Methods of measuring factor D activity and potency of factor D inhibitors
AssigneeGenentech, Inc.
InventorsLinda Orren, Aaron Miller, Xiaoqing Jia, Pin Yee Wong
Life of this patent
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Abstract
Methods of measuring the activity of Factor D, the key driver in the activation of the alternative complement pathway, methods of determining the potency of Factor D inhibitors, and methods of screening for Factor D inhibitors are provided.
Claims48
A method of measuring the activity of Factor D in a sample derived from a subject, comprising performing a proximity-based measurement assay, wherein the proximity-based measurement assay measures the cleavage of Factor B and comprises:
- (a) adding to the sample an anti-Factor Ba antibody labeled with a first moiety and an anti-Factor Bb antibody labeled with a second moiety, wherein one of the moieties is a donor, and the other moiety is an acceptor, and wherein a proximity signal generated by the acceptor and/or the donor indicates that Factor Ba and Factor Bb are in proximity;
- and
- (b) measuring the value of the proximity signal from the sample;
- wherein the activity of Factor D in the sample is negatively correlated with the value of the proximity signal.
A method of measuring the activity of Factor D in a sample derived from a subject, comprising performing a proximity-based measurement assay that comprises:
- (a) adding to the sample an anti-Factor C3b antibody labeled with a first moiety and an anti-Factor Ba antibody labeled with a second moiety, wherein one of the moieties is a donor, and the other moiety is an acceptor, and wherein a proximity signal generated by the acceptor and/or the donor indicates that Factor C3b and Factor Ba are in proximity;
- and
- (b) measuring the value of the proximity signal from the sample;
- wherein the activity of Factor D in the sample is negatively correlated with the value of the proximity signal.
A method of determining the potency of a Factor D inhibitor in inhibiting Factor D activity, comprising performing a proximity-based measurement assay, wherein the proximity-based measurement assay measures the cleavage of Factor B and comprises:
- (a) mixing the Factor D inhibitor with Factor D, Factor B, and Factor C3b in a test reaction mix;
- (b) incubating the test reaction mix;
- (c) adding to the test reaction mix an anti-Factor Ba antibody labeled with a first moiety and an anti-Factor Bb antibody labeled with a second moiety after the step of (b), wherein one of the moieties is a donor, and the other moiety is an acceptor, and wherein a proximity signal generated by the acceptor and/or the donor indicates that Factor Ba and Factor Bb are in proximity;
- and
- (d) measuring the value of the proximity signal from the text reaction mix;
- wherein the potency of the Factor D inhibitor is positively correlated with the value of the proximity signal.
A method of determining the potency of a Factor D inhibitor in inhibiting Factor D activity, comprising performing a proximity-based measurement assay, wherein the proximity-based measurement assay measures the cleavage of Factor B and comprises:
- (a) mixing the Factor D inhibitor with Factor D, Factor B and Factor C3b in a test reaction mix;
- (b) incubating the test reaction mix;
- (c) adding to the test reaction mix an anti-Factor C3b antibody labeled with a first moiety and an anti-Factor Ba antibody labeled with a second moiety after the step of (b), wherein one of the moieties is a donor, and the other moiety is an acceptor, and wherein a proximity signal generated by the acceptor and/or the donor indicates that Factor C3b and Factor Ba are in proximity;
- and
- (d) measuring the value of the proximity signal from the test reaction mix;
- wherein the potency of the Factor D inhibitor is positively correlated with the value of the proximity signal.
A method of screening for a Factor D inhibitor from a plurality of Factor D inhibitor candidates, comprising performing a proximity-based measurement assay, wherein the proximity-based measurement assay measures the cleavage of Factor B and comprises:
- (a) mixing a Factor D inhibitor candidate with Factor D, Factor B and Factor C3b in a test reaction mix;
- (b) incubating the test reaction mix;
- (c) adding to the test reaction mix an anti-Factor Ba antibody labeled with a first moiety and an anti-Factor Bb antibody labeled with a second moiety after the step of (b), wherein one of the moieties is a donor, and the other is an acceptor, and wherein a proximity signal generated by the acceptor and/or the donor indicates that Factor Ba and Factor Bb are in proximity;
- and
- (d) measuring the value of the proximity signal from the test reaction mix;
- wherein the degree of Factor D activity inhibition by a Factor D candidate is positively correlated with the value of the proximity signal.
A method of screening for a Factor D inhibitor from a plurality of Factor D inhibitor candidates, comprising performing a proximity-based measurement assay, wherein the proximity-based measurement assay measures the cleavage of Factor B and comprises:
- (a) mixing a Factor D inhibitor candidate with Factor D, Factor B and Factor C3b in a test reaction mix;
- (b) incubating the test reaction mix;
- (c) adding to the test reaction mix an anti-Factor C3b antibody labeled with a first moiety and an anti-Factor Ba antibody labeled with a second moiety after the step of (b), wherein one of the moieties is a donor, and the other is an acceptor, and wherein a proximity signal generated by the acceptor and/or the donor indicates that Factor C3b and Factor Ba are in proximity;
- and
- (d) measuring the value of the proximity signal from the test reaction mix;
- wherein the degree of Factor D activity inhibition by a Factor D candidate is positively correlated with the value of the proximity signal.
Dependencies are inferred from the claim text.