Methods to identify macromolecule binding and aggregation prone regions in proteins and uses therefor

Patent metadata

Field	Value
Publication number	US-9922164-B2
Application number	US-201615249161-A
Country	US
Kind code	B2
Filing date	Aug 26, 2016
Priority date	Jun 20, 2008
Publication date	Mar 20, 2018
Grant date	Mar 20, 2018

Abstract

The present invention provides methods and computational tools based, at least in part, on computer simulations that identify macromolecule binding regions and aggregation prone regions of a protein. Substitutions may then be made in these aggregation prone regions to engineer proteins with enhanced stability and/or a reduced propensity for aggregation. Similarly, substitutions may then be made in these macromolecule binding regions to engineer proteins with altered binding affinity for the macromolecule.

First claim

We claim:

1. An in vitro method of making a protein variant which exhibits a reduced propensity for aggregation and/or an altered binding affinity for a macromolecule, comprising
replacing or deleting at least one amino acid residue within an aggregation prone region and/or a molecular binding region in the protein by site directed mutagenesis, wherein the aggregation prone region and/or a molecular binding region is identified using Spatial-Aggregation-Propensity (SAP) scores, wherein the SAP for a particular atom in the protein is calculated by:
(a) providing a computer-readable structural model of the protein;
(b) mapping, onto the structural model of the protein the SAP, wherein the SAP is calculated for a particular atom by:
(i) identifying one or more atoms or amino acid residues in a structural model representing the protein, wherein the one or more atoms are within a defined spatial region centered on or within 30 Å of the particular atom or the one or more amino acid residues have at least one atom within a defined spatial region centered on or within 30 Å of the particular atom;
(ii) calculating, for each of the one or more atoms in the defined spatial region, a ratio of the solvent accessible area (SAA) of each of the one or more atoms to the SAA of a corresponding atom in an identical residue which is fully exposed;
(iii) multiplying each ratio by the atom hydrophobicity of the one or more atoms; and,
(iv) summing the products of step (iii), wherein the sum of the products of step (iii) consists of contributions from the one or more atoms within the defined spatial region;

whereby the sum is the SAP for the particular atom; and
wherein, optionally, the SAP for the particular atom is calculated by conducting a computational molecular dynamics simulation on the computer-readable structural model of the protein prior to step (i) and repeating steps (i)-(iv), each time conducting a further molecular dynamics simulation at a plurality of time steps, thereby producing multiple sums as in step (iv), and calculating the average of the sums;
whereby the calculated average is the SAP for the particular atom; and
(c) identifying a region within the protein having a plurality of atoms having a SAP that exceeds a set SAP threshold;
wherein the aggregation prone region and/or the molecular binding region comprises the amino acids comprising said plurality of atoms, and
wherein, if the amino acid residue is replaced and the region is the aggregation prone region, it is replaced with an amino acid residue which is more hydrophilic, such that the propensity for aggregation of the variant is reduced.

2. The method of claim 1 , wherein the defined spatial region is a sphere having a radius of between 1-30 Å.

3. The method of claim 2 , wherein the radius is 5 Å.

4. The method of claim 1 , wherein the molecular dynamics simulation is performed using a simulation package selected from the group consisting of ABINIT, AMBER, Ascalaph, CASTEP, CPMD, CHARMM, DL_POLY, FIREBALL, GROMACS, GROMOS, LAMMPS, MDynaMix, MOLDY, MOSCITO, NAMD, Newton-X, ProtoMol, PWscf, SIESTA, VASP, TINKER, YASARA, ORAC, and XMD.

5. The method of claim 1 , where the identifying comprises plotting the SAP values; calculating, for peaks in the plot, the area under the curve (AUC); and identifying one or more protein regions with a positive AUC, wherein the aggregation prone region and/or the molecular binding region comprises the identified protein regions.

6. The method of claim 1 , wherein at least two amino acid residues within the aggregation prone region and/or the molecular binding region are replaced.

7. The method of claim 1 , wherein at least one residue is replaced within more than one aggregation prone regions and/or more than one molecular binding regions within the protein.

8. The method of claim 1 , wherein the protein is selected from the group consisting of an antibody, a Fab fragment, a Fab′ fragment, an Fd fragment, an Fv fragment, an F(ab′) 2 fragment, and an Fc fragment.

Assignees

• Novartis Ag
• Massachusetts Inst Technology

Inventors

• Chennamsetty Naresh
• Helk Bernhard
• Trout Bernhardt
• Kayser Veysel
• Voynov Vladimir

Classifications

• A61P37/00

  Drugs for immunological or allergic disorders · CPC title

• G16C10/00

  Computational theoretical chemistry, i.e. ICT specially adapted for theoretical aspects of quantum chemistry, molecular mechanics, molecular dynamics or the like · CPC title

• G16B20/00

  ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• G16C99/00

  Subject matter not provided for in other groups of this subclass · CPC title