U.S. patent application number 14/897459 was filed with the patent office on 2016-05-05 for obtaining an improved therapeutic ligand.
The applicant listed for this patent is UCB BIOPHARMA SPRL. Invention is credited to Terence Seward Baker, Alastair David Griffiths Lawson, Xiaofeng Liu, Jiye Shi.
Application Number | 20160125124 14/897459 |
Document ID | / |
Family ID | 48914497 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160125124 |
Kind Code |
A1 |
Shi; Jiye ; et al. |
May 5, 2016 |
Obtaining an Improved Therapeutic Ligand
Abstract
Methods and associated apparatus involving designing a ligand ab
initio that will bind to a binding site of a macromolecular target,
or of identifying a modification to a ligand for improving the
affinity of the ligand to a binding site of a macromolecular
target, comprising using information about non-bonding,
intra-molecular or inter-molecular atom to atom contacts extracted
from a database of biological macromolecules to identify favoured
regions adjacent to the binding site for particular atom types and
modifying a candidate ligand to increase the intersection between
atoms of the candidate ligand and the favoured regions. One or more
steps of the methods may be performed by a computer.
Inventors: |
Shi; Jiye; (Brussels,
BE) ; Baker; Terence Seward; (Slough, Berkshire,
GB) ; Lawson; Alastair David Griffiths; (Slough,
Berkshire, GB) ; Liu; Xiaofeng; (Slough, Berkshire,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCB BIOPHARMA SPRL |
Brussels |
|
BE |
|
|
Family ID: |
48914497 |
Appl. No.: |
14/897459 |
Filed: |
June 13, 2014 |
PCT Filed: |
June 13, 2014 |
PCT NO: |
PCT/EP2014/062478 |
371 Date: |
December 10, 2015 |
Current U.S.
Class: |
506/24 ;
530/389.2; 702/19 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 2317/55 20130101; G16C 20/50 20190201; G16C 20/90 20190201;
C07K 16/244 20130101; G16B 15/00 20190201; G16B 5/00 20190201 |
International
Class: |
G06F 19/12 20060101
G06F019/12; C07K 16/24 20060101 C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 2013 |
GB |
1310544.0 |
Claims
1. A method for designing a ligand ab initio that will bind to a
binding site of a macromolecular target, or of identifying a
modification to a ligand for improving the affinity of the ligand
to a binding site of a macromolecular target, comprising: a)
identifying a target list of atoms forming the surface of the
target binding site; b) identifying each atom, hereinafter referred
to as a theta atom, in the target list, as a particular theta atom
type; c) extracting from a structural database of biological
macromolecules, information about non-bonding, intra-molecular or
inter-molecular atom to atom contacts, where the first atom in a
contacting pair of atoms is of a particular theta atom type and the
opposing, second atom of the pair, hereinafter referred to as an
iota atom, is of a particular iota atom type, said information
comprising spatial and/or contextual data about the iota atom
relative to the theta atom, and said data collected for a plurality
of contacts of the given theta atom type from the said database is
hereinafter referred to as a theta contact set; d) for each theta
atom identified in the target list in b), superimposing in or
around the target binding site data relating to a given iota atom
type, or a predetermined group of related iota atom types, from the
corresponding theta contact set extracted in c); e) combining
and/or parsing the superimposed data in such a way as to predict
one or more favoured regions of the binding site where the given
iota atom type, or the predetermined group of related iota atom
types, has high theoretical propensity; and f) with a candidate
ligand notionally docked into the binding site, comparing the type
and position of one or more of the atoms of the candidate ligand
with the predicted favoured regions for the respective iota atom
types, to identify a modification to the candidate ligand, in terms
of alternate and/or additional candidate ligand atoms, that will
produce a greater intersection between the alternate and/or
additional candidate ligand atoms and the respective iota atom type
favoured regions, leading to an improvement in the affinity of the
modified candidate ligand to the binding site compared to the
unmodified candidate ligand; wherein each non-bonding
intra-molecular or inter-molecular contact in the database is
defined as a contact between opposing residues of a protein fold or
between opposing monomer units of a macromolecular fold or between
two interacting macromolecular partners and is specifically between
a theta atom on one side of the fold or first interacting partner
and an iota atom on the opposing side or second interacting
partner; in an instance where the following condition is satisfied:
s-Rw.ltoreq.t, where s is the separation between the two atoms of
the contact, Rw is the sum of the van de Waals radii of the two
atoms of the contact, and t is a predetermined threshold distance;
and wherein the theta atom type is identified uniquely in b) such
that there is no intersection between the data of a theta contact
set extracted in c) for a given theta atom type and the data of any
other theta contact set extracted in c) for any other theta atom
type, apart from data concerning contacts involving the given theta
atom as the iota atom.
2. The method according to claim 1, wherein for each non-bonding
intra-molecular contact extracted from the structural database of
protein members in c) the following condition is also satisfied:
the theta atom and the iota atom of the contact are on different
residues separated by at least four residues along the linear
polypeptide or are on separate polypeptide chains.
3. The method according to claim 1, wherein the theta atom type is
identified as being one and only one of: the 167 non-hydrogen atoms
present in the 20 natural amino acids of proteins; the 82
non-hydrogen atoms present in the 4 nucleotides of the
deoxyribonucleic acid polymer (DNA); the 42 non-hydrogen atoms
present in the methylated DNA nucleotides, cytidine phosphate and
adenosine phosphate; the 85 non-hydrogen atoms present in the 4
nucleotide phosphates of the ribonucleic acid polymer (RNA); the 89
non-hydrogen atoms present in 2-O'-methylated ribose nucleotide
phosphates of RNA; the over 400 non-hydrogen atoms present in the
commonest post-transcription base modified RNA.
4. The method according to claim 1, wherein the information
extracted in c) is collected in a secondary database comprising one
and only one theta contact set for each of the theta atom
types.
5. The method according to claim 4, wherein each of said secondary
database theta contact sets is sub-divided into a plurality of
non-overlapping iota atom types or non-overlapping groups of
related iota atom types.
6. The method according to claim 1, wherein the iota atom type is
identified as being one and only one of: the 167 non-hydrogen atoms
present in the 20 natural amino acids of proteins; the oxygen atom
present in protein bound, structurally relevant, water molecules;
the 82 non-hydrogen atoms present in the 4 nucleotides of the
deoxyribonucleic acid polymer (DNA); the 42 non-hydrogen atoms
present in the methylated DNA nucleotides, cytidine phosphate and
adenosine phosphate; the 85 non-hydrogen atoms present in the 4
nucleotide phosphates of the ribonucleic acid polymer (RNA); the 89
non-hydrogen atoms present in 2-O'-methylated ribose nucleotide
phosphates of RNA; and/or the over 400 non-hydrogen atoms present
in the commonest post-transcription modified bases of RNA.
7. The method according to claim 1, wherein said predetermined
group of related iota atom types is one of a plurality of
non-overlapping groups obtained by sorting the 167 non-hydrogen
atoms present in the 20 natural amino acids of proteins into groups
of similar chemical type.
8. The method according to claim 7, wherein the iota atom types are
sorted into the plurality of non-overlapping groups according to
one or more of the following factors: elemental nature of the atom
type, hybridisation state of the atom type.
9. The method according to claim 7, wherein the iota atom types are
sorted into a plurality of non-overlapping groups comprising the
following: C sp.sup.3, C sp.sup.2(aromatic), C
sp.sup.2(non-aromatic), N sp.sup.3, N sp.sup.2, O sp.sup.3, O
sp.sup.2, S.
10. The method according to claim 1, wherein: said spatial data
extracted in c) defines the position of each iota atom specified in
the theta contact set by geometrical reference to the position of
the theta atom and to the positions of third and fourth atoms; the
third atom is covalently bonded to the theta atom; and the fourth
atom is covalently bonded to the third atom.
11. The method according to claim 10, wherein: for each iota atom
specified in the theta contact set, said spatial data extracted in
c) defines the position of fifth and sixth atoms by geometrical
reference to the position of the theta atom and to the positions of
the third and fourth atoms; the fifth atom is covalently bonded to
the iota atom; and the sixth atom is covalently bonded to either
the fifth atom or the iota atom.
12. The method according to claim 11, wherein the superimposition
in or around the target site of (d) comprises: parsing the theta
contact set to extract spatial data for contacts comprising the
given iota atom type or one or more of the predetermined group of
related iota atom types; and plotting this spatial data to
determine theoretical locations representing where each iota atom
type, or each of the one or more of the predetermined group of
related iota atom types, would be located if: i) the theta atom of
the contact were located at the position of the corresponding theta
atom in the target binding site; and ii) the third and fourth atoms
of the contact were located at the positions of the third and
fourth atoms of the corresponding theta atom in the target binding
site.
13. The method according to claim 12, wherein: the extracted
spatial data is parsed against said contextual data before said
plotting step.
14. The method according to claim 12, wherein a region in which a
density of theoretical locations for the given iota atom type, or
for the one or more of the predetermined group of related iota atom
types, is above a predetermined threshold is identified as one of
the favoured regions.
15. The method according to claim 12, wherein theoretical locations
for the given iota atom type, or for one or more of the
predetermined group of related iota atom types, are determined for
a plurality of theta atoms on the target list and a region in which
a density of the cumulative theoretical locations is above the
predetermined threshold is identified as one of the favoured
regions.
16. The method according to claim 12, wherein: if the theoretical
location of an individual iota atom intersects with the location of
an atom of the target macromolecule closer than Rw-0.2 angstroms
then the said iota atom is excluded from subsequent analysis.
17. The method according to claim 11, wherein: the third and fourth
atoms are chosen uniquely for each specified theta atom type.
18. The method according to claim 11, wherein: the fifth and sixth
atoms are chosen uniquely for each specified iota atom type.
19. The method according to claim 11, wherein: for each favoured
region, vectors are derived to describe the position of the fifth
atom relative to its respective iota atom and analysis is carried
out on said vectors in order to identify a favoured bond vector
representing a prediction of the covalent attachment of a
theoretical consensus iota atom in the said region, said identified
favoured bond vector being used to refine the design of the
candidate ligand or modification of the candidate ligand.
20. The method according to claim 1, wherein: said contextual data
extracted in (c), contains contextual information concerning the
local environment of each contact pair in the theta contact set,
including one or more of the following in any combination:
secondary structure, amino acid types or other monomer types
comprising the contact pair, adjacent monomer units and/or local
geometry thereof in a polymer chain either side of the contact,
adjacent amino acids in a polypeptide chain on either side of the
contact, local geometry of the said adjacent monomer units or amino
acids, temperature factor of the theta atom, temperature factor of
the iota atom, accessible surface area of the theta atom,
accessible surface area of the iota atom, the number of different
iota atom contacts for the particular theta atom and the number of
other theta atoms on the same monomer unit as the theta atom.
21. The method according to claim 1, wherein (f) comprises:
identifying a modification of the candidate ligand that increases a
degree of overlap between one or more atoms of the candidate ligand
and a predicted favoured region or regions for an iota atom type or
predetermined group of related iota atom types in the binding
site.
22. The method according to claim 1, wherein a plurality of
modifications to the candidate ligand are identified in (f) and the
method further comprises selecting a subset of the identified
modifications based on one or both of the following: 1) the extent
to which the intersection between the alternate and/or additional
candidate ligand atoms and the respective iota atom type favoured
regions is greater compared to the unmodified candidate ligand; and
2) the extent to which one or more factors contributing to the
total energy of the complex formed by the binding of the modified
candidate ligand to the binding site is/are reduced compared to the
case where the unmodified candidate ligand is bound.
23. The method according to claim 1, wherein t=2.5 angstroms
24. The method according to claim 1, wherein t=0.8 angstroms
25. The method according to claim 1, further comprising:
out-putting data representing the modification identified in
(f).
26. The method according to claim 1, wherein the ligand is a
protein.
27. The method according to claim 26, wherein the ligand is an
antibody.
28. The method according to claim 26, wherein (f) comprises
replacing each of one or more of the amino acid residues of the
ligand that is/are in direct contact with the target binding site,
or in close proximity to the target binding site, with each of one
or more alternative residues chosen from the other 19 natural amino
acids, each replacement being referred to as a residue replacement,
wherein for each residue replacement that does not cause conflict
between the replacement residue and adjacent atoms of the ligand or
target, the type and position of each atom of the replacement
residue is compared with the respective iota atom type favoured
regions to identify whether they will produce a greater
intersection than the atoms of the original residue.
29. The method according to claim 28, further comprising:
outputting a list of the residue replacements that are identified
as producing a greater intersection than atoms of the original
residue; for each listed residue replacement, using mutation of the
candidate ligand to produce a modified ligand that incorporates the
residue replacement; testing the affinity of each of the modified
ligands to the target binding site in order to determine which
residue replacements result in an affinity improvement that is
above a predetermined threshold.
30. The method according to claim 29, further comprising: modifying
the candidate ligand to incorporate a plurality of the residue
replacements that have been determined to result in an affinity
improvement that is above the predetermined threshold.
31. A computer readable medium or signal comprising computer
readable instructions for causing a computer to carry out the
method of claim 1.
32. The medium or signal according to claim 31, wherein the
computer is caused to carry out at least (c)-(e).
33. The medium or signal according to claim 31, wherein the
computer is caused to carry to carry out at least (f).
34. A method of manufacturing a therapeutic ligand, comprising:
designing a therapeutic ligand according to the method of claim 1;
and manufacturing the therapeutic ligand thus designed.
35. A therapeutic ligand manufactured according to the method of
claim 34.
36. The method or ligand according to claim 34, wherein the ligand
is a protein.
37. The method or ligand according to claim 36, wherein the protein
is an antibody.
38. A method of generating a database for use in a method for
designing a ligand ab initio that will bind to a binding site of a
macromolecular target, or of identifying a modification to a ligand
for improving the affinity of the ligand to a binding site of a
macromolecular target, comprising: analysing the relative positions
of atoms in each of a plurality of proteins or other biological
macromolecules in order to identify instances of a non-bonding
intra-molecular contact between a first atom, referred to as a
theta atom, and a second atom, referred to as an iota atom, of the
protein or macromolecule; and generating a database that for each
identified contact specifies: the type of the theta atom, the type
of the iota atom, and the position of the iota atom relative to the
theta atom; wherein a non-bonding intra-molecular contact is
defined as an instance where the following conditions are
satisfied: s-Rw.ltoreq.t, where s is the separation between the
theta and iota atoms, Rw is the sum of the van de Waals radii of
the theta and iota atoms, and t is a predetermined threshold
distance of typically 2.5 angstroms and preferably 0.8 angstroms;
and wherein in the case of proteins, the theta and iota atoms are
on amino acid residues separated from each other by at least four
residues on a linear polypeptide or are on separate polypeptide
chains.
39. The method according to claim 38, wherein the method comprises
sub-dividing the database to form groups of identified contacts in
which the theta atom is one and only one of the 167 non-hydrogen
atoms present in the 20 natural amino acids of proteins and the
iota atom is in one and only one of a plurality of non-overlapping
groups obtained by sorting the 167 non-hydrogen atoms present in
the 20 natural amino acids of proteins into groups based on
chemical similarity.
40. A method of generating a database for use in a method for
designing a ligand ab initio that will bind to a binding site of a
macromolecular target, or of identifying a modification to a ligand
for improving the affinity of the ligand to a binding site of a
macromolecular target, comprising: analysing the relative positions
of atoms in each of a plurality of proteins or other biological
macromolecules in order to identify instances of a non-bonding
intra-molecular contact between a first atom referred to as a theta
atom, and a second atom, referred to as an iota atom, of the
protein or macromolecule; and generating a database that for each
identified contact specifies: the type of the theta atom, the type
of the iota atom, and the position of the iota atom relative to the
theta atom; wherein a non-bonding intra-molecular contact is
defined as an instance where the following condition is satisfied:
s-Rw.ltoreq.t, where s is the separation between the theta and iota
atoms, Rw is the sum of the van de Waals radii of the theta and
iota atoms, and t is a predetermined threshold distance of
typically 2.5 angstroms and preferably 0.8 angstroms; and wherein
the method comprises sub-dividing the database to form groups of
identified contacts in which the theta atom is one and only one of
the 167 non-hydrogen atoms present in the 20 natural amino acids of
proteins and the iota atom is in one and only one of a plurality of
non-overlapping groups obtained by sorting the 167 non-hydrogen
atoms present in the 20 natural amino acids of proteins into groups
based on chemical similarity.
41. The method according to claim 39, wherein: for each contact,
the position of the iota atom is defined by geometrical reference
to the position of the theta atom and to the positions of third and
fourth atoms, the third atom being covalently bonded to the theta
atom and the fourth atom being covalently bonded to the third atom,
the method further comprising: normalizing the coordinates of the
iota atom, theta atom, third atom, and fourth atom of each contact
as a group to generate a normalized coordinate group; for each of
one or more of the theta atom types, using the normalized
coordinate groups for a plurality of contacts involving the theta
atom type and a given iota atom type to generate a two-dimensional
polar plot that represents a distribution of directions of the
given iota atom, in terms of latitude and longitude, relative to
the theta atom; repeating the above for different iota atom types;
comparing the resultant two-dimensional polar plots to identify
groups of iota atom types that yield similar distributions of
directions and using those groups as the groups based on chemical
similarity to sort the 167 non-hydrogen atoms present in the 20
natural amino acids of proteins into the plurality of
non-overlapping groups.
42. The method of generating a database according to claim 38,
comprising: extracting contact information from at least 2000
proteins or other biological macromolecules, the extracted
information containing information about at least two million
contact atom pairs.
43. The method of generating a database according to claim 38,
comprising: extracting contact information from at least 10000
proteins or other biological macromolecules, the extracted contact
information containing information about at least ten million
contact atom pairs.
44. The computer readable medium storing a database generated
according to claim 1.
45. The method according to claim 1, wherein for a given
antibody-antigen complex, specific mutations to amino acid residues
in or around the antibody binding site are predicted to produce
higher binding affinity of the antibody to the antigen.
Description
[0001] The present invention relates to obtaining an improved
therapeutic ligand, in particular by determining how an existing or
candidate ligand can be modified to improve binding of the ligand
at a binding site on a target protein or by aiding the de novo
design of a candidate ligand as a precursor to a therapeutic.
[0002] Therapeutic molecules (ligands) fall into two distinct
classes: chemical entities (or novel chemical entities, NCEs) and
biologicals. The former are low molecular weight organic compounds,
typically of molecular weight of 500 Daltons or less, that have
been chemically synthesized or isolated from natural products.
These are typically derived from starting chemicals or `hits` that
are discovered by screening chemical or natural product libraries.
Such hits typically have sub-optimal binding affinity for the
target and considerable trial and error in chemical modification is
required in order to obtain better affinity for the target
(typically of affinity constant (K.sub.D) low micromolar or less).
It is preferable that the hit has a lower molecular weight, say 300
Daltons or less, so that subsequent chemical modification does not
exceed the 500 Dalton limit. These hits are often referred to as
`fragments`. Optimisation of the hit to obtain a candidate
therapeutic or lead molecule is greatly enhanced by structural
information; for instance by obtaining an x-ray crystallographic
structure of the protein in co-complex with the hit molecule or
fragment. Such data provides insight into where on the target
protein the small molecule binds and importantly indicates how
atomic interactions between the two account for binding.
Furthermore the topographical nature of the protein surface
immediately surrounding the bound hit is revealed; and particularly
if it is a cleft or a pocket, the structure will suggest how the
hit might be elaborated to better fill the space within the pocket
and how to make further interactions with the protein and hence
improve binding affinity and specificity.
[0003] There are a number of computer based algorithms available to
assist the medicinal chemist in making rational choices for
chemical elaboration of the hit. These are either physics based
methods that attempt to calculate the free energy of binding
between the small molecule and protein from first principles (e.g.
Schrodinger.RTM. suite of software) or are statistical potential
methods that rely on a database of atomic interactions extracted
from collections of protein--small molecule structures (e.g.
SuperStar).
[0004] Biologicals are large peptide or protein molecules (of
molecular weight greater than 1000 Daltons). They are often
antibodies or antibody like molecules that recognize and bind to a
target molecule, usually with better affinity and specificity
compared to NCEs (K.sub.D low nanomolar or less). They may also be
other types of protein molecules such as hormones, cytokines,
growth factors or soluble receptors.
[0005] The binding of a candidate biological therapeutic molecule
to a binding site can be modified by mutating the candidate
therapeutic molecule. This may be required to improve the binding
affinity or alter the binding specificity. However, it is
relatively time-consuming to perform the mutation and to test the
binding efficiency of the mutated molecule. Many different
mutations may be required before improvements in binding efficiency
are obtained.
[0006] It is known to use computers to predict what kind of
modifications might be most effective. However, a given molecule
can be modified in a vast number of ways and it is difficult to
configure a computer so that the prediction can be achieved
reliably in a practical period of time.
[0007] Laskowski R A, Thornton J M, Humblet C & Singh J (1996)
"X-SITE: use of empirically derived atomic packing preferences to
identify favourable interaction regions in the binding sites of
proteins", Journal of Molecular Biology, 259, 175-201 discloses a
computer-based method for identifying favourable interaction
regions for different atom types at the surface of a protein, such
as at a dimer interface or at a molecular-recognition or binding
site. The Laskowski et al predictions are based on a database of
empirical data about non-bonding intra-molecular contacts observed
in high-resolution protein structures.
[0008] In the approach of Laskowski et al, the 20 amino acids are
broken up to yield a total of 488 possible 3-atom fragments. Taking
chemical similarities into account these are reduced to a set of
163 fragment types that sub-divide the database. Each fragment
contains a first atom (referred to as "position 3") with two
further atoms defining triangulation (or spatial normalization)
positions. A density function is derived by recording the various
positions at which an atom (which may be referred to as a "second
atom") is found to be in a non-bonding intra-molecular contact with
the first atom of a 3-atom fragment.
[0009] A predicted favourable interaction region for a given atom
type is obtained in Laskowski et al by transplanting density
functions into the binding site. Each density function is
transplanted such that the coordinates of the three atoms of the
3-atom fragment corresponding to the density function are
superimposed on the coordinates of a corresponding 3-atom fragment
in the binding site. Where density functions from different 3-atom
fragments in the binding site overlap, an average "density" is used
to predict the favourable interaction region.
[0010] The approach of Laskowski et al is relatively complex and
discards potentially useful data. The density functions of
Laskowski et al are obtained by populating a 3-D grid with the
positions of second atom contacts for each fragment type. A
different grid is used for each of the 163 fragment types. Data for
each second atom type is then mathematically transformed to give
the density function. Using the fragment definitions of Laskowski
et al, fragment type can be shared by different atoms on the same
residue and by atoms on different residues. When the Laskowski et
al database has been built on these fragment types there is an
over-abundance of main chain fragments that requires a
down-weighting at the stage of transplanting the density functions
into the binding site. Furthermore, a given fragment type can
include several actual fragments with subtle differences in bond
lengths and angles and concomitant differences in second atom
distributions which become masked when combined in these
divisions.
[0011] Short range secondary structure in the proteins used for
deriving the empirical data in Laskowski et al can lead to bias and
reduces the efficiency with which the empirical data indicates
favourable interaction regions.
[0012] It is an object of the invention to address at least one of
the problems with the art discussed above.
[0013] According to an aspect of the invention, there is provided a
method for designing a ligand ab initio that will bind to a binding
site of a macromolecular target, or of identifying a modification
to a ligand for improving the affinity of the ligand to a binding
site of a macromolecular target, comprising:
a) identifying a target list of atoms forming the surface of the
target binding site; b) identifying each atom, hereinafter referred
to as a theta atom, in the target list, as a particular theta atom
type; c) extracting from a structural database of biological
macromolecules, information about non-bonding, intra-molecular or
inter-molecular atom to atom contacts, where the first atom in a
contacting pair of atoms is of a particular theta atom type and the
opposing, second atom of the pair, hereinafter referred to as an
iota atom, is of a particular iota atom type, said information
comprising spatial and/or contextual data about the iota atom
relative to the theta atom, and said data collected for a plurality
of contacts of the given theta atom type from the said database is
hereinafter referred to as a theta contact set; d) for each theta
atom identified in the target list in step b), superimposing in or
around the target binding site data relating to a given iota atom
type, or a predetermined group of related iota atom types, from the
corresponding theta contact set extracted in step c); e) combining
and/or parsing the superimposed data in such a way as to predict
one or more favoured regions of the binding site where the given
iota atom type, or the predetermined group of related iota atom
types, has high theoretical propensity; and f) with a candidate
ligand notionally docked into the binding site, comparing the type
and position of one or more of the atoms of the candidate ligand
with the predicted favoured regions for the respective iota atom
types, to identify a modification to the candidate ligand, in terms
of alternate and/or additional candidate ligand atoms, that will
produce a greater intersection between the alternate and/or
additional candidate ligand atoms and the respective iota atom type
favoured regions, leading to an improvement in the affinity of the
modified candidate ligand to the binding site compared to the
unmodified candidate ligand;
[0014] wherein each non-bonding intra-molecular or inter-molecular
contact in the database is defined as a contact between opposing
residues of a protein fold or between opposing monomer units of a
macromolecular fold or between two interacting macromolecular
partners and is specifically between a theta atom on one side of
the fold or first interacting partner and an iota atom on the
opposing side or second interacting partner; in an instance where
the following condition is satisfied:
[0015] s-Rw.ltoreq.t, where s is the separation between the two
atoms of the contact, Rw is the sum of the van de Waals radii of
the two atoms of the contact, and t is a predetermined threshold
distance; and
[0016] wherein the theta atom type is identified uniquely in step
b) such that there is no intersection between the data of a theta
contact set extracted in step c) for a given theta atom type and
the data of any other theta contact set extracted in step c) for
any other theta atom type, apart from data concerning contacts
involving the given theta atom as the iota atom.
[0017] Thus, each target atom type in the binding site is
classified uniquely and is associated with information about a set
of contacts extracted from the structural database that is unique
and which does not overlap with the set of contacts associated with
any other atom type (apart from those contacts which involve the
target atom type itself as the iota atom). This means that a
distribution of theoretical locations for a given iota atom type,
or a predetermined group of related iota atom types, determined
based on one target atom in the binding site may be combined (e.g.
by summing) more efficiently (e.g. without weighting) with a
distribution of theoretical locations for an iota atom type, or
predetermined group of related iota atom types, determined based on
another target atom in the binding site, for example to provide an
improved prediction of one or more favoured regions for the iota
atom type or predetermined group of related iota atom types. Also
because each target atom in the binding site is classified
uniquely, there are no variations in bond lengths or angles to
consider and hence the theoretical location of a given iota atom is
more precise.
[0018] In an embodiment, simple rules are applied to uniquely
identify the neighbouring atoms for the purposes of triangulation.
No assumptions need to be made about the chemical nature of
neighbouring atoms, which is necessary for example where contact
types are characterized in terms of the 163 3-atom fragment types
of Laskowski et al.
[0019] In an embodiment, the spatial data extracted in step c)
defines the position of each iota atom specified in the theta
contact set by geometrical reference to the position of the theta
atom and to the positions of third and fourth atoms, wherein the
third atom is covalently bonded to the theta atom and the fourth
atom is covalently bonded to the third atom. In an example of such
an embodiment, for each iota atom specified in the theta contact
set, said spatial data extracted in step c) further defines the
position of fifth and sixth atoms by geometrical reference to the
position of the theta atom and to the positions of the third and
fourth atoms, wherein the fifth atom is covalently bonded to the
iota atom and the sixth atom is covalently bonded to either the
fifth atom or the iota atom.
[0020] In an embodiment, the superimposition in or around the
target site of step (d) comprises: parsing the theta contact set to
extract spatial data for contacts comprising the given iota atom
type or one or more of the predetermined group of related iota atom
types; and plotting this spatial data to determine theoretical
locations representing where each iota atom type, or each of the
one or more of the predetermined group of related iota atom types,
would be located if: i) the theta atom of the contact were located
at the position of the corresponding theta atom in the target
binding site; and ii) the third and fourth atoms of the contact
were located at the positions of the third and fourth atoms of the
corresponding theta atom in the target binding site. In an
embodiment, the spatial data is parsed against the contextual data
before the plotting step.
[0021] In an embodiment, a region in which a density of theoretical
locations for the iota atom type (or one or more of the
predetermined groups of related iota atom types) is above a
predetermined threshold is identified as one of the favoured
regions. In an example of such an embodiment, theoretical locations
for the given iota atom type, or for one or more of the
predetermined group of related iota atom types, are determined for
a plurality of theta atoms on the target list and a region in which
a density of the cumulative theoretical locations is above the
predetermined threshold is identified as one of the favoured
regions.
[0022] Thus, theoretical locations are combined cumulatively from
different atoms in the binding site before the density of
theoretical locations is obtained for the purposes of predicting
favoured regions. This results in a more accurate statistical
representation of the probability of a given iota atom type, or in
a given group of related iota atom types, being positioned at a
given location because it takes into account the contributions from
all relevant atom types in the binding site in a proportionate and
unbiased manner. In Laskowski et al., in contrast, the density
functions are derived for groups of 3-atom fragments. Each atom may
be associated with several different groups of 3-atom fragments and
so it is not possible simply to add together density functions in a
manner comparable with embodiments of the present invention.
Instead, it is necessary to perform weighting and/or averaging
before combining density functions, which increases complexity
and/or reduces accuracy.
[0023] In an embodiment only contacts between atoms that are
separated from each other by four residues or more are used for
identifying favoured regions. This significantly reduces or avoids
bias due to short range secondary structure. In an embodiment, the
contact data predominantly represents long-range, across-fold
protein data.
[0024] According to a further aspect of the invention, there is
provided a method of generating a database for use in a method for
designing a ligand ab initio that will bind to a binding site of a
macromolecular target, or of identifying a modification to a ligand
for improving the affinity of the ligand to a binding site of a
macromolecular target, comprising:
[0025] analysing the relative positions of atoms in each of a
plurality of proteins or other biological macromolecules in order
to identify instances of a non-bonding intra-molecular contact
between a first atom, referred to as a theta atom, and a second
atom, referred to as an iota atom, of the protein or macromolecule;
and
[0026] generating a database that for each identified contact
specifies: the type of the theta atom, the type of the iota atom,
and the position of the iota atom relative to the theta atom;
[0027] wherein a non-bonding intra-molecular contact is defined as
an instance where the following conditions are satisfied:
[0028] s-Rw.ltoreq.t, where s is the separation between the theta
and iota atoms, Rw is the sum of the van de Waals radii of the
theta and iota atoms, and t is a predetermined threshold distance
of typically 2.5 angstroms and preferably 0.8 angstroms; and
[0029] wherein in the case of proteins, the theta and iota atoms
are on amino acid residues separated from each other by at least
four residues on a linear polypeptide or are on separate
polypeptide chains.
[0030] According to a further aspect of the invention, there is
provided a method of generating a database for use in a method for
designing a ligand ab initio that will bind to a binding site of a
macromolecular target, or of identifying a modification to a ligand
for improving the affinity of the ligand to a binding site of a
macromolecular target, comprising:
[0031] analysing the relative positions of atoms in each of a
plurality of proteins or other biological macromolecules in order
to identify instances of a non-bonding intra-molecular contact
between a first atom referred to as a theta atom, and a second
atom, referred to as an iota atom, of the protein or macromolecule;
and
[0032] generating a database that for each identified contact
specifies: the type of the theta atom, the type of the iota atom,
and the position of the iota atom relative to the theta atom;
[0033] wherein a non-bonding intra-molecular contact is defined as
an instance where the following condition is satisfied:
[0034] s-Rw.ltoreq.t, where s is the separation between the theta
and iota atoms, Rw is the sum of the van de Waals radii of the
theta and iota atoms, and t is a predetermined threshold distance
of typically 2.5 angstroms and preferably 0.8 angstroms; and
[0035] wherein the method comprises sub-dividing the database to
form groups of identified contacts in which the theta atom is one
and only one of the 167 non-hydrogen atoms present in the 20
natural amino acids of proteins and the iota atom is in one and
only one of a plurality of non-overlapping groups obtained by
sorting the 167 non-hydrogen atoms present in the 20 natural amino
acids of proteins into groups based on chemical similarity.
[0036] Embodiments of the invention will now be described, by way
of example only, with reference to the accompanying drawings in
which corresponding reference symbols represent corresponding
parts, and in which:
[0037] FIG. 1 is a schematic illustration of an example
nomenclature for atoms at a non-bonding intra-molecular or
inter-molecular contact and neighbouring atoms that are used for
coordinate normalization;
[0038] FIGS. 2-6 illustrate a process of coordinate normalization
for an atom in a non-bonding intra-molecular or inter-molecular
contact;
[0039] FIG. 7 is a flow chart illustrating steps in a method of
designing a ligand ab initio that will bind to a binding site of a
macromolecular target, or of identifying a modification to a ligand
for improving the affinity of the ligand to a binding site of a
macromolecular target;
[0040] FIG. 8 is a computer generated visualization depicting a
light chain threonine 30 to arginine 30 mutation;
[0041] FIG. 9 is a computer generated visualization depicting a
light chain arginine 54 to serine 54 mutation;
[0042] FIG. 10 is a computer generated visualization depicting a
light chain serine 56 to isoleucine 56 mutation;
[0043] FIG. 11 is a computer generated visualization depicting a
light chain serine 60 to aspartate 60 mutation;
[0044] FIG. 12 is a computer generated visualization depicting a
light chain threonine 72 to arginine 72 mutation;
[0045] FIG. 13 is a computer generated visualization depicting the
combination of 5 mutations in antibody 496i light chain resulting
in a 180-fold improved affinity to IL17F;
[0046] FIG. 14 is a flow chart illustrating steps in a method of
predicting the effects of point mutations at the VH-VL interface of
a Fab;
[0047] FIG. 15 is a computer generated visualisation depicting a
heavy chain threonine 71 to arginine 71 mutation;
[0048] FIG. 16 is a computer generated visualisation depicting a
light chain serine 107 to glutamic acid 107 mutation;
[0049] FIG. 17 is a computer generated visualisation depicting a
light chain threonine 109 to isoleucine 109 mutation;
[0050] FIG. 18 is a computer generated visualisation depicting the
combination of three mutations in Fab X resulting in a Tm of
81.2.degree. C.
[0051] The Worldwide Protein Data Bank (wwPDB) maintains an archive
of macromolecular structural data that is freely and publicly
available to the global community. By May 2013 this dataset had
reached the milestone of 90 000 structures. Most of these
macromolecules are proteins of which the majority have been
determined by X-ray crystallography. Deposited data thus contains
three dimensional data at the atomic level in the form of Cartesian
coordinates of individual atoms that make up the respective protein
structure.
[0052] The inventors hypothesised that it is possible to extract
useful information from this archive which could be applied to aid
the design of novel therapeutics. The polypeptide chains of a
nascent protein fold into complex three dimensional tertiary and
quaternary structures in a remarkably reproducible manner to yield
the mature protein. Interactions affecting the formation of the
secondary structure of proteins, elements such as helices,
beta-sheets and turns, are known. However, rules predicting the
higher orders of protein folding are poorly understood.
Nonetheless, the inventors have realised that there must be precise
rules that govern the interaction of non-bonding but "contacting"
atoms, either within the same molecular, for example on opposing
faces of a protein fold, or on different molecules.
[0053] In an embodiment, a structural database of biological
macromolecules (e.g. the wwPDB) is analysed to extract such rules,
and the rules are applied to facilitate drug discovery. An example
of such a process is described below.
[0054] In an embodiment, non-bonding pairs of contact atoms
(referred to respectively as "theta" and "iota" atoms) are
identified for each macromolecule (e.g. protein), or a subset of
fewer than all of the macromolecules, in the structural database of
macromolecules (e.g. the wwPDB). Such contacts may occur for
example between opposing residues of a protein fold or between
opposing monomer units of a macromolecular fold (between separate
chains of a macromolecular structure) or between two interacting
macromolecular partners. Each contact is classified as being
between a theta atom on one side of the fold or first interacting
partner and an iota atom on the opposing side or second interacting
partner.
[0055] In an embodiment, the non-bonding intra-molecular or
inter-molecular contacts are defined as an instance where the
following condition is satisfied: 1) s-Rw.ltoreq.t, where s is the
separation between the two atoms of the contact, Rw is the sum of
the van de Waals radii of the two atoms of the contact, and t is a
predetermined threshold distance; and, optionally, the following
condition also: 2) the two atoms of the contact are separated from
each other by at least four residues along a linear polypeptide
chain or are on separate polypeptide chains. In an embodiment, the
predetermined distance is 2.5 angstroms. In another embodiment, the
predetermined distance is 1.5 angstroms. In another embodiment, the
predetermined distance is 1.0 angstroms. In another embodiment, the
predetermined distance is 0.8 angstroms.
[0056] In the description below, any reference to "contact" is
understood to mean "non-bonding intra-molecular contact or
inter-molecular contact" according to the definition given
above.
[0057] Databases such as the wwPDB may have information about
proteins that are very similar to each other and/or which have
related structures. In an embodiment, the database is parsed in
order to avoid/reduce bias caused by such
similarities/relationships. In an embodiment, the parsing is
performed based on primary sequence homology, for example such that
only one representative structure of each family of similar/related
proteins is selected for analysis. Additionally or alternatively,
one or more further selection criteria may be used, for example
high resolution and low temperature factor structures may be
incorporated.
[0058] In an embodiment, a secondary database is constructed
starting from the (primary) structural database of biological
macromolecules (e.g. the wwPDB). The secondary database comprises
information about the non-bonding intra-molecular or
inter-molecular contacts. In an embodiment, the secondary database
comprises information about more than 1 million contact pairs,
optionally more than 5 million contact pairs, optionally more than
11 million contact pairs. In one embodiment, the secondary database
comprises information from more than 15 million contact atom pairs,
extracted from around 20 000 non-homologous proteins.
[0059] In an embodiment, the secondary database contains
information about the precise atom types of the contact pair. In an
embodiment, the secondary database contains spatial data defining
the three dimensional relationship of the theta atom to the iota
atom. In an embodiment, the secondary database also contains
contextual data concerning the local environment of the contact. In
an embodiment, the contextual data contains information concerning
the local environment of each contact pair, including one or more
of the following in any combination: secondary structure, amino
acid types or other monomer types comprising the contact pair,
adjacent monomer units and/or local geometry thereof in a polymer
chain either side of the contact, adjacent amino acids in a
polypeptide chain on either side of the contact, local geometry of
the said adjacent monomer units or amino acids, temperature factor
of the theta atom, temperature factor of the iota atom, accessible
surface area of the theta atom, accessible surface area of the iota
atom, the number of different iota atom contacts for the particular
theta atom and the number of other theta atoms on the same monomer
unit as the theta atom.
[0060] In an embodiment, the 3-D coordinates of the contact pair
and covalently attached adjacent atoms are normalized, as a group,
to a common database reference frame as described below. This
simplifies subsequent analysis of potential underlying contact
patterns or rules and application of any such rules to drug
design.
[0061] In an embodiment, the theta atom type is identified as being
one and only one of: the 167 covalent atom types (excluding
hydrogen) that make up the 20 natural amino acid building blocks of
proteins (in this case the secondary database may be divided
accordingly and comprise information about up to 27889,
167.times.167, different contact types); and/or the 82 non-hydrogen
atoms present in the 4 nucleotides of the deoxyribonucleic acid
polymer (DNA); and/or the 42 non-hydrogen atoms present in the
methylated DNA nucleotides, cytidine phosphate and adenosine
phosphate; and/or the 85 non-hydrogen atoms present in the 4
nucleotide phosphates of the ribonucleic acid polymer (RNA); and/or
the 89 non-hydrogen atoms present in 2-O'-methylated ribose
nucleotide phosphates of RNA; and/or the over 400 non-hydrogen
atoms present in the commonest post-transcription base modified
RNA.
[0062] In an embodiment, the iota atom type is identified as being
one and only one of: the 167 covalent atom types (excluding
hydrogen) that make up the 20 natural amino acid building blocks of
proteins; and/or the oxygen atom present in protein bound,
structurally relevant, water molecules (this may be useful because
crystal structures in the primary database often contain
structurally relevant water molecules, i.e. certain protein atoms
show definite interactions with bound water molecules); and/or the
82 non-hydrogen atoms present in the 4 nucleotides of the
deoxyribonucleic acid polymer (DNA); and/or the 42 non-hydrogen
atoms present in the methylated DNA nucleotides, cytidine phosphate
and adenosine phosphate; and/or the 85 non-hydrogen atoms present
in the 4 nucleotide phosphates of the ribonucleic acid polymer
(RNA); and/or the 89 non-hydrogen atoms present in 2-O'-methylated
ribose nucleotide phosphates of RNA; and/or the over 400
non-hydrogen atoms present in the commonest post-transcription base
modified RNA.
[0063] In a contact pair the opposing atom is viewed and recorded
from either side on the contact. The nomenclature in an example
embodiment is described below and illustrated schematically in FIG.
1.
[0064] The atom on the reference side of the contact is termed the
theta atom 1 whilst the opposing atom is termed the iota atom 2. In
this example, the further atoms used for normalizing the 3-D
coordinates are defined as follows. The next atom to which the
theta atom 1 is covalently bonded, in the direction of the C alpha
atom of that amino acid, is referred to as the third atom 3 and the
next atom again, the fourth atom 4. The fourth atom 4 is covalently
bonded to the third atom 3. The next atom to which the iota atom 2
is covalently bonded, in the direction of the C alpha atom of the
respective amino acid, is termed the fifth atom 5 and the next
again atom, the sixth atom 6. The sixth atom is covalently bonding
to either the fifth atom or the iota atom 2.
[0065] In an embodiment, to avoid instances of ambiguity the third
and fourth atoms are chosen uniquely for each specified theta atom
type. In an embodiment, the fifth and sixth atoms are also chosen
uniquely. In an embodiment, the following convention is applied. If
the theta atom 1 happens to be a C alpha atom, then the third and
fourth atoms are the backbone carbonyl carbon and oxygen atoms
respectively. If the theta atom 1 is a backbone carbonyl carbon,
then the third atom 3 and the fourth atom 4 are the C alpha carbon
and the backbone nitrogen respectively. If the theta atom 1 is the
backbone nitrogen, then the third atom 3 and the fourth atom 4 are
the C alpha carbon and the backbone carbonyl carbon respectively.
If the theta atom 1 is a C beta carbon atom, then the third atom 3
and the fourth atom 4 are the C alpha carbon and the backbone
carbonyl carbon respectively. In phenylalanine and tyrosine side
chains where there is a choice of two epsilon carbon atoms for the
third and fourth atom positions, then the atom closest to the
backbone nitrogen atom is selected.
[0066] In an embodiment, coordinate normalisation of each contact
is performed on the theta, iota, third and fourth atoms, optionally
also the fifth and sixth atoms, as a group so that their 3-D
relationship is maintained. The resulting normalized coordinates
may be referred to as a normalized coordinate group. In an
embodiment, this is achieved by carrying out the following steps in
sequence, as illustrated in FIGS. 2-6.
[0067] FIG. 2 illustrates a theta atom 1, third atom 3 and fourth
atom 4 of a non-bonding intra-molecular or inter-molecular contact
positioned relative to a reference frame, defined relative to x-,
y- and z-axes, according to coordinates given in a primary database
(such as the wwPDB). In a first step of an example coordinate
normalization process, the atom group coordinates are translated so
that the theta atom 1 lies at the zero coordinate (FIG. 3). Next,
the group as a whole is rotated about the z-axis until the third
atom 3 is at y=0 (FIG. 4). Next, the group is rotated about the
y-axis until the third atom is at y=0 and z=0 (FIG. 5). Next, the
group is rotated about the x-axis until the fourth atom 4 is at y=0
and the group as a whole lies in the x-y plane (all three atoms at
y=0; FIG. 6). In this manner each of the 167 first atom types can
be superimposed for that type and the secondary database
sub-divided accordingly. In turn each of the first atom divisions
can be sub-divided into 167 iota atom types, facilitating the
analysis of the spatial distribution of each iota atom type
relative to each theta atom type.
[0068] In an embodiment, the distribution patterns of iota atoms
relative to theta atoms are analysed in order to identify
similarities between the distribution patterns for nominally
different iota atom types. In this way, the unique iota atom types
(e.g. the 167 covalent atom types mentioned above) can be combined
into a number of groups (herein referred to as "predetermined
groups of related iota atom types") to simplify subsequent use of
the data. Grouping together the atom types according to the
similarity of distribution patterns reduces the computational load
associated with the method described below with reference to FIG. 7
for example, thus increasing speed and/or reducing hardware
expense.
[0069] In an embodiment, this process is simplified by using polar
coordinates rather than Cartesian coordinates (in an embodiment,
this is achieved by performing conversion processing between
Cartesian coordinates and polar coordinates, for example where the
data in the primary database is presented using Cartesian
coordinates). In an embodiment, two-dimensional polar coordinates
are used, specifying the relative positions of the theta and iota
atoms in terms only of the two polar angles .theta. (theta) and
.PHI. (phi) (corresponding to latitude and longitude on a globe).
The resulting two-dimensional latitude-longitude plots do not show
any information about variations in the distance between the theta
and iota atoms. However, it is found that this distance is
relatively constant, so that the theta-phi plots contain most of
the relevant information concerning the contact. Reducing the
analysis to a problem in two dimensions rather than three greatly
improves the efficiency of subsequent analyses. In an embodiment,
contour lines are used to illustrate variations in the relative
position of the iota atom. The contour lines may represent lines of
constant "density" or probability of a relative positioning of the
theta and iota atoms.
[0070] Analysis of such polar angle plots has revealed that a
particularly important factor governing the pattern of iota atom
frequencies is the elemental nature and hybridisation state of the
iota atoms, i.e. C sp3, C sp2(aromatic), C sp2(non-aromatic), N
sp3, N sp2, O sp3, O sp2 or S. As a result, it is possible to
improve analysis efficiency by grouping the 167 atom types
according to these identified eight groups. In other embodiments, a
different grouping may be used.
[0071] In general, environmental factors around the contact, such
as the nature of adjacent amino acids, make less difference to the
iota frequency pattern, with the exception of secondary structure.
As might be expected the frequency patterns of backbone amide
nitrogen theta atoms versus backbone oxygen iota atoms and
vice-versa are skewed by secondary structure, in particular as
regards whether or not they are from beta sheet.
[0072] In an embodiment, the secondary database tags contact data
with the local secondary structure type (helix, beta sheet or
random coil). This provides the basis for differentiating any
potential influence of secondary structure on contact patterns at a
later stage.
[0073] In an embodiment, a method is provided based on the above
that assists with the identification of modifications to a ligand
that improve the strength of binding, or affinity, of the ligand to
a binding site. In an embodiment, the method is used to assist with
NCE or biologic drug design. In respect of the former, the method
may be useful for predicting `hotspots` or pharmacophore atom
positions in potential drug binding sites of target proteins. This
can facilitate de novo drug design. In situations where there is an
available structure of chemical matter bound in a binding site, the
method can suggest atom types and positions for elaboration of the
chemistry to obtain a ligand with better binding characteristics.
In the case of protein drugs such antibodies, the method may be
used to predict mutations in the protein or antibody binding site
that would lead to improvement in binding affinity or specificity.
The method may also be used to suggest positions for modification
within a macromolecular structure to improve the properties of the
macromolecule. For example, as illustrated in the Examples section
below, the method may be used to identify point mutations within
antibody VH and VL chains in order to improve the thermal stability
of the antibody. The mutations are on separate chains, but are
still within the antibody macromolecule.
[0074] FIG. 7 illustrates an example method for designing a ligand
ab initio that will bind to a binding site of a macromolecular
target, or of identifying a modification to a ligand for improving
the affinity of the ligand to a binding site of a macromolecular
target.
[0075] In step S1, data representing the target binding site of a
target protein is obtained, for example from a local or remote
memory device 5. A target list of atoms forming the surface of the
target binding site is identified.
[0076] In step S2, each atom in the target list is identified as a
particular theta atom type.
[0077] In step S3, information is extracted from a structural
database of biological macromolecules (e.g. the wwPDB), provided
for example by a local or remote memory device 7, about
non-bonding, intra-molecular or inter-molecular contacts in which
the first atom in a contacting pair of atoms is a particular theta
atom type and the opposing, second atom of the pair is a particular
iota atom type. The extracted information comprises spatial and/or
contextual data about the iota atom relative to the theta atom. The
data is collected for a plurality of contacts of the given theta
atom type and the resulting set of data is referred to as a theta
contact set. In an embodiment, the theta contact set comprises data
collected for all of the available contacts of the given theta atom
type. The extracted information may form a database that is an
example of the "secondary database" discussed above. In an
embodiment, the information extracted in step S3 is collected in a
secondary database that comprises one and only one theta contact
set for each of the theta atom types. In an example of such an
embodiment, the theta contact sets of the secondary database are
subdivided into a plurality of non-overlapping iota atom types or
non-overlapping groups of related iota atom types. In an example of
such an embodiment, the database is sub-divided to form groups of
identified contacts in which the first atom is one and only one of
the 167 non-hydrogen atoms present in the 20 natural amino acids of
proteins and the second atom is in one and only one of a plurality
of non-overlapping groups obtained by sorting the 167 non-hydrogen
atoms present in the 20 natural amino acids of proteins into groups
based on chemical similarity.
[0078] In step S4, for each theta atom identified in the target
list in step S2, data relating to a given iota atom type, or a
predetermined group of related iota atom types, from the
corresponding theta contact set extracted in step S3 is
superimposed in or around the target binding site. In an
embodiment, the superimposition comprises: parsing the theta
contact set to extract spatial data for contacts comprising the
given iota atom type or one or more of the predetermined group of
related iota atom types; and plotting this spatial data to
determine theoretical locations representing where each iota atom
type, or each of the one or more of the predetermined group of
related iota atom types, would be located if: i) the theta atom of
the contact were located at the position of the corresponding theta
atom in the target binding site; and ii) the third and fourth atoms
of the contact were located at the positions of the third and
fourth atoms of the corresponding theta atom in the target binding
site. Where determined theoretical locations conflict with binding
site atoms and/or are buried within the target protein, these may
be removed from further analysis. For example if it is determined
that the theoretical location of an individual iota atom intersects
with the location of an atom of the target macromolecule closer
than Rw-0.2 angstroms then the iota atom is excluded from
subsequent analysis.
[0079] In step S5, the superimposed data is combined and/or parsed
in such a way as to predict one or more favoured regions of the
binding site where the given iota type, or the predetermined group
of related iota atom types, has high theoretical propensity.
[0080] In step S6, a candidate ligand is notionally docked into the
binding site. Data defining the candidate ligand may be provided
for example from a local or remote memory device 9. A comparison is
then made between the type and position of one or more of the atoms
of the candidate ligand with the predicted favoured regions for the
respective iota atom types. On the basis of the comparison,
modifications to the candidate ligand, in terms of alternate or
additional candidate ligand atoms, are identified that will produce
a greater intersection between the alternate and/or additional
candidate ligand atoms and the respective iota atom type favoured
regions, leading to an improvement in the affinity of the modified
candidate ligand to the binding site compared to the unmodified
candidate ligand.
[0081] In step S7, the modified candidate ligand is output either
as a proposed improvement to an existing ligand or as part of an ab
initio design of a new ligand. Optionally steps S7 and S6 can be
iterated to further modify the ligand. The local or remote memory
devices 5, 7 and 9 may be implemented in a single piece of hardware
(e.g. a single storage device) or in two or more different,
separate devices.
[0082] In an embodiment, the modified candidate ligand is output to
an output memory device for storage or transmission and/or to a
display for visualization.
[0083] In an embodiment, the type of a given theta atom is
identified uniquely in step S2 such that there is no intersection
between the group of contacts for which information is extracted in
step S3 for the given theta atom and the group of contacts for any
other theta atom type (with the exception of contacts involving the
given theta atom type as the iota atom).
[0084] In an embodiment, step S5 comprises determining one or more
favoured regions for each of a plurality of different iota atom
types and/or predetermined groups of related iota atom types. In
such an embodiment, the comparison step S6 may be repeated for each
of the plurality of different iota atom types and/or predetermined
groups of related iota atom types, in order to identify potential
modifications that involve the different iota atoms types or
predetermined groups of related iota atom types.
[0085] In an embodiment, steps S2-S7 are performed for a plurality
of different atoms in the binding site. In an embodiment, as
described below, favoured regions may be determined more accurately
by cumulatively combining (e.g. summing) the distributions of
determined theoretical locations of the iota atom types as derived
for a plurality of different atoms in the binding site.
[0086] In an embodiment, the analysis is extended such that, for
each favoured region, vectors are derived that describe the
position of the fifth atom relative to its respective iota atom.
Analysis is carried out on the vectors to identify a favoured bond
vector representing a prediction of the covalent attachment of a
theoretical consensus iota atom in the region. The identified
favoured bond vector can then be used to refine the design of the
candidate ligand and/or to refine the modification of the candidate
ligand, as applicable. The identified favoured bond vector may be
used for example to indicate how iota atoms in different favoured
regions might be bonded together, thus assisting with the
identification of modifications involving plural additions or
exchanges of atoms. In an embodiment the analysis is a cluster
analysis.
[0087] The distribution of theoretical locations gives a measure or
propensity of how a particular iota atom type (or predetermined
group of related iota atom types) will be favoured at different
locations in the binding site. In an embodiment, a region in which
a density of the theoretical locations is above a predetermined
threshold is identified as one of the favoured regions. The density
of theoretical locations is a measure of the number of determined
theoretical locations that occur in a given spatial volume for
example. In an embodiment, iota atom theoretical locations are
determined for a plurality of target atoms in the binding site and
a region in which a density of the cumulative theoretical locations
for the iota atom (or predetermined group of related iota atom
types) for the plurality of target atoms is above the predetermined
threshold is identified as one of the favoured regions. The
theoretical locations determined for different target atoms in the
binding site may be summed, for example, in order to obtain the
cumulative theoretical locations. This approach to taking into
account the effects of different atoms in the binding site is
computationally efficient and minimizes loss of information about
the interaction between the candidate ligand and the binding site.
The approach is facilitated by the characterization of contacts in
terms of pairs of simple atom types or simple atom types in
combination with atoms of predetermined groups of simple atom
types. Such an approach is not valid when contacts are
characterized in terms of 3-atom fragments, such as is the case in
Laskowski et al. for example.
[0088] The obtained distributions of theoretical locations can be
transformed in various ways to create probability density
functions, i.e. a statistical potential for the preference of a
given iota atom type at a given position in the binding site. In
turn, probability density functions can be treated in an analogous
way to electron density and converted into ccp4 files which are a
standard way of visualising such maps within molecular graphics
software, e.g. Pymol.
[0089] In an embodiment, in step S5, the one or more favoured
regions is/are expressed in polar coordinates, optionally
comprising only the polar and azimuthal angles, optionally wherein
the reference frame is normalized by reference to the third and
fourth atoms 3,4.
[0090] In an embodiment, step S6 comprises: identifying a
modification of the candidate ligand that increases a degree of
overlap between an atom of the candidate ligand (whether present
before the modification or not) and a predicted favoured region for
an atom of the same type in the binding site. In an embodiment, the
generated distributions of theoretical locations and/or favoured
regions are inspected, for example by computer software or
manually, as superimpositions on the target macromolecular
structure in complex with the respective candidate ligand. If for
instance the candidate ligand relates to an antibody, the interface
between the antibody and target macromolecule may be examined to
determine the degree of overlap between antibody atoms and the
respective iota atom theoretical location distributions and/or
favoured regions identified for that atom. In some cases the degree
of overlap will already be high. However, in other regions of the
interface the overlap may be low. It is in these regions where
mutations in the adjacent amino acid residue of the antibody may be
most effectively identified/proposed. In an embodiment each of the
19 other natural amino acids is considered in turn at this
position, in all of their respective rotamer conformations and in
each case the degree of overlap with the relevant iota atom
theoretical location distributions and/or favoured regions is
examined, with the aim of selecting those residues with the maximum
degree of overlap for proposed mutations. In this manner, a
rational means of selecting mutations is provided that may generate
affinity improvements in the chosen antibody. Individual point
mutations in different regions of the antibody-target protein
interface may be generated; those that lead to affinity improvement
can be tested in combinations of two or more that may give
synergistic increases in affinity.
[0091] In an embodiment, step S6 comprises replacing each of one or
more of (optionally all of) the amino acid residues of the ligand
that is/are in direct contact with the target binding site, or in
close proximity to the target binding site, with each of one or
more of (optionally all of) the residues chosen from the other 19
natural amino acids. Each such replacement, is referred to herein
as a "residue replacement" and involves the modification of a
ligand by a single replacement of one residue with a different
residue.
[0092] In an embodiment, for each such residue replacement that
does not cause conflict with adjacent atoms of the ligand or target
(e.g overlap between one or more atoms of the replacement residue
and one or more other atoms of the target or ligand), the type and
position of each atom of the replacement residue is compared with
the respective iota atom type favoured regions to identify whether
they will produce a greater intersection than the atoms of the
original residue. In an embodiment a list is then output of the
residue replacements that are identified as producing a greater
intersection than atoms of the original residue. In an embodiment,
for each of the listed residue replacements, the candidate ligand
is then mutated to produce a modified, single-residue-mutated
ligand that incorporates the residue replacement. The affinity of
each modified ligand to the target binding site can then be tested
by experiment in order to identify those modifications which
provide the greatest affinity improvement for the candidate ligand.
For example, a group of residue replacements may be identified that
yield a residue replacement that is greater than a predetermined
threshold. In an embodiment, the predetermined threshold may be
zero so that the selected group consists only of residue
replacements that improve the affinity to some extent. More
advanced modifications of the candidate ligand can then be carried
out based on this information. For example, in an embodiment the
candidate ligand may be modified to incorporate a plurality of
residue replacements, for example a plurality of those residue
replacements that, individually, were determined as providing the
greatest affinity improvements. In this way it is possible to
design a ligand that has an affinity that is improved even more
than is possible by replacing only a single residue.
[0093] In some embodiments, lists of residue replacements may be
produced that satisfy providing a greater intersection than atoms
of the original residue (have a .DELTA.IOTAscore of less than
zero). The lists may additionally be ranked based on other
criteria. For example, lists may also be filtered based on
.DELTA..DELTA.G scores (see below). Residue replacements with
.DELTA..DELTA.G scores of less than zero imply stronger
interactions compared with the original residue. Therefore a list
may be produced where residues satisfy both criteria of a
.DELTA.IOTAscore of less than zero (a negative .DELTA.IOTAscore),
and a .DELTA..DELTA.G of less than zero (a negative
.DELTA..DELTA.G). This is illustrated in Example 2 below.
[0094] In the case of chemical matter, for instance a crystal
structure of a low molecular weight chemical fragment bound in a
pocket of a target protein, iota atom theoretical location
distributions and/or favoured regions displayed in the binding site
may suggest atom types and vectors of chemical bonds for fragment
growth that may yield a prototype NCE of higher potency.
[0095] In an embodiment, a plurality of modifications to the
candidate ligand are identified. In this case, the method may
further comprise selecting a subset of the identified
modifications, for example to identify the modifications which are
likely to be most effective in terms of improving affinity. The
selection may be carried out based on the extent to which the
intersection between the alternate and/or additional candidate
ligand atoms and the respective iota atom type favoured regions is
greater compared to the unmodified candidate ligand. For example,
modifications that result in an increase in the intersection that
is above a predetermined threshold may be selected and
modifications that result in an increase in the intersection that
is below a predetermined threshold may be discarded. An example of
such a selection process is discussed below in the context of
"Example 2". The ".DELTA.IOTAScore" is an example of a measure of
the extent to which the intersection between the alternate and/or
additional candidate ligand atoms and the respective iota atom type
favoured regions is greater compared to the unmodified candidate
ligand. Alternatively or additionally, the selection may be carried
out based on the extent to which one or more factors contributing
to the total energy of the complex formed by the binding of the
modified candidate ligand to the binding site is/are reduced
compared to the case where the unmodified candidate ligand is
bound. For example, modifications that result in a decrease in the
one or more factors (e.g. a decrease in a sum of the one or more
factors) that is above a predetermined threshold may be selected
and modifications that result in a decrease in the one or more
factors (e.g. a decrease in a sum of the one or more factors) that
is below a predetermined threshold may be discarded. An example of
such a selection process is discussed below in the context of
"Example 2". The "Rosetta .DELTA..DELTA.G score" is an example of a
measure of the extent to which one or more factors contributing to
the total energy of the complex formed by the binding of the
candidate ligand to the binding site is/are reduced. Examples of
factors contributing to the total energy of the complex include a
Lennard-Jones term, an implicit solvation term, an
orientation-dependent hydrogen bond term, sidechain and backbone
torsion potentials derived from the PDB, a short-ranged
knowledge-based electrostatic term, and reference energies for each
of the 20 amino acids that model the unfolded state, as discussed
below.
[0096] In an embodiment, the method of identifying a modification
to a candidate ligand is a computer-implemented method. In an
embodiment, any one or more of the steps S1-S7 is/are performed on
a computer. In an embodiment, all of the steps S1-S7 is/are
performed on a computer. In addition, any one or more of the steps
S101-S109 of FIG. 14 (illustrating a workflow for predicting point
mutations at the VH-VL interface of an antibody) may be automated.
Any one or more of the steps S101-S109 may be performed on a
computer. In one embodiment, all of the steps S101-S109 are
automated. All of the steps S101-S109 may be performed on a
computer.
[0097] A wide range of standard computing configurations, well
known to the person skilled in the art, could be used as platforms
to implement the method. The method is not limited to any
particular hardware configuration, operating system or means for
storing or transmitting software necessary for defining and/or
implementing the method steps. In an embodiment, a computer
readable medium or signal is provided that comprises computer
readable instructions (e.g. code in a computer programming
language) for causing a computer to carry out the method.
[0098] In an embodiment, a method of manufacturing a therapeutic
ligand is provided. In an embodiment, the method of manufacturing
comprises designing a new ligand or modifying an existing ligand
according to one or more of the embodiments described above.
EXAMPLE 1
Affinity Maturation of a Fab Fragment of an Anti-IL17F Antibody
Introduction
[0099] In vitro methods of antibody affinity maturation are well
known (see U.S. Pat. No. 8,303,953 B2 column 13 lines 19 to 33). In
a recent example Fujino et at (Fujino et at (2012) "Robust in vitro
affinity maturation strategy based on interface-focused
high-throughput mutationalscanning", Biochem. Biophys. Res. Comm.,
428, 395-400) report a high throughput mutational scanning strategy
based on ribosome display panning of single point mutant
single-chain Fab libraries at each of 50 identified antigen
interface residues of the antibody, followed combinatorial ribosome
display of enhanced binders that resulted in identification of a
Fab with over 2000-fold affinity improvement. Such methods require
a large investment in laboratory based resources and therefore
various groups (reviewed by Kuroda et at (2012) "Computer-aided
antibody design", Prot. Eng. Design & Selection, 25, 507-521)
have investigated in silico methods that predict improvements in
antibody affinity so as to reduce or eliminate the need for
screening large numbers of mutated antibody variants for improved
affinity. These computer-aided antibody design protocols are either
knowledge-based; i.e. using statistical potentials derived from
observational data or physics-based, i.e. using and energy
functions derived from models of the underlying physical
interactions. Lippow et at (2007) (Lippow et at (2007),
"Computational design of antibody-affinity improvement beyond in
vivo maturation", Nat Biotech., 25, 1171-1176) have achieved
moderate success with the latter approach based on electrostatic
interactions, but our understanding of parameterisation of such
methods is still far from complete. Knowledge-based methods to date
tend to identify individual antibody residues for random
mutagenesis (e.g. Barderas et at (2008) "Affinity maturation of
antibodies assisted by in silico modelling", PNAS, 105, 9029-9034),
which still entail considerable laboratory based effort.
[0100] In this Example, a knowledge-based approach was applied to
affinity mature a Fab fragment of the anti-IL17F antibody described
in U.S. Pat. No. 8,303,953 B2. The final affinity matured antibody
is described in WO 2012/095662 A1 as a full length IgG1 molecule.
However, the method by which this antibody was affinity matured is
not disclosed in the latter publication.
Methods
Identification of Target (Theta) Atoms Comprising the IL17F
Epitope
[0101] Using the coordinates of the co-crystal IL17F/Fab 496
complex structure described in WO 2009/130459 A2, all IL17F atoms
within 6 .ANG. of any Fab 496 atom were identified as epitope atoms
and are listed in Table 1. Of this list of 209 theta atoms there
are 86 specific theta atom types.
TABLE-US-00001 TABLE 1 List of atoms comprising the IL17F epitope
where the notation (1)-(2)-(3)- (4) designates (1) the respective F
and I chains of the IL17 homodimer, (2) the amino acid residue and
(3) residue number and (4) the atom type. F-ASN-53-CG F-ASN-89-C
F-ILE-129-CA I-VAL-38-N F-ASN-53-OD1 F-ASN-89-CA F-ILE-129-CB
I-VAL-38-O F-ALA-70-C F-ASN-89-CB F-ILE-129-CD1 I-SER-39-C
F-ALA-70-O F-ASN-89-CG F-ILE-129-CG1 I-SER-39-CA F-GLN-71-C
F-ASN-89-N F-ILE-129-CG2 I-SER-39-CB F-GLN-71-CA F-ASN-89-ND2
F-ILE-129-N I-SER-39-N F-GLN-71-CB F-ASN-89-OD1 F-ILE-129-O
I-SER-39-OG F-GLN-71-CD F-SER-90-C F-HIS-130-C I-MET-40-C
F-GLN-71-CG F-SER-90-CA F-HIS-130-N I-MET-40-N F-GLN-71-N
F-SER-90-CB F-HIS-130-O I-MET-40-O F-GLN-71-NE2 F-SER-90-N
F-HIS-131-C I-SER-41-C F-GLN-71-O F-SER-90-O F-HIS-131-CA
I-SER-41-CA F-GLN-71-OE1 F-SER-90-OG F-HIS-131-CE1 I-SER-41-CB
F-CYS-72-C F-VAL-91-C F-HIS-131-N I-SER-41-N F-CYS-72-CA
F-VAL-91-CA F-HIS-131-ND1 I-SER-41-O F-CYS-72-CB F-VAL-91-CB
F-HIS-131-O I-SER-41-OG F-CYS-72-N F-VAL-91-CG1 F-VAL-132-C
I-ARG-42-C F-CYS-72-O F-VAL-91-CG2 F-VAL-132-CA I-ARG-42-CA
F-CYS-72-SG F-VAL-91-N F-VAL-132-CB I-ARG-42-CB F-ARG-73-C
F-VAL-91-O F-VAL-132-CG1 I-ARG-42-CD F-ARG-73-CA F-PRO-92-C
F-VAL-132-CG2 I-ARG-42-CG F-ARG-73-CB F-PRO-92-CA F-VAL-132-N
I-ARG-42-CZ F-ARG-73-CG F-PRO-92-CB F-VAL-132-O I-ARG-42-N
F-ARG-73-N F-PRO-92-CD F-GLN-133-C I-ARG-42-NE F-ARG-73-O
F-PRO-92-CG F-GLN-133-CA I-ARG-42-NH1 F-ASN-74-C F-PRO-92-N
F-GLN-133-CB I-ARG-42-NH2 F-ASN-74-CA F-PRO-92-O F-GLN-133-CD
I-ARG-42-O F-ASN-74-CB F-GLN-94-CD F-GLN-133-CG I-ASN-43-N
F-ASN-74-CG F-GLN-94-NE2 F-GLN-133-N I-ASN-43-OD1 F-ASN-74-N
F-GLN-94-OE1 F-GLN-133-O I-ILE-44-CA F-ASN-74-ND2 F-GLU-114-OE1
F-GLN-133-OE1 I-ILE-44-CB F-ASN-74-O F-LEU-117-CB F-GLN-133-OXT
I-ILE-44-CD1 F-LEU-75-C F-LEU-117-CD1 I-ILE-32-CG2 I-ILE-44-CG1
F-LEU-75-CA F-LEU-117-CD2 I-ASN-33-CB I-ILE-44-CG2 F-LEU-75-CB
F-LEU-117-CG I-ASN-33-CG I-ARG-47-CD F-LEU-75-CD1 F-THR-119-CB
I-ASN-33-ND2 I-ARG-47-CZ F-LEU-75-CD2 F-THR-119-CG2 I-ASN-33-OD1
I-ARG-47-NE F-LEU-75-CG F-THR-119-OG1 I-GLN-36-C I-ARG-47-NH1
F-LEU-75-N F-VAL-125-CB I-GLN-36-CA I-ARG-47-NH F-LEU-75-O
F-VAL-125-CG1 I-GLN-36-CB F-GLU-84-OE1 F-VAL-125-CG2 I-GLN-36-CD
F-ILE-86-C F-PRO-127-C I-GLN-36-NE2 F-ILE-86-CA F-PRO-127-CA
I-GLN-36-OE1 F-ILE-86-CB F-PRO-127-CB I-ARG-37-C F-ILE-86-CD1
F-PRO-127-CD I-ARG-37-CA F-ILE-86-CG1 F-PRO-127-CG I-ARG-37-CB
F-ILE-86-CG2 F-PRO-127-N I-ARG-37-CD F-ILE-86-O F-PRO-127-O
I-ARG-37-CG F-SER-87-C F-VAL-128-C I-ARG-37-CZ F-SER-87-CA
F-VAL-128-CA I-ARG-37-N F-SER-87-N F-VAL-128-CB I-ARG-37-NE
F-SER-87-O F-VAL-128-CG1 I-ARG-37-NH1 F-MET-88-C F-VAL-128-CG2
I-ARG-37-NH2 F-MET-88-CA F-VAL-128-N I-ARG-37-O F-MET-88-N
F-VAL-128-O I-VAL-38-C F-MET-88-O F-ILE-129-C I-VAL-38-CA
Creation of IOTA Database
[0102] A secondary database of over 11 million intra-molecular
atomic contact data was extracted from over 20000 non-homologous
protein structures where resolution was .ltoreq.2 .ANG.. Contacts
were defined as any two atoms on opposing sides of a protein fold
separated by a distance of 1 .ANG.+the sum of their respective Van
de Waals radii or less, and were limited to atoms on residues at
least 4 residues apart on the linear peptide sequence. The first
atom of the contacting pair was designated the theta atom and the
second atom, the iota atom. The database was divided into 167
contact sets according to the theta atom type, there being 167
non-hydrogen atom types within the 20 natural amino acid residues
comprising proteins. Within a contact set the relative coordinates
of each iota atom position was recorded after normalisation of the
theta-iota atom pair coordinates. The latter was achieved by
setting the theta atom to x,y,z=0,0,0; the next covalently attached
atom (3.sup.rd atom) to the theta atom (in the direction of the
peptide backbone) to x,y,z=x',0,0 and the next again covalently
attached atom (4.sup.th atom) to x,y,z=x'',0,z'. A consistent
convention was employed to defined 3.sup.rd and 4.sup.th atoms.
[0103] Each theta contact set was further sub-divided into 167 iota
atom types, but for convenience these were concatenated into 26
sub-groups according to chemical type based on the definition of
Engh and Huber (Engh and Huber (1991) "Accurate Bond and Angle
Parameters for X-ray Protein Structure Refinement", Acta Cryst.,
A47, 392-400).
[0104] In this Example the following iota sub-groups were
employed:
TABLE-US-00002 Description Atoms Carbonyl O all backbone O, Asn
O.delta.1, Gln O.epsilon.1 Tetrahedral CH.sub.2 all C.beta. 9except
Ala, Ile, Thr, Val) Arg C.delta., C.gamma., Gln C.gamma., Glu
C.gamma., Ile C.gamma.1, Lys C.gamma., C.delta., C.epsilon., Met
C.gamma. Tetrahedral CH.sub.3 Ala C.beta., Ile C.delta.1,
C.gamma.2, Leu C.delta.1, C.delta.2, Met C.epsilon., Thr C.gamma.2,
Val C.gamma.1, C.gamma.2 NH all backbone N (except Pro), Arg
N.epsilon., His N.delta.1, N.epsilon.2, Trp N.epsilon.1 Hydroxyl O
Ser O.gamma., Thr O.gamma.1, Tyr O.eta. Carboxyl O Asp O.delta.1,
O.delta.2, Glu O.epsilon.1, O.epsilon.2 NH.sub.2 Asn N.delta.2, Gln
N.epsilon.2
Superimposition of Iota Data Over the IL17F Epitope Surface
[0105] For each of the 209 theta atoms comprising the IL17F
epitope, the corresponding theta contact set was selected from the
IOTA database and from that, an appropriate iota sub-group was
selected e.g. carbonyl oxygen. The relative iota coordinates from
this sub-group were transposed relative to the reference frame of
the given theta atom of the IL17F epitope (Table 2 illustrates
example data). An iota dataset for a given sub-group was thus
accumulated over the whole IL17F epitope. In cases where the
location of a given iota data point intersected with an atom of
IL17F, closer than the sum of their respective Van de Waals radii
minus 0.2 .ANG., then these data points were excluded from the
dataset. The process was repeated for all relevant iota sub-groups
to produce a series of iota datasets for the IL17F epitope.
TABLE-US-00003 TABLE 2 id out01:0000098 out01:0000127 out01:0000249
out01:0000280 out01:0000282 out01:0000283 out01:0000346
out01:0000366 out01:0000527 out01:0000542 pdbcode 16vpA 16vpA 1a0cA
1a0pA 1a0pA 1a0pA 1a0sP 1a0sP 1a12A 1a12A res 2.1 2.1 2.5 2.5 2.5
2.5 2.4 2.4 1.7 1.7 rval 0.26 0.26 0.177 0.287 0.287 0.287 0.228
0.228 0.219 0.219 org Herpes simplex Herpes simplex Thermoanaero
Escherichia coli Escherichia coli Escherichia coli Salmonella typ
Salmonella typ Homo sapiens Homo sapiens Tchain A A A A A A P P A A
Taaind 96 235 36 126 137 137 85 138 257 12 Taanum 142 281 36 145
156 156 155 208 277 32 Taaname THR THR THR THR THR THR THR THR THR
THR Taass C E E H E E H E C C Taaphi -135.7 -129.6 -71.7 -87.1
-105.2 -105.2 -118.5 -123.9 -114.7 -127.1 Taapsi 163.3 160 157.7
-34.9 174 174 18.9 132.4 1 -10.7 Tomg -179.5 178.5 177.5 178.7
177.6 177.6 -178.3 179.3 -178.5 -171.7 Tc1 61.3 52.2 66.6 60.5 64.4
64.4 53.1 -54.5 49.2 59.2 Tc2 999.9 999.9 999.9 999.9 999.9 999.9
999.9 999.9 999.9 999.9 Tc3 999.9 999.9 999.9 999.9 999.9 999.9
999.9 999.9 999.9 999.9 Tc4 999.9 999.9 999.9 999.9 999.9 999.9
999.9 999.9 999.9 999.9 TUpAA ALA LEU LYS ALA LEU LEU SER GLY GLY
SER TDnAA ARG VAL MET GLY MET MET SER GLY GLU GLU Tnum 724 1867 288
1032 1111 1111 626 1048 1919 93 Tname C C C C C C C C C C Tbval
23.52 24.07 23.92 31.8 38.02 38.02 16.02 15.07 20.11 12.34 Tasa 0 0
0 0 0 0 1.84 0.19 0 0.21 Tcdist 0 0 0 0 0 0 0 0 0 0 Todist 1.234
1.233 1.24 1.215 1.239 1.239 1.226 1.222 1.247 1.249 Tndist 2.463
2.461 2.432 2.482 2.455 2.455 2.536 2.412 2.492 2.502 Tcadist 1.525
1.532 1.527 1.525 1.53 1.53 1.541 1.519 1.516 1.521 Icdist 3.472
3.069 3.359 3.618 3.647 3.199 3.565 3.589 3.432 3.67 Iodist 3.677
3.072 4.523 3.286 3.657 4.381 2.796 2.512 3.207 3.652 Indist 4.507
4.556 4.306 4.708 4.178 4.537 5.735 3.963 4.235 4.639 Itdist 3.472
3.069 3.359 3.618 3.647 3.199 3.565 3.589 3.432 3.67 Inum 1644 1696
244 2089 1535 1519 550 1213 1845 372 Iname CD2 OD1 O CD1 CB O NE2 N
O CG1 Ichain A A A A A A P P A A Iaaind 206 213 30 258 190 188 75
159 247 50 Iaanum 252 259 30 279 209 207 145 229 267 70 Iaaname LEU
ASP GLU TYR LEU ASP GLN GLY SER VAL Iaass H E C H C C E E C E
Iaaphi -94.9 -110.9 -60.4 -69.5 -73.2 -83.2 -125.6 -109.2 -149.7
-101.1 Iaapsi -34.2 127.4 132.7 -18.6 -32.6 -14.4 142.7 141.1 38.2
-21 Iomg -173.5 -178.2 -179.6 -177.8 -177.1 -179.8 177.8 178.2
-179.1 179.5 Ic1 -63.9 -171.8 176.2 -79.4 167.9 66 -66.4 999.9 -164
-65.2 Ic2 167.9 57.6 172.1 -57.1 69.8 -21.2 -63.5 999.9 999.9 999.9
Ic3 999.9 999.9 7 999.9 999.9 999.9 -52.7 999.9 999.9 999.9 Ic4
999.9 999.9 999.9 999.9 999.9 999.9 999.9 999.9 999.9 999.9 IUpAA
ASP CYS GLU ILE VAL ILE GLY TYR LEU VAL IDnAA PHE LEU VAL THR PHE
VAL THR ARG ASN GLN Ibval 6.09 32.79 27.18 37.22 8.29 43.39 11.84
20.76 9.22 8.96 Iasa 0.13 0 0 0 0 0 0.69 0.25 0 1.04 K1cdist 4.988
4.114 4.557 4.099 4.209 4.342 4.034 4.42 3.962 4.187 K1odist 5.113
3.819 5.728 3.526 4.6 5.553 3.425 3.283 3.395 4.575 K1ndist 5.733
5.402 5.198 5.704 4.874 5.334 5.781 4.513 5.257 4.346 K1tdist 4.988
4.114 4.557 4.099 4.209 4.342 4.034 4.42 3.962 4.187 K1num 1642
1695 243 2088 1532 1518 548 1214 1844 371 K1name CG CG C CG CA C CD
CA C CB K1aanum 252 259 30 279 209 207 145 229 267 70 K1aaname LEU
ASP GLU TYR LEU ASP GLN GLY SER VAL K2cdist NoValue NoValue NoValue
3.857 NoValue NoValue NoValue NoValue NoValue NoValue K2odist
NoValue NoValue NoValue 3.951 NoValue NoValue NoValue NoValue
NoValue NoValue K2ndist NoValue NoValue NoValue 4.353 NoValue
NoValue NoValue NoValue NoValue NoValue K2tdist NoValue NoValue
NoValue 3.857 NoValue NoValue NoValue NoValue NoValue NoValue K2num
NoValue NoValue NoValue 2091 NoValue NoValue NoValue NoValue
NoValue NoValue K2name NoValue NoValue NoValue CE1 NoValue NoValue
NoValue NoValue NoValue NoValue K2aanum NoValue NoValue NoValue 279
NoValue NoValue NoValue NoValue NoValue NoValue K2aaname NoValue
NoValue NoValue TYR NoValue NoValue NoValue NoValue NoValue NoValue
Cx 0 0 0 0 0 0 0 0 0 0 Cy 0 0 0 0 0 0 0 0 0 0 Cz 0 0 0 0 0 0 0 0 0
0 CsphR 0 0 0 0 0 0 0 0 0 0 CsphT 0 0 0 0 0 0 0 0 0 0 CsphP -3.142
0 0 3.142 0 0 0 0 0 0 CcylR 0 0 0 0 0 0 0 0 0 0 CcylT -3.142 0 0
3.142 0 0 0 0 0 0 CcylZ 0 0 0 0 0 0 0 0 0 0 Ox -0.628 -0.629 -0.648
-0.659 -0.666 -0.666 -0.609 -0.619 -0.609 -0.582 Oy 0.998 1.004
0.978 -0.754 1.028 1.028 -1.009 0.713 -1.084 -1.095 Oz 0.364 0.341
0.401 -0.689 0.182 0.182 0.341 0.777 0.085 -0.146 OsphR 1.234 1.233
1.24 1.216 1.238 1.238 1.227 1.223 1.246 1.249 OsphT 1.271 1.291
1.241 2.173 1.423 1.423 1.289 0.882 1.503 1.688 OsphP 2.132 2.13
2.156 -2.289 2.146 2.146 -2.114 2.286 -2.083 -2.059 OcylR 1.234
1.233 1.24 1.216 1.238 1.238 1.227 1.223 1.246 1.249 OcylT 2.132
2.13 2.156 -2.289 2.146 2.146 -2.114 2.286 -2.083 -2.059 OcylZ
0.364 0.341 0.401 -0.689 0.182 0.182 0.341 0.777 0.085 -0.146 Nx
2.055 2.054 2.006 2.096 2.039 2.039 2.158 1.982 2.11 2.128 Ny 1.357
1.356 1.375 1.328 1.368 1.368 1.332 1.374 1.327 1.316 Nz 0 0 0 0 0
0 0 0 0 0 NsphR 2.463 2.461 2.432 2.481 2.455 2.455 2.536 2.412
2.493 2.502 NsphT 1.571 1.571 1.571 1.571 1.571 1.571 1.571 1.571
1.571 1.571 NsphP 0.584 0.583 0.601 0.565 0.591 0.591 0.553 0.606
0.561 0.554 NcylR 2.463 2.461 2.432 2.481 2.455 2.455 2.536 2.412
2.493 2.502 NcylT 0.584 0.583 0.601 0.565 0.591 0.591 0.553 0.606
0.561 0.554 NcylZ 0 0 0 0 0 0 0 0 0 0 Cax 1.525 1.532 1.527 1.525
1.53 1.53 1.541 1.519 1.516 1.521 Cay 0 0 0 0 0 0 0 0 0 0 Caz 0 0 0
0 0 0 0 0 0 0 CasphR 1.525 1.532 1.527 1.525 1.53 1.53 1.541 1.519
1.516 1.521 CasphT 1.571 1.571 1.571 1.571 1.571 1.571 1.571 1.571
1.571 1.571 CasphP 0 0 0 0 0 0 0 0 0 0 CacylR 1.525 1.532 1.527
1.525 1.53 1.53 1.541 1.519 1.516 1.521 CacylT 0 0 0 0 0 0 0 0 0 0
CacylZ 0 0 0 0 0 0 0 0 0 0 Tx -12.209 3.16 75.678 17.53 11.229
11.229 -34.605 -58.479 39.484 5.281 Ty -17.125 -7.179 45.43 -33.077
-23.148 -23.148 -6.201 -0.92 -28.232 -11.189 Tz -3.618 -14.31
44.154 38.22 26.483 26.483 -24.486 -14.58 0.224 9.116 TsphR TsphT
TsphP TcylR TcylT TcylZ Ix -0.948 -0.804 1.174 -1.669 -0.297 0.852
-2.689 -1.363 1.533 -0.362 Iy 0.629 -0.729 -2.203 1.536 1.127 -2.85
-0.806 3.054 -2.417 -0.092 Iz -3.281 2.871 -2.247 -2.818 -3.455
-1.176 2.198 1.302 -1.893 -3.651 IsphR 3.473 3.069 3.359 3.617
3.646 3.199 3.565 3.589 3.432 3.67 IsphT 2.808 0.361 2.304 2.464
2.816 1.947 0.907 1.2 2.155 3.04 IsphP 2.556 -2.405 -1.081 2.398
1.828 -1.28 -2.85 1.991 -1.006 -2.893 IcylR 3.473 3.069 3.359 3.617
3.646 3.199 3.565 3.589 3.432 3.67 IcylT 2.556 -2.405 -1.081 2.398
1.828 -1.28 -2.85 1.991 -1.006 -2.893 IcylZ -3.281 2.871 -2.247
-2.818 -3.455 -1.176 2.198 1.302 -1.893 -3.651 K1x -1.194 -1.35
1.899 -2.979 0.085 1.65 -2.134 -1.06 0.644 0.446 K1y 1.1 -0.243
-2.896 1.098 -0.13 -3.76 -0.567 3.344 -3.182 1.142 K1z -4.717 3.878
-2.962 -2.593 -4.206 -1.413 3.376 2.69 -2.271 -4.003 K1sphR 4.989
4.113 4.557 4.099 4.209 4.342 4.034 4.421 3.962 4.187 K1sphT 2.81
0.34 2.278 2.256 3.105 1.902 0.579 0.917 2.181 2.844 K1sphP 2.397
-2.963 -0.99 2.788 -0.992 -1.157 -2.882 1.878 -1.371 1.198 K1cylR
4.989 4.113 4.557 4.099 4.209 4.342 4.034 4.421 3.962 4.187 K1cylT
2.397 -2.963 -0.99 2.788 -0.992 -1.157 -2.882 1.878 -1.371 1.198
K1cylZ -4.717 3.878 -2.962 -2.593 -4.206 -1.413 3.376 2.69 -2.271
-4.003 K2x NoValue NoValue NoValue -1.258 NoValue NoValue NoValue
NoValue NoValue NoValue K2y NoValue NoValue NoValue 2.771 NoValue
NoValue NoValue NoValue NoValue NoValue K2z NoValue NoValue NoValue
-2.37 NoValue NoValue NoValue NoValue NoValue NoValue K2sphR 3.857
K2sphT 2.232 K2sphP 1.997 K2cylR 3.857 K2cylT 1.997 K2cylZ -2.37
inter MS MS MM MS MS MM MS MM MM MS Icount 1 6 3 4 4 4 2 4 4 2
Tcount 1 1 1 1 2 2 1 1 1 1 indicates data missing or illegible when
filed
TABLE-US-00004 TABLE 2 Key Header Description Id Incremented
Identifier of a contact. Every unique identifier is a unique
theta-iota interaction. The id is eight digits with place- holding
0 s (ex. 1 is 00000001). pdbcode The PDB code that was given in the
input file. (ex 1mu4B for chain B of 1mu4, or 1mu4 for the
structure). res The resolution of the PDB structure. rval The
R-value of the PDB structure. org The organism source for the PDB
structure. Tchain The chain identifier for the theta atom. Taaind
The amino acid index for the theta amino acid (index starts at 1
for the first amino acid in the structure and increments for each
amino acid) Taanum The amino acid number for the theta atom.
Taaname The amino acid name for the theta atom. Taass The amino
acid secondary structure for the theta atom. Taaphi The amino acid
phi angle for the theta atom. Taapsi The amino acid psi angle for
the theta atom. Tomg The amino acid omega angle for the theta atom.
Tchi1 The amino acid chi 1 angle for the theta atom. Tchi2 The
amino acid chi 2 angle for the theta atom. Tchi3 The amino acid chi
3 angle for the theta atom. Tchi4 The amino acid chi 4 angle for
the theta atom. TUpAA The amino acid which is upstream of the theta
amino acid. TDnAA The amino acid which is downstream of the theta
amino acid. Tnum The atom number for the theta atom. Tname The atom
name for the theta atom. Tbval The B value or temperature factor
for the theta atom. Tasa The Accessible Surface Area of the theta
atom. Tcdist The distance from the theta atom to the backbone
Carbon. Todist The distance from the theta atom to the backbone
Oxygen. Tndist The distance from the theta atom to the backbone
Nitrogen. Tcadist The distance from the theta atom to the backbone
Alpha Carbon. Icdist The distance from the iota atom to the
backbone Carbon of the theta amino acid. Iodist The distance from
the iota atom to the backbone Oxygen of the theta amino acid.
Indist The distance from the iota atom to the backbone Nitrogen of
the theta amino acid. Itdist The distance from the iota atom to the
theta atom. hum The atom number for the iota atom. Iname The atom
name for the iota atom. Ichain The chain identifier for the iota
atom. Iaaind The amino acid index for the iota amino acid (index
starts at 1 for the first amino acid in the structure and
increments for each amino acid) Iaanum The amino acid number for
the iota atom. Iaaname The amino acid name for the iota atom. Iaass
The amino acid secondary structure for the iota atom. Iaaphi The
amino acid phi angle for the iota atom. Iaapsi The amino acid psi
angle for the iota atom. Iomg The amino acid omega angle for the
iota atom. Ichi1 The amino acid chi 1 angle for the iota atom.
Ichi2 The amino acid chi 2 angle for the iota atom. Ichi3 The amino
acid chi 3 angle for the iota atom. Ichi4 The amino acid chi 4
angle for the iota atom. IUpAA The amino acid which is upstream of
the iota amino acid. IDnAA The amino acid which is downstream of
the iota amino acid. Ibval The B value or temperature factor for
the iota atom. Iasa The Accessible Surface Area for the iota atom.
K1cdist The distance from the first kappa atom to the backbone
Carbon of the theta amino acid. K1odist The distance from the first
kappa atom to the backbone Oxygen of the theta amino acid. K1ndist
The distance from the first kappa atom to the backbone Nitrogen of
the theta amino acid. K1tdist The distance from the first kappa
atom to the theta atom. K1num The atom number for the first kappa
atom. K1name The atom name for the first kappa atom. K1aanum The
amino acid number for the first kappa atom. K1aaname The amino acid
name for the first kappa atom. K2cdist The distance from the second
kappa atom to the backbone Carbon of the theta amino acid. K2odist
The distance from the second kappa atom to the backbone Oxygen of
the theta amino acid. K2ndist The distance from the second kappa
atom to the backbone Nitrogen of the theta amino acid. K2tdist The
distance from the second kappa atom to the theta atom. K2num The
atom number for the second kappa atom. K2name The atom name for the
second kappa atom. K2aanum The amino acid number for the second
kappa atom. K2aaname The amino acid name for the second kappa atom.
Cx The Theta-superimposed x coordinate for the backbone Carbon atom
of the theta AA. Cy The Theta-superimposed y coordinate for the
backbone Carbon atom of the theta AA. Cz The Theta-superimposed z
coordinate for the backbone Carbon atom of the theta AA. CsphR The
Theta-superimposed spherical polar distance for the backbone Carbon
of the theta AA. CsphT The Theta-superimposed spherical polar
latitude angle for the backbone Carbon of the theta AA. CsphP The
Theta-superimposed spherical polar longitude angle for the backbone
Carbon of the theta AA. CcylR The Theta-superimposed cylindrical
polar distance for the backbone Carbon of the theta AA. CcylT The
Theta-superimposed cylindrical polar angle for the backbone Carbon
of the theta AA. CcylZ The Theta-superimposed cylindrical polar z
coordinate for the backbone Carbon of the theta AA. Ox The
Theta-superimposed x coordinate for the backbone Oxygen atom of the
theta AA. Oy The Theta-superimposed y coordinate for the backbone
Oxygen atom of the theta AA. Oz The Theta-superimposed z coordinate
for the backbone Oxygen atom of the theta AA. OsphR The
Theta-superimposed spherical polar distance for the backbone Oxygen
of the theta AA. OsphT The Theta-superimposed spherical polar
latitude angle for the backbone Oxygen of the theta AA. OsphP The
Theta-superimposed spherical polar longitude angle for the backbone
Oxygen of the theta AA. OcylR The Theta-superimposed cylindrical
polar distance for the backbone Oxygen of the theta AA. OcylT The
Theta-superimposed cylindrical polar angle for the backbone Oxygen
of the theta AA. OcylZ The Theta-superimposed cylindrical polar z
coordinate for the backbone Oxygen of the theta AA. Nx The
Theta-superimposed x coordinate for the backbone Nitrogen atom of
the theta AA. Ny The Theta-superimposed y coordinate for the
backbone Nitrogen atom of the theta AA. Nz The Theta-superimposed z
coordinate for the backbone Nitrogen atom of the theta AA. NsphR
The Theta-superimposed spherical polar distance for the backbone
Nitrogen of the theta AA. NsphT The Theta-superimposed spherical
polar latitude angle for the backbone Nitrogen of the theta AA.
NsphP The Theta-superimposed spherical polar longitude angle for
the backbone Nitrogen of the theta AA. NcylR The Theta-superimposed
cylindrical polar distance for the backbone Nitrogen of the theta
AA. NcylT The Theta-superimposed cylindrical polar angle for the
backbone Nitrogen of the theta AA. NcylZ The Theta-superimposed
cylindrical polar z coordinate for the backbone Nitrogen of the
theta AA. Cax The Theta-superimposed x coordinate for the Alpha
Carbon atom of the theta AA. Cay The Theta-superimposed y
coordinate for the Alpha Carbon atom of the theta AA. Caz The
Theta-superimposed z coordinate for the Alpha Carbon atom of the
theta AA. CasphR The Theta-superimposed spherical polar distance
for the Alpha Carbon of the theta AA. CasphT The Theta-superimposed
spherical polar latitude angle for the Alpha Carbon of the theta
AA. CasphP The Theta-superimposed spherical polar longitude angle
for the Alpha Carbon of the theta AA. CacylR The Theta-superimposed
cylindrical polar distance for the Alpha Carbon of the theta AA.
CacylT The Theta-superimposed cylindrical polar angle for the Alpha
Carbon of the theta AA. CacylZ The Theta-superimposed cylindrical
polar z coordinate for the Alpha Carbon of the theta AA. Tx The
Theta-superimposed x coordinate for the theta atom. Ty The
Theta-superimposed y coordinate for the theta atom. Tz The
Theta-superimposed z coordinate for the theta atom. TsphR The
Theta-superimposed spherical polar distance for the Theta atom.
TsphT The Theta-superimposed spherical polar latitude angle for the
Theta atom. TsphP The Theta-superimposed spherical polar longitude
angle for the Theta atom. TcylR The Theta-superimposed cylindrical
polar distance for the Theta atom. TcylT The Theta-superimposed
cylindrical polar angle for the Theta atom. TcylZ The
Theta-superimposed cylindrical polar z coordinate for the Theta
atom. Ix The Theta-superimposed x coordinate for the iota atom. Iy
The Theta-superimposed y coordinate for the iota atom. Iz The
Theta-superimposed z coordinate for the iota atom. IsphR The
Theta-superimposed spherical polar distance for the iota atom.
IsphT The Theta-superimposed spherical polar latitude angle for the
iota atom. IsphP The Theta-superimposed spherical polar longitude
angle for the iota atom. IcylR The Theta-superimposed cylindrical
polar distance for the iota atom. IcylT The Theta-superimposed
cylindrical polar angle for the iota atom. IcylZ The
Theta-superimposed cylindrical polar z coordinate for the iota
atom. K1x The Theta-superimposed x coordinate for the first kappa
atom. K1y The Theta-superimposed y coordinate for the first kappa
atom. K1z The Theta-superimposed z coordinate for the first kappa
atom. K1sphR The Theta-superimposed spherical polar distance for
the first kappa atom. K1sphT The Theta-superimposed spherical polar
latitude angle for the first kappa atom. K1sphP The
Theta-superimposed spherical polar longitude angle for the first
kappa atom. K1cylR The Theta-superimposed cylindrical polar
distance for the first kappa atom. K1cylT The Theta-superimposed
cylindrical polar angle for the first kappa atom. K1cylZ The
Theta-superimposed cylindrical polar z coordinate for the first
kappa atom. K2x The Theta-superimposed x coordinate for the second
kappa atom. K2y The Theta-superimposed y coordinate for the second
kappa atom. K2z The Theta-superimposed z coordinate for the second
kappa atom. K2sphR The Theta-superimposed spherical polar distance
for the second kappa atom. K2sphT The Theta-superimposed spherical
polar latitude angle for the second kappa atom. K2sphP The
Theta-superimposed spherical polar longitude angle for the second
kappa atom. K2cylR The Theta-superimposed cylindrical polar
distance for the second kappa atom. K2cylT The Theta-superimposed
cylindrical polar angle for the second kappa atom. K2cylZ The
Theta-superimposed cylindrical polar z coordinate for the second
kappa atom. inter The type of interaction for the theta & iota
contact: MM - Main chain to Main chain, MS - Main chain to Side
chain, SM . . . , SS . . . where the first letter refers to theta
and the second refers to iota. Icount The iota atom count for the
particular theta atom. Tcount The theta atom count for the
particular amino acid.
Visualisation of IL17F Iota Datasets
[0106] Each iota dataset was visualized in relation to the
IL17F/Fab 496 structure using molecular graphics computer software
such as Pymol. This could be done by direct plotting of the iota
dataset as individual points or by first mathematically
transforming the dataset into a density function and a file format
compatible for molecular graphic display e.g. ccp4, so that contour
maps of higher density could be displayed over the IL17
epitope.
Inspection of Iota Density Maps for Intersection with Fab 496
Paratope Atoms
[0107] The IL17F/Fab 496 interface was examined to determine the
degree of intersection between individual Fab 496 atoms per residue
and the corresponding iota density maps. Residues were identified
where there was no or little intersection. In these cases
alternative residues were substituted via the molecular graphics
software to determine whether better intersection could be achieved
between residue atoms and relevant iota density maps. Amino acid
substitutions producing good iota density map intersection were
short listed for in vitro production and testing as single point
mutations as intact IgG versions of Fab 496.
DNA Manipulations and General Methods
[0108] E. coli strain INV.alpha.F (Invitrogen) was used for
transformation and routine culture growth. DNA restriction and
modification enzymes were obtained from Roche Diagnostics Ltd. and
New England Biolabs. Plasmid preparations were performed using Maxi
Plasmid purification kits (Qiagen, catalogue No. 12165). DNA
sequencing reactions were performed using ABI Prism Big Dye
terminator sequencing kit (catalogue No. 4304149) and run on an ABI
3100 automated sequencer (Applied Biosystems). Data was analysed
using the program Auto Assembler (Applied Biosystems).
Oligonucleotides were obtained from Invitrogen. The concentration
of IgG was determined by IgG assembly ELISA.
Affinity Maturation of Antibody CA028_00496
[0109] CA028_0496 is a humanised neutralising antibody which binds
both IL17A and IL17F isoforms. It comprises the grafted variable
regions, termed gL7 and gH9, whose sequences are disclosed in WO
2008/047134. The wild type Fab' fragment of this antibody (Fab 496)
and mutant variants were prepared as follows: oligonucleotide
primer sequences were designed and constructed in order to
introduce single point mutations in the light chain variable region
(gL7) as per residues and positions determined in the above short
list. Each mutated light chain was separately sub-cloned into the
UCB Celltech human light chain expression vector pKH10.1, which
contained DNA encoding the human C-kappa constant region (Km3
allotype). The unaltered heavy chain variable region (gH9) sequence
was sub-cloned into the UCB Celltech expression vector pVhg1Fab6His
which contained DNA encoding human heavy chain gamma-1 constant
region, CH1. Heavy and light chain encoding plasmids were
co-transfected into HEK293 cells using the 293Fectin.TM. procedure
according to the manufacturer's instructions (InVitrogen. Catalogue
No. 12347-019). IgG1 antibody levels secreted into the culture
supernatants after 10 to 12 days culture were assessed by ELISA and
binding kinetics assessed by surface plasmon resonance (see below).
Mutants showing improved or similar binding to IL17F were then
prepared and tested in combination as double, triple, quadruple or
quintuple light chain mutations as above.
Surface Plasmon Resonance (SPR)
[0110] All SPR experiments were carried out on a Biacore 3000
system (Biacore AB) at 25'C using HBS-EP running buffer (10 mM
HEPES pH 7.4, 150 mM NaCl3 mM EDTA 0.005% (v/v) surfactant P20,
Biacore AB). Goat F(ab').sub.2 anti-IgG Fab' specific antibody
(Jackson Labs. Product code 109-006-097) was covalently attached to
the surface of a CM5 sensor chip (GE Healthcare) by the amine
coupling method, as recommended by the manufacturers. Briefly, the
carboxymethyl dextran surface was activated with a fresh mixture of
50 mM N-hydroxysuccimide and 200 mM
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide for 5 minutes at a
flow rate of 10 .mu.l/min. Anti-Fab antibody at 50 .mu.g/ml in 10
mM sodium acetate pH 5.0 buffer was injected for 60 sec at the same
flow rate. Finally the surface was deactivated with a 10 minute
pulse of 1 M ethanolamine.HCl pH8.5, leaving of 4000 to 5000
response units (RU) of immobilized antibody on the chip. A
reference flow cell was prepared on the same chip by omitting the
protein from the above procedure.
[0111] Wild type and mutated 496 Fabs were harvested from culture
supernatants in the range 3 to 30 .mu.g/ml and crude supernatants
were diluted in running buffer into the range 0.5 to 2 .mu.g/ml. In
order to evaluate binding kinetics to IL17F, each antibody was
first captured on the anti-Fab' surface by injection at 10
.mu.l/min for 60 sec to yield an additional 150 to 250 RU signal.
Recombinant human IL17F was titrated from 10 nM in running buffer
and injected at 30 .mu.l/ml, to produce an association phase over
180 sec followed by a dissociation phase of 300 sec. At the end of
each cycle the surface was regenerated with a 60 sec pulse of 40 mM
HCl followed by a 30 sec pulse of 5 mM NaOH at 10 .mu.l/min. For
each Fab' a control cycle was carried out where the IL17F injection
was replaced with an injection of running buffer.
[0112] Sensograms were corrected by subtraction of reference flow
cell signal, then by subtracting the control cycle sensogram for
the respective Fab'. Dissociation rate constants (k.sub.d) and
association rate constants (k.sub.a) were fitted to the data using
Biaevaluation software (Biacore AB). Fab affinities (K.sub.D) were
calculated as K.sub.D=k.sub.d/k.sub.a.
Results
[0113] Intersection of Iota Density Maps with Fab 496 Atoms
[0114] Inspection of that part of the surface of Fab 496 forming
the interface with IL17F revealed that in many areas there was
complete intersection between a given Fab 496 atom and the
corresponding iota density map. This was particularly the case for
amino acid residues comprising the Fab 496 heavy chain. However
there were a number of regions comprising the light chain where
there was no intersection or little intersection of Fab 496 atoms
and corresponding iota density maps (Panel A, FIGS. 8 to 13). By
substituting alternative residues into the Fab 496 structure at
these positions it was possible to achieve intersection between one
or more atoms of the substituted residue and the corresponding iota
density maps. (Panel B, FIGS. 8 to 13). Thus on Fab 496 light chain
the threonine 30 C.gamma.2 atom does not intersect with the methyl
carbon iota density map and the O.gamma.1 atom does not intersect
with the hydroxyl oxygen iota density map. However when threonine
30 is mutated to arginine, there is intersection between the NE
atom and the secondary amide iota density map (FIG. 8).
[0115] FIG. 9 shows tetrahedral methylene iota density and
secondary amide iota density dispersed around light chain arginine
54 without any intersection with respective side chain atoms of
this residue. Conversely when mutated to a serine at this position
there is good intersection of hydroxyl oxygen iota density and the
gamma oxygen atom of the serine side chain. The light chain serine
56 gamma oxygen atom does not intersect with hydroxyl oxygen iota
density. However when this residue is mutated to isoleucine there
is intersection of the delta 1 methyl atom of the isoleucine side
chain and methyl iota density (FIG. 10). In the case of serine 60,
again the gamma oxygen is too distant from adjacent hydroxyl oxygen
iota density to achieve intersection, (FIG. 11), but mutating this
residue to aspartate brings about intersection of the side chain
delta oxygen atom with carboxyl oxygen iota density.
[0116] Light chain threonine 72 is not a CDR residue but part of
framework 3, its side chain methyl atom does not intersect with
methyl carbon iota density nor its side chain oxygen atom with
hydroxyl oxygen iota density. But the arginine 72 mutation allows
intersection of both side chain delta carbon atom with methylene
carbon iota density and of side chain eta nitrogen atoms with
guanidinium nitrogen iota density.
Effects of Iota Designed Mutations on Antibody CA028_00496 on
Binding Kinetics
[0117] Five iota designed single point light chain mutations in Fab
496, T30R, R54S, S56I, S60D and T72R, showed small improvements in
binding affinity ranging from 1.7 to 3.6 fold. The improvement
observed with the non-CDR mutation, T72R, was surprising since
residues at this position do not normally contact the antigen. For
all but one of these mutations (S60D), the improvement was driven
by a reduction in dissociation rate constant (Table 3).
Combinations of these mutations in pairs resulted in a synergistic
improvement in binding, with a 3.8 to 7.5 fold reduction in
dissociation rate constant; with triple and quadruple combinations
producing further step reductions in dissociation rate constant
(Table 3).
[0118] The combination of all five light chain mutations produced
the largest improvement in binding affinity (Table 4) to give an
affinity value of 11 pM to IL17F, some 180-fold better than the
original Fab 496. An important finding was that there was no
deleterious effect on the binding to the IL17A isoform, in fact an
improvement, with affinity constant at 2 pM compared to 14 pM for
the original Fab 496. It is interesting that combinations of the
designed mutations produce a synergistic enhancement of affinity;
one explanation is that they are reasonably spaced across the light
chain paratope surface (FIG. 13) and therefore avoid negative
interaction effects.
TABLE-US-00005 TABLE 3 Effect of single residue and combined
residue light chain mutations of antibody CA028_000496 on IL17F
dissociation rate constant CA028_00496 light chain fold mutations
k.sub.d (s.sup.-1) change wt 4.2E-03 T30R 3.2E-03 1.3 R54S 2.1E-03
2.0 S56I 3.0E-03 1.4 S60D 4.1E-03 1.0 T72R 2.5E-03 1.7 T30R/R54S
7.0E-04 5.8 T30R/S56I 1.1E-03 3.8 R54S/S56I 6.2E-04 6.6 R54S/T72R
5.5E-04 7.5 S56I/T72R 8.0E-04 5.1 S60D/T72R 1.2E-03 3.6
T30R/R54S/T72R 2.5E-04 19 T30R/S56I/T72R 4.1E-04 10
T30R/S56I/S60D/T72R 3.1E-04 14 T30R/R54S/S56I/T72R 1.3E-04 35
T30R/R54S/S60D/T72R 9.3E-05 44 T30R/R54S/S56I/S60D/T72R 5.4E-05
104
TABLE-US-00006 TABLE 4 Affinity constant of antibody CA028_000496
versus variant comprising 5 light chain mutations k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (M) K.sub.D (pM) wild
type 2.0E+06 4.1E-03 2.0E-09 2000 T30R/R54S/S56I/ 2.3E+06 2.6E-05
1.1E-11 11 S60D/T72R
Conclusion
[0119] The method of creating iota density maps over the epitope
surface of IL17F in order to predict favourable mutations in the
corresponding antibody, Fab 496, has proven to be successful in
that the affinity constant has been improved 180-fold to a K.sub.D
of 11 pM. This method does not assume that only CDR mutations can
be used but demonstrates that framework region mutations are also
important for affinity maturation.
EXAMPLE 2
Utilisation of an Automated Process of the Invention in the Region
Between an Ab's Heavylight Chains to Improve Stability Methods
Identification of Target (Theta) Atoms of Both the Heavy and Light
Chains of the Fv Interface of Fab X
[0120] Using the coordinates of the crystal structure of an
antibody Fab fragment "Fab X" complexed with antigen, all heavy
chains atoms within 6 .ANG. of any light chain atoms were
identified as epitope atoms (167 theta atoms listed in Table 5).
Similarly, all light chains atoms within 6 .ANG. of any heavy chain
atoms were identified as epitope atoms (159 theta atoms listed in
Table 5).
TABLE-US-00007 TABLE 5 List of atoms comprising the heavy and light
chain epitopes where the notation (1)-(2)-(3) designates (1) the
respective H and L chains of Fab X, (2) residue number and (3) the
atom type. Binding pocket (THETA) = H Binding partner (IOTA) = L
H-47-CA H-47-CB H-47-CG1 H-47-CG2 H-49-CB H-49-CD H-49-CG H-49-NE2
H-49-OE1 H-53-C H-53-CA H-53-O H-54-C H-54-CA H-54-N H-54-O H-55-C
H-55-CA H-55-CB H-55-CD1 H-55-CD2 H-55-CG H-55-N H-55-O H-56-C
H-56-CA H-56-CB H-56-N H-56-O H-57-C H-57-CA H-57-CB H-57-CD1
H-57-CD2 H-57-CE2 H-57-CE3 H-57-CG H-57-CH2 H-57-CZ2 H-57-CZ3
H-57-N H-57-NE1 H-57-O H-60-CB H-60-CD1 H-60-CG1 H-60-CG2 H-62-CE2
H-62-CH2 H-62-CZ2 H-62-NE1 H-68-C H-68-CA H-68-CB H-68-CD1 H-68-CD2
H-68-CE1 H-68-CE2 H-68-CG H-68-CZ H-68-N H-68-O H-69-C H-69-CA
H-69-N H-69-O H-70-C H-70-CA H-70-CB H-70-N H-70-O H-71-C H-71-CA
H-71-CB H-71-CG2 H-71-N H-71-OG1 H-72-N H-104-CD1 H-104-CD2
H-104-CE1 H-104-CE2 H-104-CG H-104-CZ H-104-OH H-109-CB H-109-CG1
H-109-CG2 H-112-CD1 H-112-CE1 H-112-CE2 H-112-CZ H-112-OH H-113-C
H-113-CA H-113-CB H-113-O H-113-OG H-114-C H-114-CA H-114-CB
H-114-CG2 H-114-N H-114-O H-114-OG1 H-115-C H-115-CA H-115-CB
H-115-N H-115-O H-116-C H-116-CA H-116-CB H-116-CD H-116-CG H-116-N
H-116-O H-117-C H-117-CA H-117-CB H-117-CD1 H-117-CD2 H-117-CE1
H-117-CE2 H-117-CG H-117-CZ H-117-N H-117-O H-117-OH H-118-C
H-118-CA H-118-CB H-118-CD1 H-118-CD2 H-118-CE1 H-118-CE2 H-118-CG
H-118-CZ H-118-N H-118-O H-119-C H-119-CA H-119-CB H-119-CG H-119-N
H-119-O H-121-C H-121-CA H-121-CB H-121-CD1 H-121-CD2 H-121-CE2
H-121-CE3 H-121-CG H-121-CH2 H-121-CZ2 H-121-CZ3 H-121-N H-121-NE1
H-121-O H-122-C H-122-CA H-122-N H-122-O H-123-C H-123-CA H-123-O
Binding pocket (THETA) = L Binding partner (IOTA) = H L-11-OD1
L-41-O L-42-C L-42-CA L-42-CB L-42-O L-43-C L-43-CA L-43-CB
L-43-CD1 L-43-CD2 L-43-CE1 L-43-CE2 L-43-CG L-43-CZ L-43-N L-43-O
L-44-C L-44-CA L-44-N L-44-O L-45-C L-45-CA L-45-CB L-45-N L-45-O
L-45-OG L-47-CB L-47-CD1 L-47-CD2 L-47-CE1 L-47-CE2 L-47-CG L-47-CZ
L-47-OH L-49-CB L-49-CD L-49-CG L-49-NE2 L-49-OE1 L-52-C L-52-O
L-53-C L-53-CA L-53-N L-53-O L-54-C L-54-CA L-54-CB L-54-N L-54-O
L-55-C L-55-CA L-55-CB L-55-CD L-55-CG L-55-N L-55-O L-56-C L-56-CA
L-56-CB L-56-N L-56-O L-57-C L-57-CA L-57-CB L-57-CD1 L-57-CD2
L-57-CG L-57-N L-59-C L-59-O L-60-C L-60-CA
L-60-CB L-60-CD1 L-60-CD2 L-60-CE1 L-60-CE2 L-60-CG L-60-CZ L-60-N
L-60-O L-61-C L-61-CA L-61-CB L-61-CD L-61-CG L-61-N L-61-O
L-61-OE1 L-61-OE2 L-62-N L-64-NZ L-66-CG2 L-98-CB L-98-CD1 L-98-CD2
L-98-CE1 L-98-CE2 L-98-CG L-98-CZ L-98-OH L-100-C L-100-CA L-100-N
L-100-O L-101-C L-101-CA L-101-N L-101-O L-102-C L-102-CA L-102-N
L-105-O L-106-C L-106-CA L-106-CB L-106-CD1 L-106-CG1 L-106-CG2
L-106-N L-106-O L-107-C L-107-CA L-107-CB L-107-N L-107-O L-107-OG
L-108-C L-108-CA L-108-CB L-108-CG L-108-N L-108-OD1 L-108-OD2
L-109-C L-109-CA L-109-CB L-109-CG2 L-109-N L-109-O L-109-OG1
L-110-CA L-110-N L-111-CA L-111-CB L-111-CD1 L-111-CD2 L-111-CE1
L-111-CE2 L-111-CG L-111-CZ L-111-N L-111-O L-112-O L-113-C
L-113-CA L-113-O
Superimposition of Iota Data Over the Heavy and Light Chain
Interface Surface
[0121] For each of the 167 theta atoms comprising the heavy chain
epitope, the corresponding theta contact set was selected from the
IOTA database and from that, an appropriate iota sub-group was
selected e.g. carbonyl oxygen. The relative iota coordinates from
this sub-group were transposed relative to the reference frame of
the given theta atom of the heavy chain epitope. An iota dataset
for a given sub-group was thus accumulated over the whole heavy
chain epitope. In cases where the location of a given iota data
point intersected with an atom of the heavy chain, closer than the
sum of their respective Van de Waals radii minus 0.2 .ANG., then
these data points were excluded from the dataset. The process was
repeated for all relevant iota sub-groups to produce a series of
iota datasets for the heavy chain epitope.
[0122] For each of the 159 theta atoms comprising the light chain
epitope, the corresponding theta contact set was selected from the
IOTA database and from that, an appropriate iota sub-group was
selected e.g. carbonyl oxygen. The relative iota coordinates from
this sub-group were transposed relative to the reference frame of
the given theta atom of the heavy chain epitope. An iota dataset
for a given sub-group was thus accumulated over the whole light
chain epitope. In cases where the location of a given iota data
point intersected with an atom of the light chain, closer than the
sum of their respective Van de Waals radii minus 0.2 .ANG., then
these data points were excluded from the dataset. The process was
repeated for all relevant iota sub-groups to produce a series of
iota datasets for the light chain epitope.
Inspection of Iota Density Maps for Intersection with Heavy-Light
Chain Atoms
[0123] The whole process was automatically performed with an
internal customised Rosetta python library script tailored for
mutable positions identification, single point mutant generation,
low-energy rotamer state enumeration, quantitative IOTA score
computation, VH-VL chains binding energy estimation, and point
mutants prioritisation.
[0124] Two scoring methods were used for mutants ranking:
1. .DELTA.IOTAScore
[0125] Aforementioned IOTA density maps generated were used to
compute the spatial intersection values between each heavy atom of
residue at each mutable position and the density critical points in
the corresponding type of maps nearby. IOTAScore is the sum of the
volumetric overlaps between the heavy atoms of one residue with the
maximum of IOTA densities with the corresponding type definitions,
which reflects the degree of intersection between individual Fab X
atoms per residue and the corresponding iota density maps.
IOTAScore is negative numerically, where lower values imply more
intersection. .DELTA.IOTAScore is the change of IOTAScores between
the mutant residue and the wildtype one; similarly, the more
negative the .DELTA.IOTAScore value the greater the implication
that the mutant is more favoured than the wildtype one.
2. Rosetta .DELTA..DELTA.G score
[0126] The Rosetta energy function is a linear combination of terms
that model interaction forces between atoms, solvation effects, and
torsion energies. More specifically, Score12, the default full atom
energy function in Rosetta is composed of a Lennard-Jones term, an
implicit solvation term, an orientation-dependent hydrogen bond
term, sidechain and backbone torsion potentials derived from the
PDB, a short-ranged knowledge-based electrostatic term, and
reference energies for each of the 20 amino acids that model the
unfolded state. The binding strength between two binding partners,
or .DELTA.G, can be computed by subtracting the Rosetta scores of
the individual partners alone with that of the complex structure
formed by the two partners. Lower .DELTA.G implies stronger
binding. .DELTA..DELTA.G is the change of .DELTA.G between the
mutant complex and the wildtype one; the more negative the
.DELTA..DELTA.G value the greater the implication that the mutant
binding affinity is higher than the wildtype one.
[0127] FIG. 14 illustrates the workflow for in silico predicting
point mutation at the VH-VL interface of the Fab X structure.
[0128] In step S101, all residues on the heavy chain with at least
one heavy atom within 8 .ANG. of any light chain heavy atoms were
identified as mutable positions. Similarly, all the residues on
light chain with at least one heavy atom within 8 .ANG. of any
heavy chain heavy atoms were identified as mutable positions.
[0129] In step S102, for the wildtype Fab X crystal structure, the
residue-wise IOTAScores and binding energy .DELTA.G are computed,
respectively. In step S102.1, the IOTAScore for the wildtype
residue on the current mutable position with the corresponding IOTA
density maps nearby is computed, termed as (IOTAScore.sub.wt,
Position); in step S102.2, the binding energy of wildtype Fab X VH
and VL chains is computed with Rosetta score12 function, termed as
.DELTA.G.sub.wt.
[0130] In step S103, the wildtype residue on the current mutable
position identified in step S101 are replaced (mutated) by the
other amino acid types. Out of the 20 natural amino acid types,
proline and cysteine are excluded from mutation. All the other 18
types (alanine, arginine, asparagine, aspartic acid, glutamic acid,
glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, serine, threonine, tryptophan, tyrosine,
valine) except the wildtype itself are mutated on each mutable
position one by one.
[0131] In step S104, for each mutated residue type at each mutable
position, the top 100 lowest-energy (in terms of Rosetta scoring
function) rotamer states are generated using Rosetta. The other
high energy rotamer states are discarded.
[0132] In step S105, for each rotamer state of mutant residue
generated in S104, the IOTAScore is computed in the same way of
step S102, termed as (IOTAScore.sub.mutant, Position.sub.j,
Type.sub.k, Rotamer.sub.i).
[0133] In step S106, the .DELTA.IOTAScore for the current
combination of rotamer state, mutant residue type, and mutable
position is computed by subtracting (IOTAScore.sub.wt, Position)
with (IOTAScore.sub.mutant, Position.sub.j, Type.sub.k,
Rotamer.sub.i), which is termed as (.DELTA.IOTAScore,
Position.sub.j, Type.sub.k, Rotamer.sub.i). Steps S105 and S106
were repeated to compute all of the .DELTA.IOTAScores for each
rotamer states for the current mutant residue type and mutable
position.
[0134] In step S107, the optimal rotamer state of the current
mutant residue type and mutable position is determined with the
lowest .DELTA.IOTAScore value, as shown in step S107.1. The binding
energy of the mutant with the optimal rotamer state is computed in
step S107.2 in the same way as step S102.2, termed as
(.DELTA.G.sub.mutant, Position.sub.j, Type.sub.k). In step S107.3,
the change of binding energies .DELTA..DELTA.G between mutant and
wildtype is calculated by subtraction of .DELTA.G.sub.wt with
.DELTA.G.sub.mutant. After the optimal rotamer state is
prioritised, steps S103 to S107 were repeated for the next mutant
amino acid type at the current mutable position.
[0135] In step S108, for the current mutable position, only the
candidate mutants satisfying the criteria of both
.DELTA.IOTAScore<0 and .DELTA..DELTA.G<0 are kept for later
ranking. The rest are discarded. Steps S102 to S108 were repeated
to go through all the mutable positions and generate all candidate
mutants satisfying the same criteria.
[0136] In step S109, all the candidate mutant structures were
outputed for later visualisation analysis. The final list of
candidate mutants were sorted and ranked by the lowest
.DELTA.IOTAScores.
[0137] The running command and parameters used were as below:
[0138] For light chain mutations prediction, the command was:
"python multiRotamersFabInterfaceIOTAScan.py --pdb FabX.pdb
--only_chains L --region all --useIOTA --IOTAtype 167
--output_mutant"
[0139] For heavy chain mutations prediction, the command was:
"python multiRotamersFabInterfaceIOTAScan.py --pdb FabX.pdb
--only_chains H --region all --useIOTA --IOTAtype 167
--output_mutant"
[0140] Extra Rosetta relevant parameters were initialized by adding
the following code to the
"multiRotamersFabInterfaceIOTAScan.py":
"init(extra_options="-ex1 -ex2 -score:weights score12
-no_his_his_pairE -constant_seed -edensity:mapreso 3.0 -correct
-mute all"
DNA Manipulations and General Methods
[0141] E. coli strain INV.alpha.F (Invitrogen) was used for
transformation and routine culture growth. DNA restriction and
modification enzymes were obtained from Roche Diagnostics Ltd. and
New England Biolabs. Plasmid preparations were performed using Maxi
Plasmid purification kits (Qiagen, catalogue No. 12165). DNA
sequencing reactions were performed using ABI Prism Big Dye
terminator sequencing kit (catalogue No. 4304149) and run on an ABI
3100 automated sequencer (Applied Biosystems). Data was analysed
using the program Auto Assembler (Applied Biosystems).
Oligonucleotides were obtained from Invitrogen. The concentration
of IgG was determined by IgG assembly ELISA.
Thermostability Improvement of Fab X Through Affinity Maturation of
the Heavy-Light Chain Interface
[0142] The wild type Fab fragment of Fab X and mutant variants were
prepared as follows: oligonucleotide primer sequences were designed
and constructed in order to introduce single point mutations in
both the heavy and light chain variable regions as per residues and
positions determined in the above short list. Each mutated light
chain was separately sub-cloned into the UCB Celltech human light
chain expression vector pKH10.1, which contained DNA encoding the
human C-kappa constant region (Km3 allotype). Each mutated heavy
chain variable region sequence was separately sub-cloned into the
UCB Celltech expression vector pVhg1Fab6His which contained DNA
encoding human heavy chain gamma-1 constant region, CH1. Heavy and
light chain encoding plasmids were co-transfected into HEK293 cells
using the 293Fectin.TM. procedure according to the manufacturer's
instructions (InVitrogen. Catalogue No. 12347-019). IgG1 Fab
antibody levels secreted into the culture supernatants after 10 to
12 days culture were assessed by ELISA and binding kinetics
assessed by surface plasmon resonance (see below).
[0143] Mutants showing improved thermostability were then prepared
and tested in combination as double, or triple mutations as
above.
Surface Plasmon Resonance (SPR)
[0144] All SPR experiments were carried out on a BIAcore T200 (GE
Healthcare). Affinipure F(ab').sub.2 Fragment goat anti-human IgG,
F(ab').sub.2 fragment specific (Jackson ImmunoResearch) was
immobilised on a CM5 Sensor Chip via amine coupling chemistry to a
capture level of .apprxeq.5000 response units (RUs). HBS-EP buffer
(10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20,
GE Healthcare) was used as the running buffer with a flow rate of
10 .mu.L/min. A 10 .mu.L injection of Fab X at 0.75 .mu.g/mL was
used for capture by the immobilised anti-human IgG-F(ab').sub.2.
Antigen was titrated over the captured Fab X at various
concentrations (50 nM to 6.25 nM) at a flow rate of 30 .mu.L/min.
The surface was regenerated by 2.times.10 .mu.L injection of 50 mM
HCl, followed by a 5 .mu.L injection of 5 mM NaOH at a flowrate of
10 .mu.L/min. Background subtraction binding curves were analysed
using the T200evaluation software (version 1.0) following standard
procedures. Kinetic parameters were determined from the fitting
algorithm.
Thermostability Assay
[0145] Thermofluor assay was performed to assess the thermal
stabilities of purified molecules. Purified proteins (0.1 mg/ml)
were mixed with SYPRO.RTM. Orange dye (Invitrogen), and the mixture
dispensed in quadruplicate into a 384 PCR optical well plate.
Samples were analysed on a 7900HT Fast Real-Time PCR System
(Agilent Technologies) over a temperature range from 20.degree. C.
to 99.degree. C., with a ramp rate of 1.1.degree. C./min.
Fluorescence intensity changes per well were plotted against
temperature and the inflection points of the resulting slopes were
used to generate the T.sub.m.
Results
[0146] Intersection of Iota Density Maps with Heavy and Light Chain
Atoms of the Interface
[0147] The automated method using a Rosetta scan produced a table
of mutations ranked by IOTA score (Table 6).
Effects of Iota Designed Mutations on Fab X on Thermostability
[0148] Six iota-designed single point mutations in Fab X, H-T71R,
H-T71K, H-T71N, H-T71H, L-S107E and L-T109I, showed small
improvements in thermostability ranging from 0.5.degree. C. to
2.9.degree. C. over wild-type (Table 7). FIGS. 15, 16 and 17
provide computer generated visualisations depicting the effects of
these single point mutations. In particular, these Figures show
that whilst H-T71, L-S107 and L-T109 have no density intersection,
H-R71 intersects with the amide density, L-E107 intersects with the
carboxylate density and L4109 intersects with the methyl density.
Combinations of these mutations in pairs resulted in a synergistic
improvement in thermostability; with triple combinations producing
further step improvements in thermostability (Table 8).
[0149] The combination of H-T71R, L-S107E and L-T109I, mutations
produced the largest improvement in thermostability (Table 8) to
give a Tm of 81.2.degree. C., some 5.8.degree. C. better than the
original Fab X. This combination of the three mutations is depicted
in FIG. 18. An important finding was that there was no significant
loss on the binding to its antigen.
TABLE-US-00008 TABLE 6 Proposed mutations generated by the Rosetta
scan method and their ranking in order of IOTA score Rank (by Heavy
.DELTA.IOTA Light .DELTA.IOTA IOTAScore) chain .DELTA..DELTA.G
Score chain .DELTA..DELTA.G Score 1 T71R -0.37 -56.27 T109H -1.49
-92.37 2 V109R -0.04 -50.58 T109K -0.01 -67.33 3 V109K -0.51 -40.67
T109I -1.69 -45.17 4 T71H -1.06 -38.94 I106L -2.11 -42.11 5 T71K
-0.08 -36.09 T109L -1.58 -37.44 6 T71W -0.67 -25.95 I106H -3.5
-31.77 7 T71Y -0.67 -25.64 S107E -0.62 -18.71 8 T71N -0.03 -24.89
A53Y -0.93 -17.68 9 D119W -0.89 -23.58 A53F -1.13 -15.64 10 T71Q
-0.38 -23.28 I106N -0.47 -13.57 11 V109I -0.56 -22.64 12 V109H -0.4
-21.73
TABLE-US-00009 TABLE 7 Thermostability of mutations compared with
wild-type thermostability of 75.7.degree. C. Heavy .DELTA.IOTA
Light .DELTA.IOTA Rank chain .DELTA..DELTA.G Score Tm .degree. C.
chain .DELTA..DELTA.G Score Tm .degree. C. 1 T71R -0.37 -56.27 78.6
T109H -1.49 -92.37 75.2 2 V109R -0.04 -50.58 75.6 T109K -0.01
-67.33 ND 3 V109K -0.51 -40.67 75.6 T109I -1.69 -45.17 79.2 4 T71H
-1.06 -38.94 76.6 I106L -2.11 -42.11 74.5 5 T71K -0.08 -36.09 78.6
T109L -1.58 -37.44 77.6 6 T71W -0.67 -25.95 75.6 I106H -3.5 -31.77
72.2 7 T71Y -0.67 -25.64 75.3 S107E -0.62 -18.71 77.3 8 T71N -0.03
-24.89 76.2 A53Y -0.93 -17.68 68.8 9 D119W -0.89 -23.58 73.7 A53F
-1.13 -15.64 67.2 10 T71Q -0.38 -23.28 75.8 I106N -0.47 -13.57 71.8
11 V109I -0.56 -22.64 ND 12 V109H -0.4 -21.73 75.1 ND = Not
Determined
TABLE-US-00010 TABLE 8 Thermostability and affinity of combinations
of mutations. Rosetta Rosetta ddG dE Tm Combination (VH/VL) (Fv)
(.degree. C.) Tm SD KD (nM) H-T71R + L-S107E + -3.72 -1.73 81.2 0.3
ND L-T109I H-T71R + -2.3 -1.17 80.1 0 0.88 L-T109I H-T71R + L-S107E
-2.13 -0.93 80.1 0 3.90 H-T71K + L-S107E -0.3 0.76 79.4 0.6 3.50
H-T71K + L-S107E + -4.11 -1.41 79.3 0.3 ND L-T109L H-T71K + -1.96
-0.98 79.1 0.3 1.39 L-T109I H-T71R + -2.77 -0.95 78.0 0.6 0.96
L-T109L H-T71K + -2.2 -0.7 77.7 0.1 1.59 L-T109L H-T71R -0.37 78.0
0.3 0.80 H-T71K -0.08 77.9 0.3 1.27 L-S107E -0.62 77.3 0.5 5.52
L-T109I -1.69 77.9 0.3 1.47 L-T109L -1.58 76.9 0.5 1.73 WT 75.4 0.2
1.35 ND = Not Determined
* * * * *