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Methods and compositions with enhanced therapeutic activity

A molecular and property technology, applied in the field of improving therapeutic activity and components, can solve problems such as lack of Fc function

Inactive Publication Date: 2009-08-12
CENTOCOR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it was demonstrated that the type of sialic acid linkage can have at least some effect on the Fc function of mutant IgG3Abs, the study did not compare the Fc function of IgGs with high to low sialic acid content

Method used

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  • Methods and compositions with enhanced therapeutic activity
  • Methods and compositions with enhanced therapeutic activity
  • Methods and compositions with enhanced therapeutic activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Example 1: Lectin-based separation of antibody species with different levels of sialic acid

[0118] Agarose beads coupled with MAA (Huaihua Agglutinin) or WGA (Wheat Germ Agglutinin) were purchased from Vector Labs or EY Labs. The test antibody Ab1 is a fully human monoclonal antibody that binds human TNF. will contain 20mM CaCl 2 and 20mM MgCl 2 Ab1 (~1-10 mg) in 20 mM Tris-HCl buffer (pH 7.0) was fractionated on MAA-Sepharose or WGA-Sepharose columns. Ab1 bound to the MAA-Sepharose column was eluted with 0.5% acetic acid, neutralized with 1M Tris-HCl (pH 7.0), and then the buffer was exchanged with PBS. This substance is called Ab1 MAAB.

[0119] After loading Ab1 antibody samples onto a WGA-Sepharose column, the non-bound (T, passed) fraction was collected and the buffer was exchanged for PBS. This substance is called Ab1WGAT. The column containing bound Ab1 was washed with the same buffer as above, and a 1 ml fraction of any eluted antibody (representing weak...

Embodiment 2

[0121] Example 2: Enzyme Modification of Galactosylated and Sialylated Antibodies

[0122] To enzymatically galactosylate purified antibody samples, bovine β-1,4-galactosyltransferase (β1,4GT) from Sigma Chemical Co. (St. Louis, MO) and UDP-Gal. Recombinant rat liver α-2,3-sialyltransferase (α2,3ST), recombinant α-1,3-galactosyltransferase (α1,3GT) and CMP-Sia were from Calbiochem (San Diego, CA ). PNGase F was from New England Biolabs (Beverly, MA) or from Prozyme (San Leandro, CA) or from Selectin BioSciences (Pleasant Hill, CA). β-galactosidase and β-glucosaminidase of Diplococcus pneumoniae were obtained from ProZyme or Selectin BioSciences. Beta-galactosidase from bovine kidney and all other enzymes were obtained from ProZyme or from Selectin BioSciences. NAP-5 and HiTrap protein A columns were obtained from Pharmacia Biotech (Piscataway, NJ). All other reagents were of analytical grade.

[0123] An enzymatically deglycosylated form of Abl (termed Gno) was prepared ...

Embodiment 3

[0135] Example 3: Binding to low affinity cellular FC receptors

[0136] Of the several types of Fc receptors on effector cells, Fcγ types II and III are considered low or intermediate affinity receptors. Typically, the binding of the monomer may be with too low affinity to be detected or at a very low level. For example, binding of monomeric IgG to gamma IIA is difficult to measure. These receptors act to bind immune complexes due to their more avid, multivalent nature of binding, perhaps due to the slowed rate of the complexes.

[0137] Human K562 cells, which express FcγRIIA as the sole Fcγ receptor, were used in two types of binding assays to test whether alterations in the acid saliva content of Fc glycans affect binding to this low-affinity human Fcγ receptor . To obtain binding with sufficient affinity to FcγRIIA (which has low affinity for monomeric IgG), an immune complex was prepared by mixing the test Ab against TNF with homotrimeric TNF in a 2:1 molar ratio , t...

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Abstract

The properties of an Fc-containing protein, for example, an antibody, are controlled by altering the sialylation of the oligosaccharides in the Fc region. The modified Fc-containing proteins have therapeutic utility in diseases or conditions in which it is desirable to control the affinity for one or more of the FcgRI, FcgRIIA, and FcgRIIIA receptors, ADCC activity, macrophage or monocyte activation, serum half-life, and avidity.

Description

technical field [0001] The present invention relates to modified therapeutic proteins that interact with Fc receptors and methods of producing modified therapeutic proteins, such as antibodies, such that the composition of the oligosaccharide chains increases the affinity of the antibody for its target and alters the receptor binding affinity and thus The antibody effector function activity is altered compared to an unmodified antibody. Background technique [0002] Antibodies are soluble serum glycoproteins that play an important role in innate immunity. The carbohydrate structures of all naturally occurring antibodies vary with isotype at positions conserved in the heavy chain constant region. The various isotypes have different arrangements of N-linked oligosaccharide structures that can differently affect protein assembly, secretion, or functional activity (Wright, A., and Morrison, S.L., Trends Biotech. 15:26-32 (1997)). refer to figure 1 and 2 , the degree to which...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/395A61K38/16
CPCC07K2316/52C07K16/241C07K2317/52C07K16/00C07K2317/92C07K2317/21C07K2317/732A61K2039/505C07K2317/41A61P1/00A61P17/00A61P17/06A61P19/02A61P27/02A61P29/00A61P35/00
Inventor A·蔡C·吴T·S·拉于B·斯卡伦
Owner CENTOCOR
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