Methods of qualitatively and/or quantitatively analyzing properties of activatable antibodies and uses thereof
The invention provides methods and kits for qualitatively and/or quantitatively analyzing activation and other properties of activatable antibody therapeutic in biological samples, including tissues and/or biofluid samples. The invention also relates to methods of using a capillary-based immunoassay platform to qualitatively and/or quantitatively analyze levels of activation in biological samples, including tissues and/or biofluid samples.
1 . A method of quantitating a level of activation of an activatable antibody, the method comprising:
i) contacting a loaded capillary or population of loaded capillaries with a biological sample comprising one or more components selected from the group consisting of an uncleaved activatable antibody, a cleaved activated antibody, and a combination thereof,
wherein the uncleaved activatable antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds a target, a masking moiety (MM) coupled to the AB, and a cleavable moiety (CM) coupled to the AB,
wherein the MM inhibits the binding of the AB to the target,
wherein the CM is a polypeptide that functions as a substrate for a protease,
wherein protease cleavage of the CM generates the cleaved activated antibody;
wherein the loaded capillary or population of loaded capillaries is/are pre-loaded with a stacking matrix and a separation matrix;
ii) separating one or more high molecular weight (MW) components of the biological sample from one or more low molecular weight (MW) components of the biological sample within each capillary, wherein at least one high MW component comprises the uncleaved activatable antibody and wherein at least one low MW component comprises the cleaved activated antibody;
iii) immobilizing the high MW components and the low MW components within each capillary;
iv) immunoprobing each capillary with at least a first reagent that is specific for at least one activatable antibody, wherein the first reagent comprises an anti-idiotypic antibody or antigen-binding fragment thereof,
wherein when the MM and CM are conjugated to a heavy chain, the anti-idiotypic antibody or antigen-binding fragment thereof binds to a variable heavy chain region of the uncleaved activatable antibody and the cleaved activated antibody, and
wherein when the MM and CM are conjugated to a light chain, the anti-idiotypic antibody or antigen-binding fragment thereof binds to a variable light chain region of the uncleaved activatable antibody and the cleaved activated antibody; and
v) detecting and quantitating a level of the first reagent in each capillary or population of capillaries to determine relative levels of cleaved activated antibody and uncleaved activatable antibody in each capillary or population of capillaries, thereby determining the level of activation of the activatable antibody.
2 . The method of claim 1 , further comprising, prior to step i), loading at least one capillary or a population of capillaries with a stacking matrix and a separation matrix to generate the at least one loaded capillary or a population of loaded capillaries.
3 . The method of claim 1 , wherein the separating step is carried out for a separation time of at least about 35 minutes, at least about 36 minutes, at least about 37 minutes, or at least about 38 minutes.
4 . The method of claim 1 , wherein step iii) comprises using UV light to immobilize the high MW components and the low MW components of the biological sample.
5 . The method of claim 1 , wherein the activatable antibody is selected from the group consisting of a conjugated activatable antibody, a multispecific activatable antibody, and a conjugated multispecific activatable antibody.
6 . The method of claim 1 , wherein the anti-idiotypic antibody or antigen-binding fragment thereof, binds to a variable light chain (VL) CDR of the at least one activatable antibody, conjugated activatable antibody, multispecific activatable antibody, conjugated multispecific activatable antibody, or combination thereof, wherein the VL CDR is selected from the group consisting of VL CDR1, VL CDR2, and VL CDR3.
7 . The method of claim 1 , wherein the first reagent is a detectable reagent.
8 . The method of claim 1 , wherein step iv) further comprises loading each capillary with a second reagent that specifically binds to the first reagent.
9 . The method of claim 8 , wherein the second reagent comprises a secondary antibody.
10 . The method of claim 8 , wherein the secondary reagent comprises a detectable label.
11 . The method of claim 9 , wherein the second reagent comprises a secondary antibody conjugated to a detectable label.
12 . The method of claim 9 , wherein the secondary antibody is not conjugated to a detectable label.
13 . The method of claim 12 , wherein the secondary antibody is conjugated to a first binding tag of a set of a first binding tag and a second binding tag, wherein the first binding tag is capable of binding to the second binding tag.
14 . The method of claim 13 , wherein step iv) further comprises loading each capillary with a third reagent that specifically binds to the second reagent.
15 . The method of claim 14 , wherein the third reagent comprises a reporter enzyme conjugated to the second binding tag.
16 . The method of claim 15 , wherein the reporter enzyme is selected from the group consisting of horseradish peroxidase and alkaline phosphatase.
17 . The method of claim 15 , wherein the first and second binding tags are selected from the group consisting of biotin and streptavidin; streptavidin and biotin; biotin and avidin; and avidin and biotin; respectively.
18 . The method of claim 14 , wherein the third reagent comprises a detectable tertiary antibody.
19 . The method of claim 1 , wherein step iv) further comprises loading each capillary with a substrate selected from the group consisting of a chemiluminescent substrate and a colorimetric substrate.
20 . The method of claim 19 , wherein the substrate is a chemiluminescent substrate, and step v) comprises detecting a level of chemiluminescence in each capillary or population of capillaries.
21 . The method of claim 20 , wherein the chemiluminescent substrate is luminol, and wherein step iv) further comprises loading each capillary with peroxide.
22 . The method of claim 1 wherein step i) comprises loading approximately 1-500 ng of biological sample.
23 . The method of claim 22 , wherein step i) comprises loading approximately 5-40 ng of biological sample.
24 . The method of claim 1 , wherein the biological sample is a bodily fluid.
25 . The method of claim 24 , wherein the bodily fluid is selected from the group consisting of blood, plasma, and serum.
26 . The method of claim 1 , wherein the biological sample is a diseased tissue.
27 . The method of claim 26 , wherein the diseased tissue is a lysate.
28 . The method of claim 27 , wherein the disease tissue is tumor tissue.
29 . The method of claim 1 , wherein the uncleaved activatable antibody has the structure from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM.
30 . The method of claim 29 , wherein the AB is linked directly to the CM, the CM is linked directly to the AB, or both.
31 . The method of claim 29 , wherein the AB is linked to the CM via a linking peptide, the CM is linked to the AB via a linking peptide, or both.
32 . The method of claim 29 , wherein the uncleaved activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the uncleaved activatable antibody has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
33 . The method of claim 32 , wherein the two linking peptides need not be identical to each other.
34 . The method of claim 32 , wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.