US20160125124A1 - Obtaining an Improved Therapeutic Ligand - Google Patents

Obtaining an Improved Therapeutic Ligand Download PDF

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US20160125124A1
US20160125124A1 US14/897,459 US201414897459A US2016125124A1 US 20160125124 A1 US20160125124 A1 US 20160125124A1 US 201414897459 A US201414897459 A US 201414897459A US 2016125124 A1 US2016125124 A1 US 2016125124A1
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atom
theta
iota
atoms
ligand
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Jiye Shi
Terence Seward Baker
Alastair David Griffiths Lawson
Xiaofeng Liu
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UCB Biopharma SRL
UCB Celltech Ltd
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • G06F19/12—
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/50—Molecular design, e.g. of drugs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/90—Programming languages; Computing architectures; Database systems; Data warehousing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • 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.
  • Therapeutic molecules 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 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.
  • 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.
  • 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.
  • Biolistics 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 D low nanomolar or less). They may also be other types of protein molecules such as hormones, cytokines, growth factors or soluble receptors.
  • 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.
  • Laskowski R A, Thornton J M, Humblet C & Singh J 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.
  • 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.
  • 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.
  • an average “density” is used to predict the favourable interaction region.
  • 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.
  • fragment type can be shared by different atoms on the same residue and by atoms on different residues.
  • 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.
  • 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.
  • 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:
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the spatial data is parsed against the contextual data before the plotting step.
  • 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.
  • 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.
  • the contact data predominantly represents long-range, across-fold protein data.
  • 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:
  • 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;
  • non-bonding intra-molecular contact is defined as an instance where the following conditions are satisfied:
  • 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
  • t is a predetermined threshold distance of typically 2.5 angstroms and preferably 0.8 angstroms
  • 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.
  • 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:
  • 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;
  • non-bonding intra-molecular contact is defined as an instance where the following condition is satisfied:
  • 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
  • t is a predetermined threshold distance of typically 2.5 angstroms and preferably 0.8 angstroms
  • 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.
  • 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;
  • FIGS. 2-6 illustrate a process of coordinate normalization for an atom in a non-bonding intra-molecular or inter-molecular contact
  • 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;
  • FIG. 8 is a computer generated visualization depicting a light chain threonine 30 to arginine 30 mutation
  • FIG. 9 is a computer generated visualization depicting a light chain arginine 54 to serine 54 mutation
  • FIG. 10 is a computer generated visualization depicting a light chain serine 56 to isoleucine 56 mutation
  • FIG. 11 is a computer generated visualization depicting a light chain serine 60 to aspartate 60 mutation
  • FIG. 12 is a computer generated visualization depicting a light chain threonine 72 to arginine 72 mutation
  • 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;
  • 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
  • FIG. 15 is a computer generated visualisation depicting a heavy chain threonine 71 to arginine 71 mutation
  • FIG. 16 is a computer generated visualisation depicting a light chain serine 107 to glutamic acid 107 mutation
  • FIG. 17 is a computer generated visualisation depicting a light chain threonine 109 to isoleucine 109 mutation
  • FIG. 18 is a computer generated visualisation depicting the combination of three mutations in Fab X resulting in a Tm of 81.2° C.
  • the Worldwide Protein Data Bank 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.
  • a structural database of biological macromolecules e.g. the wwPDB
  • wwPDB biological macromolecules
  • non-bonding pairs of contact 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).
  • macromolecule e.g. protein
  • iota iota atoms
  • 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.
  • the non-bonding intra-molecular or inter-molecular contacts are defined as an instance where the following condition is satisfied: 1) s ⁇ Rw ⁇ 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.
  • 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.
  • any reference to “contact” is understood to mean “non-bonding intra-molecular contact or inter-molecular contact” according to the definition given above.
  • Databases such as the wwPDB may have information about proteins that are very similar to each other and/or which have related structures.
  • the database is parsed in order to avoid/reduce bias caused by such similarities/relationships.
  • 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.
  • 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.
  • 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.
  • the secondary database comprises information from more than 15 million contact atom pairs, extracted from around 20 000 non-homologous proteins.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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-tran
  • 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.
  • DNA deoxyribonucleic acid polymer
  • RNA ribonucleic acid polymer
  • RNA ribonucleic acid polymer
  • 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 .
  • 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 .
  • the third and fourth atoms are chosen uniquely for each specified theta atom type.
  • the fifth and sixth atoms are also chosen uniquely.
  • 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.
  • theta atom 1 is a C beta carbon atom
  • the third atom 3 and the fourth atom 4 are the C alpha carbon and the backbone carbonyl carbon respectively.
  • the atom closest to the backbone nitrogen atom is selected.
  • 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 .
  • 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).
  • a primary database such as the wwPDB.
  • the atom group coordinates are translated so that the theta atom 1 lies at the zero coordinate ( FIG. 3 ).
  • each of the 167 first atom types can be superimposed for that type and the secondary database sub-divided accordingly.
  • 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.
  • 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.
  • the unique iota atom types e.g. the 167 covalent atom types mentioned above
  • predetermined groups of related iota atom types e.g. the 167 covalent atom types mentioned above
  • 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.
  • 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).
  • 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) and ⁇ (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.
  • 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.
  • 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.
  • 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.
  • the method is used to assist with NCE or biologic drug design.
  • 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.
  • the method can suggest atom types and positions for elaboration of the chemistry to obtain a ligand with better binding characteristics.
  • 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.
  • 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.
  • step S 1 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.
  • each atom in the target list is identified as a particular theta atom type.
  • step S 3 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.
  • 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.
  • the information extracted in step S 3 is collected in a secondary database that comprises one and only one theta contact set for each of the theta atom types.
  • 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.
  • 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.
  • step S 4 for each theta atom identified in the target list in step S 2 , 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 S 3 is superimposed in or around the target binding site.
  • 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.
  • step S 5 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.
  • 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.
  • 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.
  • step S 7 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.
  • steps S 7 and S 6 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.
  • the modified candidate ligand is output to an output memory device for storage or transmission and/or to a display for visualization.
  • the type of a given theta atom is identified uniquely in step S 2 such that there is no intersection between the group of contacts for which information is extracted in step S 3 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).
  • step S 5 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.
  • the comparison step S 6 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.
  • steps S 2 -S 7 are performed for a plurality of different atoms in the binding site.
  • 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.
  • 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.
  • the analysis is a cluster analysis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • step S 5 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 .
  • step S 6 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • step S 6 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.
  • 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.
  • a list is then output of the residue replacements that are identified as producing a greater intersection than atoms of the original residue.
  • the candidate ligand is then mutated to produce a modified, single-residue-mutated ligand that incorporates the residue replacement.
  • 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.
  • lists of residue replacements may be produced that satisfy providing a greater intersection than atoms of the original residue (have a ⁇ IOTAscore of less than zero).
  • the lists may additionally be ranked based on other criteria. For example, lists may also be filtered based on ⁇ G scores (see below). Residue replacements with ⁇ 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 ⁇ IOTAscore of less than zero (a negative ⁇ IOTAscore), and a ⁇ G of less than zero (a negative ⁇ G). This is illustrated in Example 2 below.
  • 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.
  • a plurality of modifications to the candidate ligand are identified.
  • 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 “ ⁇ 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.
  • 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 ⁇ 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.
  • 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.
  • the method of identifying a modification to a candidate ligand is a computer-implemented method.
  • any one or more of the steps S 1 -S 7 is/are performed on a computer.
  • all of the steps S 1 -S 7 is/are performed on a computer.
  • any one or more of the steps S 101 -S 109 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 S 101 -S 109 may be performed on a computer. In one embodiment, all of the steps S 101 -S 109 are automated. All of the steps S 101 -S 109 may be performed on a computer.
  • 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.
  • a method of manufacturing a therapeutic ligand comprises designing a new ligand or modifying an existing ligand according to one or more of the embodiments described above.
  • a secondary database of over 11 million intra-molecular atomic contact data was extracted from over 20000 non-homologous protein structures where resolution was ⁇ 2 ⁇ .
  • Contacts were defined as any two atoms on opposing sides of a protein fold separated by a distance of 1 ⁇ +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.
  • a consistent convention was employed to defined 3 rd and 4 th atoms.
  • 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).
  • 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.
  • 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 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 theta AA.
  • Cz The Theta-superimposed z coordinate for the backbone Carbon atom of 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 theta AA.
  • CsphP The Theta-superimposed spherical polar longitude angle for the backbone Carbon of theta AA.
  • CcylR The Theta-superimposed cylindrical polar distance for the backbone Carbon of 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 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 theta AA.
  • Nz The Theta-superimposed z coordinate for the backbone Nitrogen atom of theta AA.
  • NsphR The Theta-superimposed spherical polar distance for the backbone Nitrogen of theta AA.
  • NsphT The Theta-superimposed spherical polar latitude angle for the backbone Nitrogen of theta AA.
  • NsphP The Theta-superimposed spherical polar longitude angle for the backbone Nitrogen of theta AA.
  • NcylR The Theta-superimposed cylindrical polar distance for the backbone Nitrogen of theta AA.
  • NcylT The Theta-superimposed cylindrical polar angle for the backbone Nitrogen of theta AA.
  • NcylZ The Theta-superimposed cylindrical polar z coordinate for the backbone Nitrogen of 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 theta AA.
  • Caz The Theta-superimposed z coordinate for the Alpha Carbon atom of 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 theta AA.
  • CacylT The Theta-superimposed cylindrical polar angle for the Alpha Carbon of theta AA.
  • CacylZ The Theta-superimposed cylindrical polar z coordinate for the Alpha Carbon of 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.
  • 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.
  • molecular graphic display e.g. ccp4
  • 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.
  • E. coli strain INV ⁇ 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.
  • 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 293FectinTM 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.
  • 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 ⁇ l/min.
  • Anti-Fab antibody at 50 ⁇ g/ml in 10 mM sodium acetate pH 5.0 buffer was injected for 60 sec at the same flow rate.
  • 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.
  • Wild type and mutated 496 Fabs were harvested from culture supernatants in the range 3 to 30 ⁇ g/ml and crude supernatants were diluted in running buffer into the range 0.5 to 2 ⁇ g/ml.
  • each antibody was first captured on the anti-Fab′ surface by injection at 10 ⁇ 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 ⁇ l/ml, to produce an association phase over 180 sec followed by a dissociation phase of 300 sec.
  • 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 d ) and association rate constants (k a ) were fitted to the data using Biaevaluation software (Biacore AB).
  • 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.
  • 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.
  • 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 ).
  • 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.
  • 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 ⁇ , 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.
  • 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 ⁇ , 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.
  • 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.
  • ⁇ IOTAScore is the change of IOTAScores between the mutant residue and the wildtype one; similarly, the more negative the ⁇ IOTAScore value the greater the implication that the mutant is more favoured than the wildtype one.
  • 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 ⁇ 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 ⁇ G implies stronger binding.
  • ⁇ G is the change of ⁇ G between the mutant complex and the wildtype one; the more negative the ⁇ G value the greater the implication that the mutant binding affinity is higher than the wildtype one.
  • FIG. 14 illustrates the workflow for in silico predicting point mutation at the VH-VL interface of the Fab X structure.
  • step S 101 all residues on the heavy chain with at least one heavy atom within 8 ⁇ 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 ⁇ of any heavy chain heavy atoms were identified as mutable positions.
  • step S 102 for the wildtype Fab X crystal structure, the residue-wise IOTAScores and binding energy ⁇ G are computed, respectively.
  • step S 102 . 1 the IOTAScore for the wildtype residue on the current mutable position with the corresponding IOTA density maps nearby is computed, termed as (IOTAScore wt , Position); in step S 102 . 2 , the binding energy of wildtype Fab X VH and VL chains is computed with Rosetta score12 function, termed as ⁇ G wt .
  • step S 103 the wildtype residue on the current mutable position identified in step S 101 are replaced (mutated) by the other 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
  • the wildtype itself are mutated on each mutable position one by one.
  • step S 104 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.
  • step S 105 for each rotamer state of mutant residue generated in S 104 , the IOTAScore is computed in the same way of step S 102 , termed as (IOTAScore mutant , Position j , Type k , Rotamer i ).
  • step S 106 the ⁇ IOTAScore for the current combination of rotamer state, mutant residue type, and mutable position is computed by subtracting (IOTAScore wt , Position) with (IOTAScore mutant , Position j , Type k , Rotamer i ), which is termed as ( ⁇ IOTAScore, Position j , Type k , Rotamer i ). Steps S 105 and S 106 were repeated to compute all of the ⁇ IOTAScores for each rotamer states for the current mutant residue type and mutable position.
  • step S 107 the optimal rotamer state of the current mutant residue type and mutable position is determined with the lowest ⁇ IOTAScore value, as shown in step S 107 . 1 .
  • the binding energy of the mutant with the optimal rotamer state is computed in step S 107 . 2 in the same way as step S 102 . 2 , termed as ( ⁇ G mutant , Position j , Type k ).
  • step S 107 . 3 the change of binding energies ⁇ G between mutant and wildtype is calculated by subtraction of ⁇ G wt with ⁇ G mutant .
  • steps S 103 to S 107 were repeated for the next mutant amino acid type at the current mutable position.
  • step S 108 for the current mutable position, only the candidate mutants satisfying the criteria of both ⁇ IOTAScore ⁇ 0 and ⁇ G ⁇ 0 are kept for later ranking. The rest are discarded. Steps S 102 to S 108 were repeated to go through all the mutable positions and generate all candidate mutants satisfying the same criteria.
  • step S 109 all the candidate mutant structures were outputed for later visualisation analysis.
  • the final list of candidate mutants were sorted and ranked by the lowest ⁇ IOTAScores.
  • E. coli strain INV ⁇ 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.
  • 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 293FectinTM 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).
  • a 10 ⁇ L injection of Fab X at 0.75 ⁇ g/mL was used for capture by the immobilised anti-human IgG-F(ab′) 2 .
  • Antigen was titrated over the captured Fab X at various concentrations (50 nM to 6.25 nM) at a flow rate of 30 ⁇ L/min.
  • the surface was regenerated by 2 ⁇ 10 ⁇ L injection of 50 mM HCl, followed by a 5 ⁇ L injection of 5 mM NaOH at a flowrate of 10 ⁇ 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.
  • Thermofluor assay was performed to assess the thermal stabilities of purified molecules.
  • Purified proteins 0.1 mg/ml
  • SYPRO® Orange dye Invitrogen
  • Samples were analysed on a 7900HT Fast Real-Time PCR System (Agilent Technologies) over a temperature range from 20° C. to 99° C., with a ramp rate of 1.1° 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 m .
  • the automated method using a Rosetta scan produced a table of mutations ranked by IOTA score (Table 6).
  • FIGS. 15, 16 and 17 provide computer generated visualisations depicting the effects of these single point mutations.
  • 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
  • L4109 intersects with the methyl density.

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