Screening methods and uses thereof
A subtraction and equation technique, applied in compound screening, allergic diseases, instrumentation, etc., to solve problems such as the limited number of unique antibody sequences
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Embodiment 1
[0185] Example 1 - Differential biopanning combined with subsequent high-throughput sequencing yields an unprecedented number of unique antibody clones specific for differentially expressed target cell surface receptors
[0186] Generation of antibody pools by differential biopanning of cancer B cells against Jurkat T cells ("B not T")
[0187] In this experiment, from the 10 2x10 of the high diversity n-CoDeR library of genotype unique binders 13 Phage particles were mixed with whole B lymphoma cell line Ramos cells (positive selection) and plasma membrane or coarse membrane vesicles from T leukemia cell line Jurkat (negative selection). Binders specific for antigens uniquely expressed on the B-lymphoma cell line Ramos were selectively isolated compared to the T-cell leukemia cell line Jurkat.
[0188] Calculation of positive and negative cell numbers for screening
[0189] Cell numbers for different screening rounds were calculated as taught in WO2004 / 023140. The reactio...
Embodiment 2
[0285] Example 2 - Combining Differential Biopanning and Subsequent High-Throughput Sequencing Generates a Qualitative Pool of Unique Antibody Clones - Including Those Specific for Lower Expressed Differentially Expressed Surface Receptors
[0286] Computer calculations and application of the law of mass action as described in WO2004 / 023140 and Frendéus, (2006) taught that when differential biopanning is applied as in the present application (as exemplified in Example 1 with cancer B cells or prostate cancer cells as targets), the frequency of antibody clones recovered in the pool of antibodies screened will be a direct function of a) the absolute and relative expression of the receptors they target on the surface of target versus non-target cells and b) Their respective affinities for the targeted surface receptors.
[0287] These calculations further determined that, contrary to the prevailing belief (Hoogenboom, 2002) (Liu et al., 2004; Mutuberria et al., 1999; Osbourn et a...
Embodiment 3
[0421] Example 3 - Derivation of Differential Biopanning Equations
[0422] By applying the general law of mass action (LMA), the number of ligands required to isolate antiligands against lowly expressed ligands and / or differentially expressed ligands from high diversity display libraries can be calculated.
[0423] LMA shows that non-covalent (hydrogen bonding, electrostatic, van der Waals or hydrophobic forces), reversible binding between anti-ligand A and its target ligand T and their complex AT is caused by equilibrium interactions Take the equilibrium dissociation constant or affinity K d =[A][T] / [AT] is given.
[0424] The equilibrium interaction between anti-ligands with the same specificity for the target ligand (T) can be described as
[0425] Bound A (bA) Free A(fA)+Free T(fT)
[0426] by
[0427]
[0428] Total A or T is known to be the sum of free and bound A or T
[0429]That is, [A] (total A) = [fA] + [bA], and [T] (total T) = [fT] + [bA]
[0430] The...
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Abstract
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