [Figure 6A-6B] Figures 6A-6D are graphs showing PBMC-mediated cytotoxicity as a function of panitumumab non-fucosylated 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. [Figures 7A-7B]Figures 7A-7D are graphs showing PBMC-mediated cytotoxicity as a function of high-mannose levels of panitumumab 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. [Figure 8C-8D] Same as above. [Figure 9A-9B] Figure 9A shows a representative panitumumab dose curve for PBMC cytotoxic activity. Figure 9B is a plot showing FcγR blocking using antibodies against the indicated receptor or control. [Figure 10A-10B] Figures 10A-10C are SPR sensorograms showing that panitumumab or control binds to (A) FcγRI with up to 10 μM of antibody; to (B) FcγRIIIa-158F with up to 10 μM of antibody; and to (C) FcγRIIa-131H with up to 10 μM of antibody. [Figure 10C] Same as above. [Figures 11A-11B] Figures 11A and 11B show the SPR equilibrium binding curves of panitumumab, where (A) the fucose-concentrated sample and (B) the non-fucose-concentrated sample bind to huFcgRIIa-131H with apparent KD values ​​of approximately 7.9 μM and 8 μM, respectively. [Modes for carrying out the invention]

[0075] 1. Overview IgG1 and IgG2, the most common human antibody subclasses used in biotherapy, differ significantly in their immunological properties. Common effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), may be important mechanisms of action for IgG1 antibodies. Previously, it was not known that IgG2 antibodies exhibited effector function. However, in recent years, panitumumab has been shown to mediate cytotoxic effects similar to ADCC by binding to FcγRIIa. This is in contrast to conventional ADCC mediated by IgG1 binding to FcγRIIIa.

[0076] Panitumumab is a human IgG2 monoclonal antibody that binds to the human epidermal growth factor receptor (EGF receptor, also known as 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 Sequence ID No. 1 and No. 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 in Sequence ID No. 1.

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

[0078] For example, galactosylation at the N-297 site showed a positive correlation with the reporter gene, while non-fucosylation levels and high mannose levels at the N-297 site showed an inverse correlation with cell toxicity. Therefore, panitumumab-mediated FcγR cytotoxicity may 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, panitumumab-mediated FcγR cytotoxicity may 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 modified mammalian (Chinese hamster ovary) cells. During the recombinant production process, the glycan moiety is bound to the antibody by post-translational modification. The findings made herein by the inventors provide a quantifiable relationship between the glycoform profile of panitumumab and its cytotoxicity. This finding can be used to modulate the glycosylation pattern during the CHO cell production process so that the cytotoxicity level meets a desired reference level.

[0080] 2.Definition "Panitumumab" (trade name Vectibix®) refers to a human monoclonal antibody containing a heavy chain with SEQ ID NO: 1 and a light chain with 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 monosaccharides 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 bound to the N-glycosylation site of Asn-297 (Kabat EU numbering) and generally consists of a heptasaccharide core with an outer arm constructed by variable additions 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 refers to a core GlcNAc and mannose oligosaccharide structure having 0, 1, or 2 terminal galactose molecules, respectively. G1 may have two additional structures, abbreviated as G1a and G1b, where G1a or G1b indicates whether the terminal galactose group is bound to the 6-arm or 3-arm of the core structure. See Figures 1B and 1C. When fucosylated (i.e., when a fucose group is bound to the coreglycan structure), 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," for example, G2FS2 refers to a glycan having two galactose, fucose, and two sialic acid groups. Further glycans linked to antibodies may also exist, including high-mannose (HM) structures formed by incorporating additional mannose groups (e.g., "Man5" or "M5" as shown in Figure 1C and Table 2).As used herein, the terms “glycan” or “glycans” refer to any of the monosaccharide oligomers described herein, or any other monosaccharide oligomer 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 scheme. A complete chart comparing different numbering schemes 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 glycans containing one or two galactose molecules linked to an IgG antibody at an N-glycosylation site (Asn-297) via an N-acetylglucosamine moiety bound to a core mannose structure. Exemplary glycans containing “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 glycan may or may not contain core fucose.

[0087] The terms "core fucose" or "fucosylated species" refer to glycans containing fucose molecules (alpha-1-6) linked to an IgG antibody at an N-glycosylation site (Asn-297) via an N-acetylglucosamine moiety bound to a core mannose structure. Exemplary glycans containing "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 “non-fucosylated,” “non-fucosylated glycan,” or “non-fucosylated” refer to the removal or absence of core fucose on an antibody. Exemplary non-fucosylated glycans are shown in Figures 1B and 1C. In some embodiments, antibodies lacking core fucose may or may not contain other glycans (including terminal β-galactose and / or high-mannose glycans). Examples of non-fucosylated glycoforms include, but are not limited to, A1G0, A1G1a, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. See, for example, Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015).

[0089] The terms "high mannose," "high mannose glycan," or "HM" refer to glycans containing more than three mannose molecules linked to an IgG antibody at an N-glycosylation site (Asn-297). Exemplary high mannose antibodies, including "Man-5 high mannose glycan" containing two additional mannose molecules, are shown in Figure 1C and Table 2. High mannose glycans encompass glycans containing 5, 6, 7, 8, or 9 mannose residues, respectively, abbreviated as Man5 or M5, Man6 or M6, Man7 or M7, Man8 or M8, and Man9 or M9. 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 involved in inducing phagocytosis in opsonized cells or microorganisms. See, for example, 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 for FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcγRIIIB can be found in many sequence databases, such as 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,” as well as similar reference subjects, relating to the description of this disclosure (particularly the claims below), should be interpreted as encompassing both singular and plural unless otherwise indicated herein or unless explicitly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open terms unless otherwise specified (i.e., “comprising, but not limited to,” allowing for the presence of one or more features or components). The terms “a” (or “an”), and “one or more” and “at least one” can be used interchangeably herein. Furthermore, “and / or,” as used herein, should be interpreted as each of two specific features or components in a specific disclosure (whether or not there are other features or components). Therefore, when the term "and / or" is used in this specification in phrases such as "A and / or B," it is intended to encompass "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it is intended to encompass 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 (alone); B (alone); and C (alone).

[0093] The term “about” used in relation to numerical values ​​throughout the specification and claims indicates an interval of precision that is well known and accepted by those skilled in the art. Generally, such an interval of precision is ±10%.

[0094] 3. Posttranslational glycosylation and FcγR-mediated cytotoxicity 3.1 Post-translational glycosylation Many secreted proteins undergo post-translational glycosylation, a process in which a sugar moiety (e.g., glycans, sugars) is covalently bonded to specific amino acids in the protein. In eukaryotic cells, two types of glycosylation occur: (1) N-linked glycosylation, in which glycans are linked to asparagine at the recognition sequence Asn-X-Thr / Ser (where "X" is any amino acid other than proline), and (2) O-linked glycosylation, in which glycans are linked to serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), the glycan structures that can be bound to each site (O or N) are wide-ranging, resulting in minute heterogeneity in protein glycoforms. For IgG2 antibodies, N-linked glycosylation occurs at the asparagine-297 (N-297) site (Eu numbering system). In the case of panitumumab, the actual location of this asparagine occurs at residue number 295, but nevertheless, the N-glycosylation site is generally referred to as N-297 to match 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) type consisting of two N-acetylglucosamine (GalNAc) moieties and numerous (e.g., 5, 6, 7, 8, or 9) mannose (Man) residues; (B) complex type containing three or more GlcNAc moieties and any number of other sugar types; or (C) hybrid type containing Man residues on one branch and GlcNAc at the base of the complex branch. Figure 1A (Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2 nd (Based on ed., Cold Spring Harbor Laboratory Press; 2009) shows three types of N-glycans.

[0096] N-linked glycans typically contain one or more monosaccharides, including 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 glycan structures vary, particularly depending on the glycosylation mechanism in the ER and Golgi apparatus, the accessibility of enzymes in that mechanism to the glycan structure, the order of action of each enzyme, and the stage at which the protein is released from the glycosylation mechanism. Since the glycan structure that binds to the Fc domain affects the interaction with FcγR, which mediates cell damage, controlling glycan structure is important in the recombinant production of therapeutic monoclonal antibodies.

[0098] 3.2 Glycans that affect FcγR-mediated cytotoxicity This disclosure elucidates the effects of various glycans (e.g., β-galactose, core-fucose, and / or high-mannose) on FcγR-mediated cytotoxicity by IgG2 antibodies such as panitumumab. Therefore, this disclosure provides methods for modulating Fc-gamma receptor (FcγR)-mediated cytotoxicity by IgG2 antibodies (such as panitumumab) or antibody-containing compositions (antibody compositions). In exemplary embodiments, the methods include modulating the amounts of (a) galactosylated glycans of the antibody, (b) non-fucosylated glycans of the antibody, (c) high-mannose glycans of the antibody, or (d) combinations thereof. While not bound by any particular theory, the methods disclosed herein are considered to provide means for tailor-made compositions containing specific glycoforms of a given antibody in specific amounts that exhibit a target level of FcγR-mediated cytotoxicity. Particularly relevant glycan structures are shown in Figure 1C.

[0099] In exemplary embodiments, the methods provided by the Disclosure relate to the modification of an IgG2 antibody (such as panitumumab) or a composition comprising an antibody (antibody composition), wherein the modification involves a step of achieving a desired or predetermined level of the glycoform of the IgG2 antibody such that the antibody or antibody composition exhibits a desired or predetermined reference level of FcγR-mediated cytotoxicity. In exemplary embodiments, the methods include modifying (increasing or decreasing) antibody-induced or stimulated FcγR-mediated cytotoxicity by modifying (increasing or decreasing) the amount of (a) galactosylated glycan, (b) non-fucosylated glycan, (c) high-mannose glycan, or (d) a combination thereof of the IgG2 antibody (such as panitumumab). In exemplary embodiments, the method includes modulating (increasing or decreasing) antibody-induced or stimulated FcγR-mediated cytotoxicity by adjusting (increasing or decreasing) the amount of a glycoform, for example, (a) a galactosylated glycoform, (b) a non-fucosylated glycoform, (c) a high-mannose glycoform, or (d) a combination thereof.

[0100] The term "quantity" refers to the relative percentage of a specific glycan in N-297 compared to the total amount of glycans in N-297, especially when referring to the amount of glycans (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). Since it is impractical / impossible to count glycan species at the individual molecular level, the glycan content amounts described herein are generally calculated based on relative percentages obtained by commonly used analytical methods. For example, as illustrated in Example 2.2, an enzyme is used to release all N-glycans from the protein, and then the glycans are separated by hydrophobic interaction liquid chromatography (HILIC). HILIC yields various peaks, each representing a glycan species. The amount of a particular glycan is calculated as a relative percentage based on the area of ​​that peak within the total area of ​​all peaks. Therefore, unless otherwise specified, the amount of glycan refers to the relative percentage of that particular glycan species among all N-glycans at the N-297 site, using one of the general analytical methods (HPAEC, CE-SDS, HILIC, or LC-MS, etc.).

[0101] Methods for measuring and determining the quantity or relative percentage of glycans (including, for example, 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. These references are incorporated herein by reference for all purposes. In some embodiments, the amount may be determined or calculated as an incorporated mole percentage.

[0102] In some embodiments, the methods disclosed herein involve adjusting the amount of terminal β-galactose, core fucose, or high mannose, or a combination thereof, bound to a specific IgG2 molecule (such as panitumumab).

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

[0104] In other exemplary embodiments, the method may include increasing the amount of core fucose on IgG2 (e.g., panitumumab) to increase FcγR-mediated cytotoxicity. FcγR-mediated cytotoxicity may be increased by increasing the amount of antibody molecules containing fucosylated glycans at the N-297 site or by decreasing the amount of antibody molecules containing non-fucosylated glycans at the N-297 site. Alternatively, the method may include decreasing the amount of core fucose on IgG2 (e.g., panitumumab) to decrease FcγR-mediated cytotoxicity. FcγR-mediated cytotoxicity may be decreased by decreasing the amount of antibody molecules containing fucosylated glycans at the N-297 site or by increasing the amount of antibody molecules containing non-fucosylated glycans at the N-297 site.

[0105] In other exemplary embodiments, the method may include increasing FcγR-mediated cytotoxicity by reducing the amount of high-mannose (e.g., Man-5) on IgG2 (e.g., panitumumab). FcγR-mediated cytotoxicity may be increased by reducing the amount of antibody molecules containing high-mannose glycan at the N-297 site. Alternatively, the method may include decreasing FcγR-mediated cytotoxicity by increasing the amount of mannose (e.g., Man-5) on IgG2 (e.g., panitumumab). FcγR-mediated cytotoxicity may be decreased by increasing the amount of antibody molecules containing 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 ("activated FcRs") and those that inhibit activation upon co-binding ("inhibitory FcRs"). In humans, IgG has two low-affinity activated FcRs: FcγRIIa and FcγRIIIa. FcγRIIa (or FcγRIIA) is a single-chain low-affinity receptor for IgG, with an ITAM sequence in its cytoplasmic tail. It is expressed in macrophages, mast cells, monocytes, neutrophils, and some B cells. It has an ITIM sequence in its cytoplasmic domain and is expressed in B cells, macrophages, mast cells, neutrophils, and monocytes, but not in NK cells or T cells. It is 90% homologous in its extracellular domain to the human inhibitory FcRIIb molecule. FcγRIIIa (or FcγRIIIA) is an oligomeric activated receptor consisting of an ITAM containing a ligand-binding subunit and a gamma or zeta subunit. It is expressed in NK cells, macrophages, and mast cells. It is not expressed in neutrophils, B cells, or T cells. In addition, a receptor called FcRIIIb, which has more than 95% sequence identity in its extracellular domain, is found in human neutrophils as a GPI-anchored protein. Without binding to an ITAM-containing receptor such as FcRIIa, it can bind to immune complexes but cannot activate cells. FcRII and FcRIII are approximately 70% identical in their ligand-binding extracellular domains.

[0107] Therefore, in humans, IgG cytotoxic antibodies interact with four different low-affinity receptors—two of which are FcRIIa and FcRIIIa, which can activate cellular responses; one of which is inhibitory FcRIIb; and one of which is FcRIIIb, which binds to the IgG complex but does not trigger a cellular response. Macrophages express FcRIIa, FcRIIb, and FcRIIIa, neutrophils express FcRIIa, FcRIIb, and FcRIIIb, but NK cells express only FcRIIIa. Thus, the efficacy of therapeutic antitumor antibodies depends on specific interactions with activating, inhibitory, and inactive low-affinity FcR receptors, which are differentially expressed in 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, as well as an in vivo system using A431 cell xenografts in athymic mice, to study the cytotoxicity of EGFR-binding IgG1 and IgG2 antibodies.

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

[0110] In certain embodiments, FcγR-mediated cytotoxicity is FcγRIIa-mediated cytotoxicity.

[0111] In certain embodiments, the FcγR-mediated cytotoxicity 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 the FcγR receptor, an NFAT response element, and / or a reporter gene. The reporter gene may be any gene whose expression provides a measurable signal. Exemplary reporter genes include green fluorescent protein (GFP), antibiotic resistance proteins (e.g., chloramphenicol transferase), toxic 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 from the American firefly, Photinus pyralis (EC 1.13.12.7), and reniral luciferase from the sea slug, Renilla reniformis (EC 1.13.12.7). Examples include the gene encoding bacterial luciferase (EC 1.14.14.3) from Photobacterium fischeri. Various other reporter genes are well known to those skilled in the art. In exemplary embodiments, the reporter gene encodes luciferase.

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

[0113] In certain embodiments, the Disclosure provides a method for increasing FcγR-mediated cytotoxicity of an IgG2 antibody (such as panitumumab) or an antibody-containing composition compared to a control or reference value. In exemplary embodiments, the increase is at least or about 0.1% to about 100% compared to a control or reference value (e.g., at least or about 0.1%, at least or about 0.2%, at least or about 0.3%, at least or about 0.4%, at least or about 0.5%, at least or about 0.55%, at least or about 0.6%, at least or about 0.65%, at least or about 0.7%, at least or about 0.75%, at least or about 0.8%, at least or about 0.9%, at least or about 1%, at least or about 1.2%, at least or about 1.25%, at least or... An increase of approximately 1.3%, at least or approximately 1.35%, at least or approximately 1.4%, at least or approximately 1.5%, at least or approximately 2%, at least or approximately 2.5%, at least or approximately 2.7%, at least or approximately 2.75%, at least or approximately 2.8%, at least or approximately 2.85%, at least or approximately 2.9%, at least or approximately 2.95%, at least or approximately 3%, at least or approximately 4%, at least or approximately 5%, at least or approximately 6%, at least or approximately 7%, at least or approximately 8%, at least or approximately 9%, at least or approximately 9.(A 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%, for example, 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 antibody-containing composition is increased by at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, or at least about 1.9 times, compared to a control or reference value. In exemplary embodiments, FcγR-mediated cytotoxicity of an antibody or antibody-containing composition is increased by at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, or at least about 10 times compared to a control or reference value. In exemplary embodiments, FcγR-mediated cytotoxicity of an antibody or antibody-containing composition is increased by about 1.It increases by approximately 1x to 10x, approximately 1.2x to 10x, approximately 1.3x to 10x, approximately 1.4x to 10x, approximately 1.5x to 10x, approximately 1.1x to 5x, approximately 1.2x to 5x, approximately 1.3x to 5x, approximately 1.4x to 5x, or approximately 1.5x to 5x.

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

[0115] As used herein, “control” or “reference value” refers to the level of FcγR-mediated cytotoxicity of an antibody or antibody-containing composition before any experimental intervention aimed at modulating the glycan profile (e.g., the level of cytotoxicity measured initially). If an antibody or antibody-containing composition has undergone an experimental intervention aimed at modulating the glycan profile, but further modification 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 demonstrated by a commercially available panitumumab sample at the same dose (e.g., the same amount of antibody molecules). In certain embodiments, the reference value is a predetermined level that provides a therapeutic effect.

[0117] In certain embodiments, the disclosure specifies that the amount of a particular glycan in the antibody (e.g., galactosyl glycan, G1, G1a, G1b and / or G2 galactosyl glycan, fucosylated glycan, non-fucosylated glycan, core fucose, high mannose glycan, Man-5 glycan, or a combination thereof) be 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. The invention provides a method that includes adjusting (i.e., increasing or decreasing) by 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 described above, when describing a specific glycan species, the percentage generally refers to the relative percentage of the total glycan content at the N-297 site, calculated according to one of the analytical methods accepted in the art (e.g., HILIC, LC-MS). In one exemplary embodiment, the relative percentage is calculated according to the area of ​​the chromatographic peak.

[0118] In certain embodiments, the Disclosure provides a method for modulating panitumumab-mediated Fc gamma receptor (FcγR)-mediated cytotoxicity, 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 a panitumumab molecule containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0119] In certain embodiments, the Disclosure provides a method for increasing panitumumab's Fc gamma receptor (FcγR)-mediated cytotoxicity, comprising increasing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or increasing the amount of a panitumumab molecule containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. In certain embodiments, an increase of about 1 percent of 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. As mentioned above, the percentages of β-galactose, or G1, G1a, G1b, and G2 galactosylated glycans, refer to the relative percentage of each 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., changes in the percentage level of a particular glycan and the corresponding changes in cytotoxicity levels), FcγR-mediated cytotoxicity is often expressed as a relative value quantified against a standard. For example, "relative activity percentage" (relative to standard) can be used to express the level of FcγR-mediated cytotoxicity. "Relative activity percentage" can be calculated as: (i) cytotoxic activity of the sample / cytotoxic activity of the standard (" / " means division); or (ii) cytotoxic activity of the standard / cytotoxic activity of the sample (" / " means division). For example, if sample A shows a 50% cytotoxicity level compared to the standard and sample B shows a 51% cytotoxicity level compared to the same standard, then FcγR-mediated cytotoxicity can be said to have increased by 1% from sample A to sample B.

[0121] In certain embodiments, the standard is the level of FcγR-mediated cytotoxicity demonstrated by a commercially available panitumumab sample at the same dose (e.g., the same amount of antibody molecules). Therefore, in certain embodiments, the quantitative relationship is established using the relative cytotoxicity level. Referring to Figure 2A, for example, when terminal β-galactose is about 0%, the relative cytotoxicity level (calculated for a commercially available panitumumab sample at the same dose) is about 88%. When terminal β-galactose increases to about 10%, the relative cytotoxicity level (calculated for a commercially available panitumumab sample at the same dose) is about 95%. Thus, terminal β-galactose correlates with an increase of about 0.67 percent 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 percent.

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

[0123] If necessary, the relative cytotoxicity of the sample can be measured multiple times (e.g., two, three, or four times), and the results can be reported as the average of these multiple values.

[0124] In certain embodiments, the disclosure provides a method for reducing panitumumab-mediated Fc gamma receptor (FcγR) mediated cytotoxicity, comprising reducing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or reducing the amount of a panitumumab molecule containing 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 reduces 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. Furthermore, the change in cytotoxicity levels is generally calculated based on the relative cytotoxicity values ​​as described above.

[0125] In certain embodiments, the Disclosure provides a method for modulating panitumumab-mediated Fc-gamma receptor (FcγR)-mediated cytotoxicity, comprising increasing or decreasing the amount of a panitumumab molecule containing a fucosylated glycan at the N-297 site, or increasing or decreasing the amount of a panitumumab molecule containing a non-fucosylated glycan at the N-297 site.

[0126] In certain embodiments, the Disclosure provides a method for increasing panitumumab's Fc-gamma receptor (FcγR)-mediated cytotoxicity, comprising increasing the amount of a panitumumab molecule containing a fucosylated glycan at the N-297 site. In certain embodiments, an increase of about 1 percent of the fucosylated panitumumab molecule increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3.0 percent, for example, 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 for increasing panitumumab's Fc-gamma receptor (FcγR)-mediated cytotoxicity, comprising decreasing the amount of a panitumumab molecule containing a non-fucosylated glycan at the N-297 site. In certain embodiments, a reduction of approximately 1 percent in the non-fucosylated panitumumab molecule increases FcγR-mediated cytotoxicity by approximately 2.70 percent to 3.0 percent, for example, approximately 3.0 percent, 2.95 percent, 2.90 percent, 2.85 percent, or 2.70 percent. Furthermore, changes in cytotoxicity levels are generally calculated based on relative cytotoxicity values ​​as described above.

[0127] In certain embodiments, the Disclosure provides a method for reducing panitumumab's Fc-gamma receptor (FcγR)-mediated cytotoxicity, comprising reducing the amount of panitumumab molecules containing a fucosylated glycan at the N-297 site. In certain embodiments, a reduction of about 1 percent of the fucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.70 percent to about 3.0 percent, for example, 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 for reducing panitumumab's Fc-gamma receptor (FcγR)-mediated cytotoxicity, comprising increasing the amount of panitumumab molecules containing a non-fucosylated glycan at the N-297 site. In certain embodiments, an increase of approximately 1 percent in the non-fucosylated panitumumab molecule reduces FcγR-mediated cytotoxicity by approximately 2.70 percent to approximately 3.0 percent, for example, approximately 3.0 percent, approximately 2.95 percent, approximately 2.90 percent, approximately 2.85 percent, or approximately 2.70 percent. Furthermore, changes in cytotoxicity levels are generally calculated based on relative cytotoxicity values ​​as described above.

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

[0129] In certain embodiments, the Disclosure provides a method for modulating panitumumab-mediated Fc-gamma receptor (FcγR)-mediated cytotoxicity, comprising increasing or decreasing the amount of a panitumumab molecule containing a high-mannose glycan at the N-297 site. In exemplary embodiments, the high-mannose glycan may be Man-5, Man-6, Man-7, Man-8, or Man-9. In exemplary embodiments, the high-mannose glycan is Man-5.

[0130] In certain embodiments, the disclosure provides a method for increasing panitumumab's Fc-gamma receptor (FcγR)-mediated cytotoxicity, comprising reducing the amount of panitumumab molecule containing high-mannose glycan at the N-297 site. In certain embodiments, a reduction of about 1 percent in high-mannose glycan increases FcγR-mediated cytotoxicity by about 1.2 percent to about 1.4 percent, for example, about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent. The change in cytotoxicity levels is generally calculated based on the relative cytotoxicity values ​​as described above. In certain embodiments, high-mannose is mannose-5 (Man-5).

[0131] In certain embodiments, the disclosure provides a method for reducing panitumumab's Fc-gamma receptor (FcγR)-mediated cytotoxicity, comprising increasing the amount of a panitumumab molecule containing a high-mannose glycan at the N-297 site. In certain embodiments, an increase of about 1 percent in high-mannose glycan reduces FcγR-mediated cytotoxicity by about 1.2 percent to about 1.4 percent, for example, about 1.2 percent, about 1.25 percent, about 1.3 percent, about 1.35 percent, or about 1.40 percent. The change in cytotoxicity levels is generally calculated based on the relative cytotoxicity values ​​as described above. In certain embodiments, the high-mannose is mannose-5 (Man-5).

[0132] The methods provided herein also include methods for matching the 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., galactosyl glycans, terminal β-galactose, G1, G1a, G1b and / or G2 galactosyl 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 embodiments, the reference value is the level of FcγR-mediated cytotoxicity demonstrated by a commercially available panitumumab sample at the same dose (e.g., the same amount of antibody molecules). In certain embodiments, 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 reference sample using the methods described herein. In exemplary embodiments, the determination or measurement of the cytotoxic activity of the antibody sample and / or reference sample is performed (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 embodiments, the present disclosure provides a method for matching the Fc gamma receptor (FcγR)-mediated cytotoxicity of an IgG2 antibody sample (such as a panitumumab sample) to a reference value, comprising: (1) obtaining a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the IgG2 antibody sample (such as a panitumumab sample); and (3) modifying the FcγR-mediated cytotoxicity of the 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 by increasing or decreasing the amount of IgG2 molecules containing 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 approximately 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 approximately 30% or less, approximately 25% or less, approximately 20% or less, approximately 15% or less, approximately 10% or less, or approximately 5% or less. In some examples, step (1) ("obtaining a reference value for FcγR-mediated cytotoxicity") is performed before, after, or simultaneously with step (2) ("determining the FcγR-mediated cytotoxicity of the IgG2 sample or panitumumab sample") and / or step (3) ("modifying the FcγR-mediated cytotoxicity of the IgG2 sample 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 embodiments, FcγR-mediated cytotoxicity of IgG2 or panitumumab samples 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 containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. In certain embodiments, an increase of approximately 1 percent in β-galactose increases FcγR-mediated cytotoxicity by approximately 0.55 percent to approximately 0.75 percent, for example, approximately 0.55 percent, approximately 0.6 percent, approximately 0.65 percent, approximately 0.7 percent, or approximately 0.75 percent. The calculation of glycan levels and cytotoxicity levels is as described above, and generally, changes in cytotoxicity levels are generally calculated based on relative cytotoxicity values ​​as described above.

[0135] In certain embodiments, FcγR-mediated cytotoxicity of IgG2 or panitumumab samples is reduced by decreasing the amount of terminal β-galactose at the N-297 glycosylation site of the antibody, or by reducing the amount of antibody molecules containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site. In certain embodiments, a reduction of approximately 1 percent of β-galactose increases FcγR-mediated cytotoxicity by approximately 0.55 percent to approximately 0.75 percent, for example, approximately 0.55 percent, approximately 0.6 percent, approximately 0.65 percent, approximately 0.7 percent, or approximately 0.75 percent. Furthermore, changes in cytotoxicity levels are generally calculated based on relative cytotoxicity values ​​as described above.

[0136] In certain embodiments, the present disclosure provides a method for matching the Fc-gamma receptor (FcγR)-mediated cytotoxicity of an IgG2 antibody sample (such as a panitumumab sample) to a reference value, comprising: (1) obtaining a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the IgG2 antibody sample (such as a panitumumab sample); and (3) modifying the FcγR-mediated cytotoxicity of the IgG2 antibody sample (such as a panitumumab sample) by increasing or decreasing the amount of IgG2 molecules containing fucosylated glycan at the N-297 site, or by increasing or decreasing the amount of IgG2 molecules containing 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 approximately 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 approximately 30% or less, approximately 25% or less, approximately 20% or less, approximately 15% or less, approximately 10% or less, or approximately 5% or less. In some examples, step (1) ("obtaining a reference value for FcγR-mediated cytotoxicity") is performed before, after, or simultaneously with step (2) ("determining the FcγR-mediated cytotoxicity of the IgG2 sample or panitumumab sample") and / or step (3) ("modifying the FcγR-mediated cytotoxicity of the IgG2 sample 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 embodiments, FcγR-mediated cytotoxicity of an IgG2 sample or a panitumumab sample is increased by increasing the amount of panitumumab molecules containing fucosylated glycan at the N-297 site, or by decreasing the amount of panitumumab molecules containing non-fucosylated glycan at the N-297 site. In certain embodiments, an increase of about 1 percent in fucosylated panitumumab molecules increases FcγR-mediated cytotoxicity by about 2.7 percent to about 3.0 percent, for example, 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 reduction of approximately 1 percent in the non-fucosylated panitumumab molecule increases FcγR-mediated cytotoxicity by approximately 2.7 percent to 3.0 percent, for example, approximately 3.0 percent, 2.95 percent, 2.90 percent, 2.85 percent, or 2.70 percent. Furthermore, changes in cytotoxicity levels are generally calculated based on relative cytotoxicity values ​​as described above.

[0138] In certain embodiments, FcγR-mediated cytotoxicity of an IgG2 sample or a panitumumab sample is reduced by decreasing the amount of panitumumab molecules containing fucosylated glycan at the N-297 site, or by increasing the amount of panitumumab molecules containing non-fucosylated glycan at the N-297 site. In certain embodiments, a reduction of about 1 percent in fucosylated panitumumab molecules reduces FcγR-mediated cytotoxicity by about 2.7 percent to about 3.0 percent, for example, 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 approximately 1 percent in the non-fucosylated panitumumab molecule reduces FcγR-mediated cytotoxicity by approximately 2.7 percent to 3.0 percent, for example, approximately 3.0 percent, 2.95 percent, 2.90 percent, 2.85 percent, or 2.70 percent. Furthermore, changes in cytotoxicity levels are generally calculated based on relative cytotoxicity values ​​as described above.

[0139] In certain embodiments, the present disclosure provides a method for matching the Fc gamma receptor (FcγR)-mediated cytotoxicity of an IgG2 antibody sample (such as a panitumumab sample) to a reference value, comprising: (1) obtaining a reference value for FcγR-mediated cytotoxicity; (2) determining the FcγR-mediated cytotoxicity of the IgG2 antibody sample (such as a panitumumab sample); and (3) modifying the FcγR-mediated cytotoxicity of the IgG2 antibody sample (such as a panitumumab sample) by increasing or decreasing the amount of IgG2 molecules containing high-mannose glycan at the N-297 site so that the difference in FcγR-mediated cytotoxicity between the antibody sample and the reference value is approximately 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 approximately 30% or less, approximately 25% or less, approximately 20% or less, approximately 15% or less, approximately 10% or less, or approximately 5% or less. In some examples, step (1) ("obtaining a reference value for FcγR-mediated cytotoxicity") is performed before, after, or simultaneously with step (2) ("determining the FcγR-mediated cytotoxicity of the IgG2 sample or panitumumab sample") and / or step (3) ("modifying the FcγR-mediated cytotoxicity of the IgG2 sample 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 embodiments, FcγR-mediated cytotoxicity in IgG2 or panitumumab samples is increased by reducing the amount of panitumumab molecules containing high-mannose glycan at the N-297 site. In certain embodiments, a reduction of approximately 1 percent in high-mannose glycan increases FcγR-mediated cytotoxicity by approximately 1.2 percent to 2.4 percent, for example, approximately 1.2 percent, 1.25 percent, 1.3 percent, 1.35 percent, or 1.40 percent. Furthermore, changes in cytotoxicity levels are generally calculated based on relative cytotoxicity values ​​as described above.

[0141] In certain embodiments, FcγR-mediated cytotoxicity in IgG2 or panitumumab samples is reduced by increasing the amount of panitumumab molecules containing high-mannose glycan at the N-297 site. In certain embodiments, an increase of approximately 1 percent in high-mannose glycan reduces FcγR-mediated cytotoxicity by approximately 1.2 percent to 2.4 percent, for example, approximately 1.2 percent, 1.25 percent, 1.3 percent, 1.35 percent, or 1.40 percent. Furthermore, changes in cytotoxicity levels are generally calculated based on relative cytotoxicity values ​​as described above.

[0142] 3.4 Method for adjusting glycans Preferred methods for controlling the amount of glycoproteins, such as antibodies, 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), are known in the art. See, for example, Zhang et al., Drug Discovery Today 21(5):2016. Thus, in some embodiments, glycosylated competent cells (which can be used to produce glycoproteins, such as antibodies, by recombinant technology) are cultured under specific conditions to achieve desired levels of glycans.

[0143] For example, International Publication Nos. 2013 / 114164, 2013 / 114245, 2013 / 114167, 2015128793, and 2016 / 089919 describe recombinant cell culture techniques useful for regulating glycans, such as 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 glycan), such as glycosphing with an increased proportion of total non-fucosylated glycans. The following methods are provided: a method for obtaining proteins (International Publication No. 2013 / 114164), a method for obtaining glycoproteins with an increased proportion of Man5 glycans and / or non-fucosylated glycans (International Publication No. 2013 / 114245), a method for obtaining glycoproteins containing specific amounts of high-mannose glycans, non-fucosylated glycans and GF glycans (International Publication No. 2013 / 114167); a method for obtaining glycoproteins containing high-mannose glycans and reduced galactosylation and / or high-galactosylated glycans (International Publication No. 2015128793); and a method for manipulating the fucosylated glycan content on recombinant proteins (International Publication No. 2016 / 089919). The cell culture methods described in International Publication No. 2013 / 114164, International Publication No. 2013 / 114245, International Publication No. 2013 / 114167, International Publication No. 2015128793, and International Publication No. 2016 / 089919 include modifying one or more cell culture parameters such as temperature and pH to regulate specific glycans, culturing cells with manganese ions or salts thereof (e.g., 0.35 μM to about 20 μM manganese), and / or culturing cells with copper (e.g., 10 to 100 ppb) and manganese (e.g., 50 to 1000 nM).

[0144] Furthermore, International Publication No. 2015 / 140700 describes culturing cells in the presence of betaine to increase non-fucosylated glycans, or culturing cells with manganese, galactose, and betaine to obtain target levels of mannosylated glycans, galactosylated glycans, and non-fucosylated glycans. U.S. Patent Application Publication No. 2014 / 0356910 teaches a method for increasing 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 osmotic pressure level of cell culture medium and extending the culture period. Similarly, Konno et al., Cytotechnology 64:249-3+6 (2012) describes a method for controlling antibody fucose content by the osmotic pressure of culture medium. Wong et al., Biotechnology and Bioengineering 89(2):164-177 (2004) teach a method to reduce recombinant protein sialylation and increase high mannose glycan by using low glutamine-free fed-batch culture. International Publication No. 2017 / 079165 describes a method to increase or decrease the non-fucosylated or fucosylated form of recombinant proteins by using genetically engineered host cells that do not possess GMD or FX, and culturing these host cells with fucose. International Publication No. 2017 / 134667 describes culturing cells with nicotinamide and fucose to produce antibodies with reduced non-fucosylated levels. Sha et al., TIBs 34(10):835-846(2016) also outlines several methods for modulating glycans, including methods for increasing galactosylation levels on antibodies by culturing with uridine, manganese, and galactose, and methods for increasing high-mannose glycoforms by using mannose as a carbon source.

[0145] Accordingly, the methods of the present disclosure, in exemplary embodiments, include employing one or more procedures, cell culture media, and / or cell culture conditions taught in one or more of the above references or other references described herein, in order to adjust 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 method includes culturing antibody-expressing glycosylated competent cells in cell culture medium under conditions that adjust 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 method, in some embodiments, comprises culturing glycosylated competent cells expressing antibodies in a cell culture medium under conditions that control glycan levels, wherein the cell culture medium comprises fucose, or fucose and glucose.

[0146] In a method comprising maintaining or culturing cells in a cell culture, the cell culture may 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 with specific pH, temperature, cell density, culture volume, dissolved oxygen level, pressure, osmotic pressure, etc. In a typical embodiment, the cell culture is shaken in a CO2 incubator under standard humidified conditions with 5% CO2 (e.g., 70 rpm) before inoculation. In an exemplary embodiment, the method comprises culturing antibody-expressing glycosylated competent cells in a cell culture medium under conditions that control glycan levels, for example, by increasing the osmotic pressure of the cell culture medium to decrease the level of non-fucosylated glycans in the antibody, as taught by Konno et al. (above). In exemplary embodiments, the method comprises culturing antibody-expressing glycosylated competent cells in cell culture medium under conditions that control glycan levels, and controlling the pH and temperature of the cell culture as taught, for example, in International Publication No. 2013 / 114164, International Publication No. 2013 / 114245, International Publication No. 2013 / 114167, or International Publication No. 2015 / 128793 (each incorporated herein by reference).

[0147] In exemplary embodiments, the method of the present disclosure includes maintaining glycosylated competent cells in a cell culture medium at pH, temperature, osmotic pressure, and dissolved oxygen levels suitable for the production of recombinant glycosylated proteins or antibodies, as is well known in the art. In exemplary embodiments, the cell culture is maintained in a medium suitable for cell proliferation, as is well known in the art, and / or is supplied with one or more feed media according to an arbitrary preferred supply schedule.

[0148] In exemplary embodiments, glycosylated competent cells are eukaryotic cells, including, but not limited to, yeast cells, filamentous fungal cells, protist cells, algal cells, insect cells, or mammalian cells. Such host cells have been described in the art; see, for example, Frenzel, et al., Front Immunol 4:217 (2013). In exemplary embodiments, eukaryotic cells are mammalian cells. In exemplary embodiments, mammalian cells are non-human mammalian cells. In some embodiments, the cells include Chinese hamster ovary (CHO) cells and their derivatives (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 their derivatives (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, and mouse embryonic fibroblast cells NIH These include 3T3, mouse fibroblasts L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or neonatal hamster kidney (BHK) cells (Gaillet et 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 genes that encode the relevant enzymes required for glycosylation. Exemplary enzymes include, but are not limited to, oligosaccharide transferases, glycosidases, glucosidase I, glucosidase II, calnexin / calreticulin, glycosyltransferases, mannosidases, GlcNAc transferases, galactosyltransferases, and sialyltransferases.

[0150] In further or alternative embodiments, glycosylated competent cells that produce antibodies by recombinant technology are genetically engineered to regulate the antibody glycans (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 the M5 high-mannose species)). In exemplary embodiments, glycosylated competent cells are genetically engineered to alter the activity of enzymes in the de novo or salvage pathway. Optionally, glycosylated competent cells are genetically engineered to knock out genes encoding GDP-keto-6-deoxymannose-3,5-epimerase,4-reductase. In exemplary embodiments, glycosylated competent cells are genetically engineered to alter the activity of enzymes in the de novo or salvage pathway. These two pathways of fucose metabolism are well known in the art and are shown in Figure 1E. In an exemplary embodiment, glycosylated competent cells are genetically engineered to alter the activity of one or more of the following: fucosyltransferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinases, GDP-fucos pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, glycosylated competent cells are genetically engineered to knock out the gene encoding FX. In exemplary embodiments, glycosylated competent cells are genetically engineered to alter the activity of β(1,4)-N-acetylglucosaminyltransferase III (GNTIII) and / or GDP-6-deoxy-D-lyxo-4-hexose reductase (RMD). In exemplary embodiments, glycosylated competent cells are genetically engineered to overexpress GNTIII and / or RMD. In exemplary embodiments, glycosylated competent cells are genetically engineered to have modified β-galactosyltransferase activity.In some embodiments, glycosylated competent cells are genetically engineered to regulate the expression levels of genes encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase, β1-4-galactosyltransferase, and / or β1-4N-acetylgalactosaminyltransferase.

[0151] In the art, several methods are known for reducing or eliminating fucosylation of Fc-containing molecules, such as antibodies. These include recombinant expression in specific mammalian cell lines, including FUT8 knockout cell lines, mutant CHO cell line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNAs specific to the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Alternatively, Fc-containing molecules may 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 after antibody production. In exemplary embodiments, the method of the present disclosure includes chemically or enzymatically treating the antibody after it has been produced by recombinant technology. 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, kifunensin, and PNGase-F. In exemplary embodiments, the chemical or enzyme is incubated with the antibody for various times to produce an antibody having various amounts of glycan. In some embodiments, the antibody is incubated with β-1,4-galactosyltransferase as described in the examples. In some further embodiments, antibodies having varying levels of galactose can be produced by incubating the antibody with β-1,4-galactosyltransferase for a certain period of time, for example, 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 in the range of about 10 minutes to about 9 hours.

[0153] 3.5 Method for measuring glycans Various methods are known in the art for evaluating glycoforms present in glycoprotein-containing compositions, such as antibodies, or for determining, detecting, or measuring the glycoform profile of a specific sample containing glycoproteins. Preferred methods include, but are not limited to, hydrophilic interaction liquid chromatography (HILIC), liquid chromatography-tandem mass spectrometry (LC-MS), cation MALDI-TOF analysis, anion 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, as well as 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 See Chem, 2010, Vol.397:3457-3481, Kurogochi et al., PLOS One 10(7):e0132848;doi:10.1371 / journal.pone.0132848, Thomann et al., PLOS One 10(8):e0134949.Doi:10.1371 / journal.pone.0134949, Pace et al., Biotechnol.Prog.32(5):1181-1192(2016), and Geoffrey, RGet.al. Analytical Biochemistry 1996, Vol.240, pages 210-226.Furthermore, the examples shown herein describe a suitable method for evaluating glycoforms present in glycoprotein-containing compositions such as antibodies.

[0154] For example, glycan content can be measured by high pH anion exchange chromatography (HPAEC), as described in 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 the reducing end is labeled with a fluorescent tag (e.g., 2-aminobenzamide or 2-aminobenzoic acid). Fluorescent N-glycans are separated by HPAEC and detected by fluorescence detection. Generally, the separation of neutral N-glycans is based on the increasing complexity of the N-glycan structure. The 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 sample are visually compared with 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-glycan (e.g., by an enzyme such as PNGase F), (ii) labeling (e.g., by 2-aminobenzoic acid or 2-aminobenzamide), (iii) removal of the free label (e.g., by gel filtration or solid-phase extraction), (iv) separation of the glycan species by HILIC, and (v) detection (e.g., by fluorescence spectroscopy). Further details of HILIC are provided in Melmer et al., Analytical and Bioanalytical Chemistry, September 2010, Volume 398, Issue 2, pp. 905-914.

[0156] Another commonly used method is liquid chromatography-tandem mass spectrometry (LC-MS). After the release, labeling, and removal of the free label of N-glycan, the sample can be analyzed by a technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass spectrometry 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 exemplary embodiments, the composition is prepared by a method of adjusting the amount of glycans (e.g., galactosyl glycans, terminal β-galactose, G1, G1a, G1b and / or G2 galactosyl glycans, non-fucosylated glycans, fucosylated glycans, core fucose, high mannose glycans, Man-5 glycans, or combinations thereof) in the antibody. In exemplary embodiments, the recombinant glycosylated protein is an IgG2 antibody such as panitumumab. Thus, antibody compositions comprising an IgG2 antibody (e.g., panitumumab) with increased or decreased FcγR-mediated cytotoxicity are provided herein, wherein the IgG2 antibody (e.g., panitumumab) is manipulated to increase or decrease FcγR-mediated cytotoxicity compared to a control or reference value by adjusting the glycan profile as described above.

[0158] In exemplary embodiments, the antibody compositions provided herein are combined with pharmaceutically acceptable carriers, diluents, or excipients. Accordingly, pharmaceutical compositions comprising the recombinant glycosylated protein compositions described herein (e.g., antibody compositions) and pharmaceutically acceptable carriers, diluents, or excipients are provided herein. As used herein, the term “pharmaceutically acceptable carrier” includes some 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 embodiments are provided solely for illustrative purposes and are not intended to limit the scope of this disclosure. [Examples]

[0160] 1. Introduction To deepen our understanding of IgG2-mediated cytotoxicity, we developed highly sensitive cytotoxicity assays using panitumumab as a model IgG2, utilizing specific responsive cell types. We studied cytotoxic activity in FcγRIIa signaling assays using engineered cell lines and reporter genes, as well as in primary cells derived from PBMCs isolated from genotyped donor whole blood. Donors expressing common FcγRIIa and FcγRIIIa receptor allotypes were used. To understand the impact of quality characteristics that may vary as a function of the manufacturing process, we generated panitumumab strains containing a wide range of major glycan species, including galactosylated, non-fucosylated, and mannosylated strains, and evaluated their respective effects on panitumumab activity in various assays.

[0161] 2. Materials and Methods At Amgen (Thousand Oaks, CA), panitumumab was produced in CHO cells using a standard manufacturing process.

[0162] 2.1 Concentration of glycan species and enzymatic remodeling High-mannose-containing species were concentrated from mAbs using a ProSwift ConA-1S affinity column (5×50 mm, ThermoFisher, PN 074148) on an Agilent 1100 series HPLC system at a flow rate of 0.5 mL / min. The column was first maintained at initial conditions for 10.5 minutes with 100% buffer A (50 mM sodium acetate, 0.2 M NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 5.3), and then eluted for 17.5 minutes 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). Both the flow-through and eluted 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 a reaction buffer (pH 6.0) containing 50 mM sodium phosphate at 37°C for 1 hour. The reaction was stopped by flash freezing.

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

[0164] Galactose remodeling samples were generated by in vitro activity of β-1,4-galactosyltransferase (Sigma / Roche). First, fucosylated mAbs (mainly G0F) were prepared by collecting the flow-through fraction from an FcγIIIa column and removing terminal galactose by treatment with galactosidase. Then, the G0F-enriched mAbs were 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. Samples were taken from the reaction mixture at various time points (10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 9 hours), and the reaction was subsequently stopped by flash freezing to obtain MAb with varying levels of galactose.

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

[0166] 2.2 Characterization of Glycan Species Concentration and Remodeling To ensure the desired glycan properties and minimum levels of high molecular weight species, all concentrated and remodeled samples were characterized by hydrophilic interaction liquid chromatography (HILIC) and size exclusion chromatography. Glycans were released from mAbs using PNGase F (New England BioLabs) with an E / S ratio of 1 / 25 (μL / μg) and labeled with 12 mg / mL of 2-aminobenzoic acid (2-AA, Sigma-Aldrich) by incubation of the reaction mixture at 80°C for 75 minutes. The 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 a column in a high-concentration organic solvent, and then eluted by increasing the gradient of aqueous ammonium formate buffer (76% B was maintained for 5 minutes, followed by elution over 14 minutes with a gradient from 76% B to 65.5% B). Confirmation that the required procedure did not result in the formation of high molecular weight species was evaluated on an Agilent 1100 HPLC system at a flow rate of 0.5 mL / min using a size exclusion column (SEC) TSK-Gel G3000SWLXL (7.8 × 300 mm, Tosoh Bioscience). Sample loads of 20–40 μg were typically separated compositionally 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 their cell surface, similar to the luciferase reporter gene which has a response element of T cell activator (NFAT). Simultaneous binding of an antibody on target cells to the Fc domain of an antibody containing stably expressed FcγRIIa on Jurkat effector cells activates the transcription factor NFAT. Activated NFAT translocates to the nucleus of Jurkat cells and induces luciferase reporter gene expression. Addition of a luciferase substrate containing luciferin and a surfactant generates a luminescence signal, enabling the detection of FcγRIIa reporter activity. For panitumumab, to function as a dose-response curve, the reference standard, assay control, and test samples were serially diluted over eight concentration levels in RPMI1640 assay medium containing low IgG FBS, with final plate well concentrations ranging from 0.004 μg / mL to 2 μg / mL. Effector Jurkat reporter cells and target (A431) cells were mixed in a 3:2 effector-to-target (E to T) cell ratio to prepare a cell suspension. The plates were then incubated for 5.5 hours in a humidified incubator at 5% CO2 and 37°C. At the end of incubation, the cells were lysed with a surfactant in luciferase assay buffer. The luciferase reaction in luciferase assay buffer induced luminescence of the luciferin signal of its substrate, which was detected by an EnVision plate reader. Data were fitted to mean luminescence values ​​using a 4-parameter fit with SoftMaxPro, and the EC50 standard / EC50 sample was calculated and reported as an activity percentage. Each sample is tested in three independent assays, and the final result for each sample is reported as the average of the three measurements.

[0168] 2.4 Donor allotyping and cellular 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, the cells were counted, and then the DNA was isolated. DNA was extracted from the cells using the QIAGEN blood and cell culture DNA kit. The DNA was then subjected to Taqman single nucleotide polymorphism (SNP) genotyping analysis using a 7900HT real-time PCR system with qualified sets specific to each receptor (FcγRIIa and FcγRIIIa). The qPCR assay was set to 40 cycles using the master mix, DNA, and assay oligomix (fluorescently labeled probes). Each probe, if present, anneals specifically to the complementary sequence. The exonuclease activity of the DNA polymerase cleaves the probe hybridized to the target, releasing the reporter dye and increasing fluorescence. The presence of the dye indicates a specific polymorphism, as the probe does not attach during amplification if a particular sequence is absent, and therefore the dye is not released. The SDS software performs a read of each well, and the genotype is determined by the call. The software also provides an allele identification plot where clustering shows individual genotypes. The TaqMan® 5'-nuclease assay chemical method provided a way to obtain single nucleotide polymorphism (SNP) genotyping results. Each pre-designed TaqMan® SNP genotyping assay included two allele-specific TaqMan® MGB probes containing different fluorescent dyes, and a PCR primer pair for detecting a specific SNP target. These TaqMan® probe and primer sets (assays) are uniquely aligned with the genome and provide unparalleled specificity to 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 a β-galactosidase (β-gal) reporter, a component of the Eurofins DiscoverX KILR® Cytotoxicity Assay. Modified target cells were mixed with PBMCs in a 1:200 ratio in the presence of various concentrations of panitumumab or glyco-engineered samples. Upon lysis of the target cells, the tagged housekeeping protein was released into the culture medium. The tagged housekeeping protein was detected in the culture medium by adding a reagent containing another fragment of the β-gal reporter, leading to the formation of an active β-gal enzyme. When the chemiluminescent reagent was hydrolyzed in response to β-gal, luminescence was generated in a dose-dependent manner. The luminescence response data was directly proportional to the amount of cytotoxicity. In this assay, novel PBMCs from healthy donors were used as effector cells. The luminescence signal was detected using a plate reader. The luminescence response was plotted against the test concentration, and a dose-response curve was constructed.

[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 collected, washed with D-PBS, and distributed in 1.2 × 10⁶ wells per 96-well plate. 6Cells were dispensed. KILR® U2OS target cells (6,000 / well) were added to wells containing PBMCs and incubated for 12 hours while increasing the concentration of panitumumab or glycosylated samples (0.148–200 ng / mL). Dose-dependent increases in luminescence signaling were detected by reading the assay plate with a Perkin ElmerEnvision plate reader. Data analysis was performed using SoftMax Pro v5.4.1, and dose-response curves were reported.

[0171] 2.5 FcγR blocking assay for demonstrating specificity Receptor antibody blocking studies were performed by individually blocking CD16 (FcγRIIIa), CD32 (FcγRIIa), and CD64 (FcγRI) with antibodies that specifically bind to and block these receptors, and the resulting cytotoxic activity was measured. Panitumumab was used at a constant concentration of 200 mg / mL and at various concentrations (2000 ng / mL to 1 ng / mL) of 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]). 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 modified with the KILR® housekeeping gene from Eurofins DiscoverX were harvested and seeded in 96-well plates at a density of 6000 cells / well. A constant concentration of panitumumab (200 ng / mL) was mixed with a blocking reagent in a concentration range of 2000 ng / mL to 1 ng / mL and added to the target cells. Healthy donor PBMCs were used as effector cells by collecting whole blood and separating them in BD-CPT tubes. These PBMCs were then added to a mixture of target cells and antibody at a density of 1.2e6 cells / well, with an effector-to-target ratio of 200:1. After co-culturing the assay plates at 37°C for approximately 18 hours, KILR® detection reagent was added, and the luminescence signal was read using an Envision plate reader.

[0173] 2.6 FcγR binding by SPR Surface plasmon resonance (SPR) experiments were performed using an SPR T-200 instrument. His-tagged human FcγR was expressed in CHO cells and purified in mouse anti-his capture antibody immobilized at approximately 5000 RU on a Series S sensor chip CM5 (GE Healthcare) using Instrument Buffer (0.005% P20 in PBS). FcγR was diluted to 3.3–10 nM with running buffer (0.005% P20, 0.1 mg / mL BSA in PBS) and injected at 10 μL / min for 1.5 minutes 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 into the captured FcγR at 50 μL / min with a binding and dissociation time of 3 minutes. The tip surface was regenerated by injecting 10 mM glycine, pH 1.7 at a rate of 30 μL / min for 30 seconds.

[0174] 3.Results 3.1 Effects of glycan species on panitumumab-mediated cytotoxicity As previously described, panitumumab can mediate cell-mediated cytotoxic activity not previously described for therapeutic antibodies against human IgG2. To determine which product quality characteristics may influence this activity, a series of enrichments and enzymatic treatments (see Materials and Methods) were used to alter the glycan profile of panitumumab, generating a wide range of each of the major glycan species: terminal galactose, core fucose, and high mannose. Since this activity was previously attributed to FcγRIIa, a highly sensitive FcγRIIa reporter gene assay was also devised to read the influence of quality characteristics on the activity.

[0175] The first glycan species evaluated for its effect was terminal galactose. Panitumumab samples were enzymatically treated as described in the Methods section and exhibited a wide range of terminal galactose from 0.4% to 88.3%. As shown in Figures 2A–2B, across this range of galactose levels, the activity measured by the reporter gene assay was in approximately 60% range, and the activity level showed a hyperlinear response to galactose levels. We quantified the relative effect of β-galactose on FcγRIIa signaling activity by representing it with respect to the slope of the activity / characteristic correlation plot, which can be considered to represent the response coefficient. Using this approach in panitumumab, the effect on FcγRIIa signaling activity was expressed as a R of 0.98. 2 Using the values, the value for β-galactose can be calculated as 0.6681.

[0176] Next, we investigated the effect of core fucose species levels on the FcγRIIa reporter gene assay. Panitumumab also showed a linear response to various fucose (non-fucosylated) levels. Dose-response curves of FcγRIIa signaling activity for non-fucosylation are shown in Figures 3A-3B. The calculated activity yielded a hyperlinear negative response to the amount of non-fucosylation. The data show that panitumumab mediates higher cytotoxicity at lower non-fucosylated levels and lower cytotoxicity at higher non-fucosylated levels, thereby inversely correlated with the non-fucosylation percentage of the mAb. Note that this is an interesting reversal of the effect of non-fucose levels on IgG1-mediated FcγRIIa-mediated ADCC activity.

[0177] The final element of this glycan study involved testing the effect of high mannose levels on panitumumab's ability to influence the FcγRIIa reporter gene assay. The FcγRIIa signaling activity response as a function of high mannose levels is shown in Figures 4A–4B. Here again, panitumumab shows a linear but inverse response to high mannose levels.

[0178] Table 3 summarizes the effects of various glycan species.

[0179] [Table 5]

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

[0181] 3.3 PBMC cytotoxicity data Using PBMCs from donors with the HHVV, HHFF, RRFV, and HHFV allotypes of FcγRIIa and FcγRIIIa, a wide range of non-fucosylated, mannosylated, and galactosylated panitumumab samples were tested in the cytotoxicity assays described in Materials and Methods, respectively. Representative dose-response curve overlays for methods varying specific glycan levels are shown in Figure 5. Panitumumab also showed a linear inverse relationship response to the fucose (non-fucosylated) range of 0.4% to 27.4%. The calculated activity yielded a hyperlinear negative response to the amount of non-fucosylation 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 cytotoxicity and non-fucosylation percentage.

[0182] Panitumumab also showed a linear response to the high mannose range of 2.9%–75.6%. Calculated activity yielded a hyperlinear negative response to high mannose levels 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 broad 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 no substantial correlation between cytotoxic activity and varying levels of galactosylation, unlike those observed in the FcγRIIa reporter gene assay. Using this approach for panitumumab, it was not possible to quantify the relative effect of β-galactose on cellular cytotoxicity. The assays were performed using 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 only 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 to and block these receptors, and the resulting cytotoxic activity was measured. Blocking experiments were set up using PBMCs and KILR assays with 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 blocking mAb concentrations. Cells treated with panitumumab (0.1 to 200 ng / mL) without blocking antibodies mediated cellular cytotoxicity as expected in a dose-dependent manner (Figure 9A). Cells incubated with anti-FcγRI or anti-FcγRIIIa showed no difference in the percentage of cell death at any concentration of the blocking mAb, similar to isotype control mAbs (Figure 9B). This indicates that IgG2 (panitumumab)-mediated cytotoxicity is primarily due to the involvement of the FcγRIIa receptor, and not FcγRI or FcγRIIIa.

[0185] 3.5 Binding of panitumumab to FcγR To evaluate the affinity of panitumumab and various concentrated glycan species for FcγR, binding of panitumumab samples to all three human Fcγ receptors was measured by surface plasmon resonance (SPR). 10 μM panitumumab did not show detectable binding to human FcγRI or FcγRIIIa-158F, but did bind to huFcγRIIa-131H (Figures 10A-10C). Panitumumab and huIgG2 control bound to huFcγRIIa-131H, with apparent KD values ​​of approximately 20 μM and 25 μM, respectively (Figure 10C). Binding was further evaluated by equilibrium binding for fucose and non-fucose concentrated panitumumab samples (Figures 11A-11B). Two glycan concentrated samples (approximately 7.9 μM and 8 μM fucose-concentrated and non-fucose-concentrated K) were used. DThe differences in binding by SPR between the two values ​​were not as pronounced as the differences observed in FcγRIIa signaling activity or PBMC cytotoxicity assays. This is likely due to signal amplification provided by cell line assays via receptor clustering, signaling, and gene expression aspects of binding responses (see, e.g., Unkeless et al., SeminImmunol. 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] Table 3 summarizes the effects of various glycans on the cytotoxic activity of panitumumab, showing the slope values ​​and R for each donor when tested with either a non-fucosylated sample, a high-mannose sample, or a β-galactose sample, for the ability to mediate cellular cytotoxicity. 2 This indicates.

[0187] 4. Discussion The objective of this study is to understand the mechanisms and product quality characteristics that affect therapeutic human IgG2 monoclonal antibody-mediated cytotoxicity. Highly sensitive and reproducible quantitative functional assays are needed to evaluate the impact of quality characteristics. To understand the impact of quality characteristics, which may vary as a function of the manufacturing process, panitumumab strains containing a wide range of major glycan species, including galactosylated, non-fucosylated, and mannosylated strains, were generated, and their impact on the activity of panitumumab was evaluated in various assays. Through the operational process, the inventors were able to achieve a substantially wider range than possible through process modifications, in order to more accurately find the relationship between characteristics and activity. Gal levels ranged from 0.4% to 88.3%, non-fucose 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 phagocyte activity are prone to inconsistencies in the effector types present in the donor population at the time of assay, as well as in the receptor allotypes and other background genetic diversity that may affect assay activity. Furthermore, as described elsewhere, the diversity of expressed receptors generally makes it more difficult to identify relevant receptors on phagocytes (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 standpoint, assay throughput is also limited by the number of cells that can be collected. Therefore, these types of assays are unsuitable for drug development and are characterized in quality control settings. 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 above limitations.

[0189] Effector function also depends on receptor polymorphism (158V or F for FcγRIIIa, 131H or R for FcγRIIa). To further investigate the effects of quality characteristics in a more physiologically relevant setting, we evaluated whether receptor allotypes have an effect on panitumumab-mediated cytotoxicity and drew conclusions regarding the effects of glycans using PBMC donors expressing common FcγRIIa and FcγRIIIa receptor allotypes. Because the kinetics of IgG2 are much slower than those of IgG1, reliable functional assays can be developed using PBMCs that can withstand long-read assays, which allows for longer incubation times for these assays. KILR (Killing Immunolysis) by DisCoverX is a non-radioactive assay for kinetically slow cytotoxicity. The KILR assay was set up using PBMCs as U2OS target cells and effector cells coated with panitumumab overexpressing EGFR transduced with KILR housekeeping genes.

[0190] These studies are the first to demonstrate that glycans of IgG2 panitumumab have substantial and distinct effects on cellular cytotoxicity. Non-fucosylation and mannosylation were observed to adversely affect cellular cytotoxicity in both FcγRIIa signaling activity and PBMC-mediated cell killing assays, a phenomenon entirely opposite to that observed with IgG1. Increased non-fucosylation significantly reduced the antibody's cell-killing capacity, and similarly, increased high mannose content in the antibody reduced panitumumab-mediated cellular cytotoxicity. For both of these glycans, hyperlinear negative responses were observed in both primary cell and reporter gene assays. Galactosylation, on the other hand, appeared 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 should be noted that, as measured by surface plasmon resonance, the affinity of fucosylated panitumumab for FcγRIIa-H131 (KD approximately 7.9 μM) is only slightly higher than the affinity of non-fucosylated panitumumab for FcγRIIa-H131 (KD approximately 8.0 μM), therefore, there is still no clear mechanistic explanation available for the observed association between FcγRIIa and IgG2.

[0191] PMBCs are recognized as a complex and fluctuating population. To confirm that FcγRIIa mediates activity, receptor blocking studies were performed to confirm the specificity of panitumumab, showing that it mediates cytotoxicity via FcγRIIa rather than FcγRIIIa. This was achieved by using blocking antibodies against FcγRI, FcγRIIa, and FcγRIIIa. Cytotoxicity was inhibited only by increasing the concentration of anti-FcγRIIa and was not affected when blocked with other antibodies against FcγRI or FcγRIIIa. Differences in cytotoxicity levels between different donors with the same FcγRIIa allele may be due to a variety of reasons, including receptor density, membrane mobility, or interactions / cooperation with other molecules that may affect intracellular signaling and consequently cellular cytotoxicity. It remains unclear whether and how the role of the inhibitory receptor affects overall cellular cytotoxicity in PBMCs.

[0192] The involvement of FcγR was further investigated by an SPR binding assay, and 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 test also showed similar binding activity to FcγRIIa only, 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 differences in binding between the two glycan-enriched samples and between the fucose and non-fucose-enriched samples were not as dramatic as those seen in the functional assay, with the samples binding at KDs of 7.9 μM and 8 μM, respectively. The involvement of FcγR types possessing an IgG complex-mediated immune receptor tyrosine system activation motif (ITAM) initiates numerous signaling cascades leading to cellular activation and subsequent induction of effector function. Cellular responses to Fc-FcγR interactions vary among myeloid cell types; however, FcγR aggregation typically leads to rapid internalization of FcγR and activation of various signaling pathways affecting cellular 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 differences between non-fucose and fucosylated panitumumab may not be very apparent in Biacore binding assays due to these missing elements such as clustering and signal amplification that can occur in cell line assays.

[0193] This study, combined with highly responsive functional assays and broad characterization, revealed a significant impact of protected Fc glycans on novel IgG2-mediated cytotoxic activity. This understanding should be considered in the design and characterization of therapeutic IgG2 drug candidates.

[0194] All publications, patents, and patent applications cited herein are incorporated herein by reference as if each individual publication, patent, or patent application were specifically and individually incorporated by reference. While the inventions described above are described in some detail by description and examples for the purpose of clarification, it will be readily apparent to those skilled in the art that certain changes and modifications can be made thereto without departing from the spirit or scope of the disclosed embodiments, in light of the teachings of this disclosure. The headings used herein are for structural purposes only and should not be construed as limiting the subject matter described.

[0195] The enumeration of value ranges in this Specified Terms is intended only as a simplified way of referring individually to each of the distinct values ​​at each of these ranges and endpoints, unless otherwise indicated herein, and each of the distinct values ​​and endpoints is incorporated herein as if it were individually enumerated herein.

[0196] All methods described herein may be performed in any suitable order, unless otherwise specified herein or unless it is clearly inconsistent with the context. The use of any examples or exemplary terms provided herein (e.g., "etc.") is intended solely to clarify the disclosure and does not impose any limitation on the scope of the disclosure unless otherwise claimed. Nothing in this specification should be construed as indicating that any unclaimed element is essential for the practice of the disclosure.

[0197] This specification describes preferred embodiments of the Disclosure, including, for example, the best known modes for carrying out the Disclosure. Variations of these preferred embodiments will become apparent to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will adopt such variations as needed, and the inventors intend that the Disclosure will be carried out in forms other than those specifically described herein. Accordingly, this Disclosure includes all variations and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements is included herein in all possible variations unless otherwise indicated herein or unless explicitly contradicted in the context.

[0198] The present invention relates particularly to the following embodiments: 1. A method for modulating panitumumab-mediated Fc gamma receptor (FcγR)-mediated cytotoxicity, 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 a panitumumab molecule containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

[0199] 2. The method according to Embodiment 1, wherein panitumumab-mediated FcγR-mediated cytotoxicity 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 containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

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

[0201] 4. The method according to Embodiment 1, wherein the above FcγR is FcγRIIa.

[0202] 5. The method according to Embodiment 1, wherein the FcγR-mediated cytotoxicity described above is FcγRIIa-mediated cytotoxicity.

[0203] 6. A method for matching Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab samples with a reference value, wherein: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) To determine FcγR-mediated cytotoxicity in the panitumumab sample; (3) A method for modifying the FcγR-mediated cytotoxicity of a 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 a panitumumab molecule containing 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 a reference value is approximately 35% or less.

[0204] 7. The method according to Embodiment 6, wherein FcγR-mediated cytotoxicity of a 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 containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

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

[0206] 9. The method according to Embodiment 6, wherein the above FcγR is FcγRIIa.

[0207] 10. The method according to Embodiment 6, wherein the FcγR-mediated cytotoxicity described above is FcγRIIa-mediated cytotoxicity.

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

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

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

[0211] 14. The method according to Embodiment 11, wherein the above FcγR is FcγRIIa.

[0212] 15. The method according to Embodiment 11, wherein the FcγR-mediated cytotoxicity described above is FcγRIIa-mediated cytotoxicity.

[0213] 16. A method for matching Fc gamma receptor (FcγR)-mediated cytotoxicity of panitumumab samples with a reference value, wherein: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) To determine FcγR-mediated cytotoxicity in the panitumumab sample; (3) A method for modifying the FcγR-mediated cytotoxicity of the panitumumab sample by increasing or decreasing the amount of panitumumab molecules containing fucosylated glycan at the N-297 site, or by increasing or decreasing the amount of panitumumab molecules containing 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 approximately 35% or less.

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

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

[0216] 19. The method according to Embodiment 16, wherein the above FcγR is FcγRIIa.

[0217] 20. The method according to Embodiment 16, wherein the FcγR-mediated cytotoxicity described above is FcγRIIa-mediated cytotoxicity.

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

[0219] 22. The method according to Embodiment 21, wherein panitumumab-mediated FcγR-mediated cytotoxicity is increased by reducing the amount of panitumumab molecules containing high-mannose glycan at the N-297 site.

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

[0221] 24. The method according to Embodiment 21, wherein the above FcγR is FcγRIIa.

[0222] 25. The method according to Embodiment 21, wherein the FcγR-mediated cytotoxicity described above is FcγRIIa-mediated cytotoxicity.

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

[0224] 27. The method according to Embodiment 26, wherein FcγR-mediated cytotoxicity of a panitumumab sample is increased by reducing the amount of panitumumab molecules containing high-mannose glycan at the N-297 site.

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

[0226] 29. The method according to Embodiment 26, wherein the above FcγR is FcγRIIa.

[0227] 30. The method according to Embodiment 26, wherein the FcγR-mediated cytotoxicity described above is FcγRIIa-mediated cytotoxicity.

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

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

Claims

1. A method for increasing panitumumab-mediated Fc-gamma receptor (FcγR) mediated cytotoxicity, the following: i) 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; ii) Increasing the amount of panitumumab molecules containing fucosylated glycan at the N-297 site, or decreasing the amount of panitumumab molecules containing non-fucosylated glycan at the N-297 site; and / or, iii) Reduce the amount of panitumumab molecules containing high levels of mannose glycan at the N-297 site; A method that includes this.

2. A method for reducing panitumumab-mediated Fc-gamma receptor (FcγR)-mediated cytotoxicity, the following: i) Reducing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or reducing the amount of panitumumab molecules containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site; ii) Decreasing the amount of panitumumab molecules containing fucosylated glycan at the N-297 site, or increasing the amount of panitumumab molecules containing non-fucosylated glycan at the N-297 site; and / or, iii) Increase the amount of panitumumab molecules containing high levels of mannose glycan at the N-297 site; A method that includes this.

3. A method for matching panitumumab samples with Fc-gamma receptor (FcγR)-mediated cytotoxicity to a reference value, wherein: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) To determine the FcγR-mediated cytotoxicity of the panitumumab sample; and, (3) The difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is approximately 35% or less, as follows: i) 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; ii) Increasing the amount of panitumumab molecules containing fucosylated glycan at the N-297 site, or decreasing the amount of panitumumab molecules containing non-fucosylated glycan at the N-297 site; and / or, iii) Reduce the amount of panitumumab molecules containing high levels of mannose glycan at the N-297 site; This increases the FcγR-mediated cytotoxicity of the panitumumab sample, A method that includes this.

4. A method for matching panitumumab samples with Fc-gamma receptor (FcγR)-mediated cytotoxicity to a reference value, wherein: (1) To obtain a reference value for FcγR-mediated cytotoxicity; (2) To determine the FcγR-mediated cytotoxicity of the panitumumab sample; and, (3) The difference in FcγR-mediated cytotoxicity between the panitumumab sample and the reference value is approximately 35% or less, as follows: i) Reducing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or reducing the amount of panitumumab molecules containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site; ii) Decreasing the amount of panitumumab molecules containing fucosylated glycan at the N-297 site, or increasing the amount of panitumumab molecules containing non-fucosylated glycan at the N-297 site; and / or, iii) Increase the amount of panitumumab molecules containing high levels of mannose glycan at the N-297 site; This reduces the FcγR-mediated cytotoxicity of the panitumumab sample, A method that includes this.

5. The method according to claim 1 or 3, 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 containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

6. The method according to claim 2 or 4, wherein the FcγR-mediated cytotoxicity of panitumumab is reduced by reducing the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab, or by reducing the amount of panitumumab molecules containing G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site.

7. The method according to any one of claims 1 to 4, wherein the FcγR is FcγRIIa.

8. The method according to claim 1 or 2, wherein the FcγR-mediated cytotoxicity is FcγRIIa-mediated cytotoxicity.

9. A panitumumab composition comprising panitumumab having a regulated glycan profile, wherein the Fc-gamma receptor (FcγR)-mediated cytotoxicity of the panitumumab having the regulated glycan profile is increased compared to the Fc-gamma receptor (FcγR)-mediated cytotoxicity of a commercially available panitumumab sample at the same dose, i) The panitumumab having the adjusted glycan profile has an increased amount of terminal β-galactose at the N-297 glycosylation site of the panitumumab compared to the amount of terminal β-galactose at the N-297 glycosylation site of a commercially available panitumumab sample at the same dose, or has an increased amount of G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site compared to the amount of G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site of a commercially available panitumumab sample at the same dose; ii) The panitumumab having the adjusted glycan profile has an increased amount of fucosylated glycan at the N-297 site compared to the amount of fucosylated glycan at the N-297 site of a commercially available panitumumab sample at the same dose, or has a decreased amount of non-fucosylated glycan at the N-297 site compared to the amount of non-fucosylated glycan at the N-297 site of a commercially available panitumumab sample at the same dose; and / or, iii) The panitumumab having the adjusted glycan profile has a reduced amount of high mannose glycan at the N-297 site compared to the amount of high mannose glycan at the N-297 site of a commercially available panitumumab sample at the same dose, Panitumumab composition.

10. A panitumumab composition comprising panitumumab having a regulated glycan profile, wherein the Fc-gamma receptor (FcγR)-mediated cytotoxicity of panitumumab having the regulated glycan profile is increased compared to the Fc-gamma receptor (FcγR)-mediated cytotoxicity of panitumumab before glycan profile regulation, i) The panitumumab having the modified glycan profile has an increased amount of terminal β-galactose at the N-297 glycosylation site of the panitumumab compared to the amount of terminal β-galactose at the N-297 glycosylation site of the panitumumab before modification of the glycan profile, or has an increased amount of G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site compared to the amount of G1, G1a, G1b, and / or G2 galactosylated glycans at the N-297 site of the panitumumab before modification of the glycan profile; ii) Panitumumab having the adjusted glycan profile has an increased amount of fucosylated glycan at the N-297 site compared to the amount of fucosylated glycan at the N-297 site of panitumumab before adjustment of the glycan profile, or has a decreased amount of non-fucosylated glycan at the N-297 site compared to the amount of non-fucosylated glycan at the N-297 site of panitumumab before adjustment of the glycan profile; and / or, iii) Panitumumab having the adjusted glycan profile has a reduced amount of high mannose glycan at the N-297 site compared to the amount of high mannose glycan at the N-297 site of panitumumab before adjustment of the glycan profile, Panitumumab composition.

11. The panitumumab composition according to claim 9 or 10, wherein the FcγR is FcγRIIa.

12. The panitumumab composition according to any one of claims 9 to 11, wherein the Fc gamma receptor (FcγR)-mediated cytotoxicity is antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP).

13. The panitumumab composition according to any one of claims 9 to 12, wherein the Fc gamma receptor (FcγR)-mediated cytotoxicity is antibody-dependent cell phagocytosis (ADCP).

14. A panitumumab composition according to claim 9 or any one of claims 11 to 13, which is directly or indirectly dependent on claim 9, wherein: (i) The amount of terminal β-galactose at the N-297 glycosylation site of panitumumab is increased compared to the amount of terminal β-galactose at the N-297 glycosylation site of a commercially available panitumumab sample at the same dose; (ii) The amount of fucosylated glycan at the N-297 site of panitumumab is increased compared to the amount of fucosylated glycan at the N-297 site of a commercially available panitumumab sample at the same dose, or the amount of non-fucosylated glycan at the N-297 site of panitumumab is decreased compared to the amount of non-fucosylated glycan at the N-297 site of a commercially available panitumumab sample at the same dose; and / or, (iii) The amount of high-mannose glycan at the N-297 site of panitumumab is reduced compared to the amount of high-mannose glycan at the N-297 site of commercially available panitumumab samples at the same dose. Panitumumab composition.

15. A panitumumab composition according to claim 10 or any one of claims 11 to 13, which is directly or indirectly dependent on claim 10, wherein: (i) The amount of terminal β-galactose at the N-297 glycosylation site of panitumumab is increased compared to the amount of terminal β-galactose at the N-297 glycosylation site of panitumumab before glycan profile modification; (ii) The amount of fucosylated glycan at the N-297 site of panitumumab is increased compared to the amount of fucosylated glycan at the N-297 site of panitumumab before glycan profile adjustment, or the amount of non-fucosylated glycan at the N-297 site of panitumumab is decreased compared to the amount of non-fucosylated glycan at the N-297 site of panitumumab before glycan profile adjustment; and / or, (iii) The amount of high-mannose glycans at the N-297 site of panitumumab is reduced compared to the amount of high-mannose glycans at the N-297 site of panitumumab before glycan profile adjustment. Panitumumab composition.

16. The panitumumab composition according to any one of claims 9 to 15 and Pharmacopoeia-acceptable carriers, diluents, or excipients A pharmaceutical composition containing the following:

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