# US 2019/0169284 A1 · Solubility optimization of immunobinders > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0169284 A1 - Document ID: US20190169284A1 - Status: Published application - Kind: A1 - Application number: 16/267,460 - Filed: 2019-02-05 - Published: 2019-06-06 - Earliest claimed: 2008-06-25 - Assignees: Novartis AG - Applicants: Novartis AG - Inventors: Leonardo Borras; David Urech - Web page: https://patentgrep.com/patent/US20190169284A1 ## CPC - C07K 16/241: {Tumor Necrosis Factors} - A61P 17/00: Drugs for dermatological disorders - A61P 35/00: Antineoplastic agents - A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C07K 16/22: against growth factors {; against growth regulators} - C07K 2317/567: Framework region [FR] - C07K 2317/622: Single chain antibody (scFv) - C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## Abstract The invention provides methods of using sequence based analysis and rational strategies to improve the solubility of immunobinders, and in particular of single chain antibodies (scFvs). The invention provides methods of engineering immunobinders, and in particular scFvs, by performing one or more substitutions with hydrophilic residues identified by analysis of a database of selected, stable scFv sequences. The invention also provides immunobinders with optimized solubility prepared according to the engineering methods of the invention. ## Claims ### Claim 1 An immunobinder comprising one of the following solubility enhancing motifs in the heavy chain amino acid positions 12, 103 and 144 (AHo numbering): (a) Serine (S) at heavy chain amino acid position 12; Serine (S) at heavy chain amino acid position 103; and Threonine (T) at heavy chain amino acid position 144; or (b) Serine (S) at heavy chain amino acid position 12; Threonine (T) at heavy chain amino acid position 103; and Serine (S) at heavy chain amino acid position 144; or (c) Serine (S) at heavy chain amino acid position 12; Threonine (T) at heavy chain amino acid position 103; and Threonine (T) at heavy chain amino acid position 144. ### Claim 2 (depends on 1) The immunobinder of claim 1, further comprising: (a) Aspartic acid (D) at light chain amino acid position 31; (b) Glutamic acid (E) at light chain amino acid position 83; (c) Arginine (R) at heavy chain amino acid position 43; (d) Leucine (L) at heavy chain amino acid position 67; and/or (e) Alanine (A) at heavy chain amino acid position 78. ### Claim 3 A method of enhancing the solubility of an immunobinder in native state, the immunobinder comprising a heavy chain variable (VH) region, or fragment thereof, the method comprising: (a) selecting at least two amino acid positions within the VH region for mutation; and (b) mutating the at least two amino acid positions selected for mutation, wherein the at least two amino acid positions are selected from the group of heavy chain amino acid positions consisting of 12, 103 and 144 (according to AHo numbering convention) and the mutating comprises the substitution of the amino acid at the selected amino acid position against a hydrophilic amino acid. ### Claim 4 (depends on 3) The method of claim 3, wherein the hydrophilic amino acid is (a) Serine (S) at heavy chain amino acid position 12; (b) Serine (S) or Threonine (T) at heavy chain amino acid position 103; and/or (c) Serine (S) or Threonine (T) at heavy chain amino acid position 144. ### Claim 5 (depends on 3) The method of claim 3, wherein the amino acid at the amino acid position selected for mutation is a hydrophobic amino acid. ### Claim 6 (depends on 5) The method of claim 5, wherein the hydrophobic amino acid is leucine (L) or Valine (V). ### Claim 7 (depends on 3) The method of claim 3, wherein the amino acid selected for mutation of (a) the amino acid at heavy chain amino acid position 12 is Valine (V); (b) the amino acid at heavy chain amino acid position 103 is Valine (V); and (c) the amino acid at heavy chain amino acid position 144 is Leucine (L). ### Claim 8 (depends on 3) The method of claim 3, wherein thermal stability, refolding, expression yield, aggregation and/or binding activity of the immunobinder is not adversely affected by the mutating. ### Claim 9 (depends on 3) The method of claim 3, wherein the mutating results in at least a 2-fold increase in solubility. ### Claim 10 (depends on 3) The method of claim 3, wherein the mutating further comprises the step of introducing one or more mutations at an amino acid position (AHo numbering convention) selected from the group consisting of: (a) Aspartic acid (D) at light chain amino acid position 31; (b) Glutamic acid (E) at light chain amino acid position 83; (c) Arginine (R) at heavy chain amino acid position 43; (d) Leucine (L) at heavy chain amino acid position 67; and (e) Alanine (A) at heavy chain amino acid position 78. ### Claim 11 (depends on 3) An immunobinder prepared according to the method of claim 3. ### Claim 12 (depends on 1) The immunobinder of claim 1, which is an scFv antibody, a full-length immunoglobulin, a Fab fragment, a Dab or a Nanobody. ### Claim 13 (depends on 11) The immunobinder of claim 11, which is an scFv antibody, a full-length immunoglobulin, a Fab fragment, a Dab or a Nanobody. ### Claim 14 (depends on 1) A composition comprising the immunobinder of claim 1 and a pharmaceutically acceptable carrier. ### Claim 15 (depends on 11) A composition comprising the immunobinder of claim 11 and a pharmaceutically acceptable carrier.