US2014200152A1PendingUtilityA1
Method for screening anti-ligand libraries for identifying anti-ligands specific for differentially and infrequently expressed ligands
Est. expirySep 2, 2022(expired)· nominal 20-yr term from priority
Inventors:Björn Frendéus
C40B 30/04G01N 33/6845C12N 15/1037G01N 33/554
61
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
The present invention relates to screening methods and, in particular, to methods of screening anti-ligand libraries for identifying anti-ligands specific for differentially and/or infrequently expressed ligands. The method comprises the steps of providing a library of anti-ligands; providing a first subtractor ligand; providing a second target ligand; determining the amount of the first and second target ligands using one or more equations derived from the universal law of mass action; providing the determined amount of a first subtractor ligand; providing the determined amount of a second target ligand; providing separation means capable of use to isolate anti-ligand bound to the second target ligand from anti-ligand bound to the first subtractor ligand; exposing the library of to the first and second target ligands to permit binding of anti-ligands to ligands; and using the separation means to isolate the anti-ligand bound to second target ligand.
Claims
exact text as granted — not AI-modified1 - 30 . (canceled)
31 . A method of isolating at least one anti-ligand to at least one differentially expressed target ligand comprising the steps of:
(i) providing data set(s) describing a library of anti-ligands; (ii) providing data set(s) describing a population of subtractor ligand constructs, said subtractor ligand constructs comprising a ligand associated with a separation means, wherein said separation means is a physical feature of the subtractor ligand construct; (iii) providing data set(s) describing a population of target ligand constructs, said target ligand constructs comprising the same ligand as step (ii) associated with a separation means, wherein said separation means is a physical feature of the target ligand construct; (iv) determining the amounts of subtractor ligand construct and target ligand construct utilizing one or more equations derived from the universal law of mass action
[
C
]
c
[
d
]
d
[
A
]
a
[
B
]
b
=
K
eq
,
where:
A, B, C & D=are the participants in the reaction (reactants and products)
a, b, c, & d=the coefficients necessary for a balanced chemical equation to permit the determination of the amount of subtractor ligand as compared to target ligand required for isolation of at least one anti-ligand to at least one target ligand;
(v) providing the amount of the subtractor ligand construct determined in step (iv);
(vi) providing the amount of the target ligand construct determined in step (iv);
(vii) providing the library of anti-ligands described by the data-set of step (i)
(viii) exposing the anti-ligand library of (vii) to the amounts of subtractor ligand construct provided in step (v) and the amounts of the target ligand construct provided in step (vi) to permit binding of anti-ligands to ligands; and
(ix) isolating the anti-ligand bound to the target ligand construct.
32 . The method of claim 31 wherein step (iv) and at least one of steps (v) to (ix) are performed automatically.
33 . The method of claim 31 further comprising the step of separating the anti-ligand bound to the ligand of the target ligand construct.
34 . The method of claim 31 wherein steps (ii) to (ix) are repeated one or more times.
35 . The method of claim 31 wherein the amount of one of the subtractor ligand construct or target ligand construct is provided in excess of the amount of the other of the subtractor ligand construct or the target ligand construct.
36 . The method of claim 35 where the excess of either the target ligand construct or the subtract ligand construct is 10 to 100 fold.
37 . The method of claim 31 wherein the equation of (iv) is
bA
=
(
A
+
T
+
(
K
d
)
×
(
C
×
V
)
)
2
-
(
A
+
T
+
(
K
d
)
×
(
C
×
V
)
)
2
4
-
A
×
T
where
bA=Bound anti-ligand
A=Total number of anti-ligand
T=Total number of ligands of both the target ligand construct and the subtractor ligand construct
C=Avogadro's constant (6.022×10 23 particles/mole)
V=Reaction volume (litres)
K d =Equilibrium dissociation constant.
38 . The method of claim 31 wherein the equation of (iv) is:
bA
=
{
(
A
+
T
+
(
K
d
)
×
(
C
×
V
)
)
2
-
(
A
+
T
+
(
K
d
)
×
(
C
×
V
)
)
2
4
-
A
×
T
}
×
{
(
T
p
×
C
p
)
(
(
T
p
×
C
p
)
+
(
T
s
×
C
s
)
)
}
where
bA p =Bound anti-ligand
T p =The number of ligands of the target ligand constructs on C p
T s =The number of ligands of the subtractor ligand constructs on C s
C p =The number of target ligand constructs
C s =The number of subtractor ligand constructs
A=Total number of anti-ligand
T=Total number of ligands of both target ligand constructs and subtractor ligand constructs
C=Avogadro's constant (6.022×10 23 particles/mole)
V=Reaction volume (litres)
K d =Equilibrium dissociation constant.
39 . The method of claim 31 wherein the separation means is selected from the group consisting of at least one of a solid support, cell membrane and/or portions thereof, synthetic membrane, beads, and/or chemical tags.
40 . The method of claim 39 wherein the separation means is cell membranes and/or portions thereof.
41 . The method of claim 40 whereby the subtractor and target ligands are fixed to and/or incorporated within separate cell membranes and/or portions thereof.
42 . The method of claim 31 whereby the separation means of the subtractor and target ligand constructs have a different density.
43 . The method of claim 42 wherein the separation means of the subtractor ligand construct is of a lower density than the separation means of the target ligand construct.
44 . The method of claim 43 wherein the separation means of the subtractor ligand construct is a membrane vesicle.
45 . The method of claim 43 wherein the separation means of the target ligand construct is a whole cell membrane.
46 . The method of claim 31 wherein the isolating of step (viii) is performed using at least one method selected from the group consisting of density centrifugation, solid support sequestration, magnetic bead sequestration, chemical tag binding and/or aqueous phase partitioning.
47 . The method of claim 46 wherein the-isolating of step (viii) is performed by density centrifugation.
48 . The method of claim 47 wherein the density centrifugation utilizes a sucrose-polymer gradient.
49 . The method of claim 31 wherein the library of step (i) is a display library comprising a plurality of library members which display anti-ligands.
50 . The method of claim 49 wherein the library is a phage display library.
51 . The method of claim 31 wherein the subtractor and target ligands are independently selected from the group consisting of at least one of (i) antigens; (ii) receptor ligands; and/or (iii) enzyme targets selected from the group consisting of a carbohydrate; protein; peptide; lipid; polynucleotide; inorganic molecules and/or conjugated molecules.
52 . The method of claim 31 wherein the library of anti-ligands comprises one or more of the following: antibodies, and/or antigen binding variants, derivatives or fragments thereof; scaffold molecules with engineered variable surfaces; receptors; and/or enzymes.
53 . The method of claim 31 comprising a further step of exposing the ligand of the target ligand construct to a stimulus which influences the expression of said target ligand construct.Cited by (0)
No later patents cite this yet.
References (0)
No backward citations on record.