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US-12617820-B2 - Engineered coronavirus spike (s) protein and methods of use thereof

US12617820B2US 12617820 B2US12617820 B2US 12617820B2US-12617820-B2

Abstract

Provided herein are engineered Coronavirus S proteins, such as engineered SARS-CoV-2 S proteins. In some aspects, the engineered S proteins exhibit enhanced conformational stability and/or antigenicity. Methods are also provided for use of engineered proteins as diagnostics, in screening platforms and/or in vaccine compositions.

Inventors

  • Jason McLellan
  • Christy HJORTH
  • Ching-Lin Hsieh
  • Patrick Byrne
  • Nicole Johnson
  • Nianshuang Wang
  • Daniel Wrapp
  • Jennifer Maynard
  • Andrea CHASSE
  • Ilya FINKELSTEIN
  • Mohammad JAVANMARDI
  • Jeffrey SCHAUB
  • Hung-Che KUO
  • Chia-Wei Chou
  • Jory GOLDSMITH

Assignees

  • BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM
  • THE TRUSTEES OF DARTMOUTH COLLEGE

Dates

Publication Date
20260505
Application Date
20210528

Claims (20)

  1. 1 . An engineered protein, comprising an engineered coronavirus S protein ectodomain having at least 90% identity to: (a) positions 14-1208 of SEQ ID NO: 1 or 2; (b) positions 14-1160 of SEQ ID NO: 1 or 2; or (c) positions 319-1208 of SEQ ID NO: 1 or 2, said engineered protein comprising at least one mutation relative to the sequence of SEQ ID NO: 1 or 2, said at least one mutation comprising: (i) a substitution at a position corresponding to: T724, T752, T778, T961, I1013, H1058, S735, T859, I770, A1015, L727, S1021, Q901, S875, T912, H1088, L1141, V1040, L966, A766, T778, L938, V963, V911, N1108, V705, A893, N703, A672, A694, A1080, I1132, P862, T859, T547, N978, T961, S758, Q762, D1118, S659, S698, R1039, V722, A930, A903, Q913, S974, D979, P728, V951, V736, L858, S884, P807, S875, T791, A879, G799, A924, V826, A899, Q779, F817, L865, T866, A892, T912, A570, V963, T874, S1055, V729, A1022, L894, A713, L828, L822, A1056, Q965, S1003, A972, Q992, I980, A1078, V1133, T1120, I870, S1055, T1117, D1139, T1116, Y1138, I896, G885, Q901, F1103, P1112, G889, L1034, E819, S1055, I1081, N1135, Q1054, Q957, I1130, V1104, R1000, A944, T724, S730, G769, Q895, K921, L922, N978, A942, G946, S975, A890, or S1003; and/or (ii) a deletion corresponding to positions 829-851, 675-686, 673-684, 1161-1208, or 1142-1208; and/or (iii) a substitution of two amino acids for amino acid positions 673-686.
  2. 2 . The engineered protein of claim 1 , comprising an engineered disulfide bond comprising paired cysteine substitutions at positions corresponding to: S735C and T859C; I770C and A1015C; L727C and S1021C; V911C and N1108C; A672C and A694C; A1080C and I1132C; S659C and S698C; V722C and A930C; A903C and Q913C; S974C and D979C; P728C and V951C; V736C and L858C; S884C and A893C; P807C and S875C; T791C and A879C; G799C and A924C; A570C and V963C; T874C and S1055C; V729C and A1022C; L822C and A1056C; Q965C and S1003C; A972C and Q992C; I980C and Q992C; A1078C and V1133C; H1088C and T1120C; I870C and S1055C; T1117C and D1139C; T1116C and Y1138C; I896C and Q901C; G885C and Q901C; F1103C and P1112C; G889C and L1034C; E819C and S1055C; A972C and I980C; I1081C and N1135C; or E819C and Q1054C.
  3. 3 . The engineered protein of claim 1 , comprising a cavity filling substitution selected from: T724M, I1013F, H1058W, Q901M, S875F, H1088W, L1141F, V1040F, T778L, L938F, V963L, R1039F, V826L, A899F, Q779M, L894F, H1058F, H1058Y, V1040Y, H1088Y, V1104I, R1000Y, R1000W, A944F, T724I, A944Y, S730L, A890V, D1118F, or S1003V.
  4. 4 . The engineered protein of claim 1 , comprising a proline substitution selected from: F817P, L865P, T866P, A892P, A899P, T912P, A893P, Q895P, K921P, L922P, N978P, A942P, G946P, or S975P.
  5. 5 . The engineered protein of claim 1 , comprising proline substitutions at A892P; A942P; A899P; and F817P.
  6. 6 . The engineered protein of claim 1 , comprising an electrostatic interaction substitution selected from: T752K, T912R, L828K, L828R, S730R, T961D, A766E, P862E, T859K, Q957E, G769E, T778Q, A713S, or I1130Y.
  7. 7 . The engineered protein of claim 1 , further comprising K986P and V987P substitutions.
  8. 8 . The engineered protein of claim 1 , comprising a combination of at least one engineered disulfide bond, at least one cavity filling substitution, at least one proline substitution, and at least one electrostatic interaction substitution.
  9. 9 . The engineered protein of claim 1 , wherein the engineered coronavirus S protein ectodomain comprises a mutation that eliminates the furin cleavage site.
  10. 10 . The engineered protein of claim 1 , wherein the protein is fused or conjugated to a trimerization domain.
  11. 11 . The engineered protein of claim 10 , wherein the trimerization domain comprises a T4 fibritin trimerization domain.
  12. 12 . The engineered protein of claim 1 , wherein the protein is fused or conjugated to a transmembrane domain.
  13. 13 . The engineered protein of claim 12 , wherein the transmembrane domain comprises a SARS-COV-2 transmembrane domain.
  14. 14 . An engineered coronavirus trimer comprising at least one subunit comprising the engineered protein according to claim 1 .
  15. 15 . The engineered trimer of claim 14 , wherein the trimer comprises at least one engineered disulfide bond between subunits, wherein the at least one engineered disulfide bond between subunits is selected from: V705C and A893C; T547C and N978C; T961C and S758C; and/or T961C and Q762C.
  16. 16 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier; and an engineered protein of claim 1 .
  17. 17 . A nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence of an engineered protein of claim 1 .
  18. 18 . A method of preventing coronavirus infection in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 16 .
  19. 19 . A composition comprising an engineered protein of claim 1 bound to an antibody.
  20. 20 . The engineered protein of claim 1 , further comprising proline substitutions at F817P; A892P; A899P; A942P; K986P; and V987P.

Description

REFERENCE TO RELATED APPLICATIONS The present application is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2021/034713, filed May 28, 2021, which claims the priority benefit of U.S. provisional application No. 63/032,502, filed May 29, 2020, the entire contents of each of which is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant no. R01 AI127521 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING The instant application contains a Sequence Listing, which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 28, 2022, is named UTSBP1251US_ST25.txt and is 41,774 bytes in size. BACKGROUND 1. Field The present disclosure relates generally to the fields of medicine, virology, immunology and protein engineering. More particular, the disclosure relates to engineered Coronavirus S proteins and the use thereof in drug design and vaccine formulation. 2. Description of Related Art An outbreak of COVID-19, the disease caused by infection of the coronavirus SARS-CoV-2, began in December 2019 in China has resulted in millions of infections and more than 100 thousand deaths. Like the virus that caused the SARS outbreak several years prior, SARS-CoV, the SARS-CoV-2 virus use their spike proteins to bind host cellular receptor angiotensin-converting enzyme 2 (ACE2). The interaction between the receptor binding domain (RBD) of the spike glycoprotein and the full-length human ACE2 protein. Although the sequence and structure of the SARS-CoV-2 spike protein is a known (see, e.g., Wrapp et al. 2020) there remains a need for stabilized S proteins that could be used for identifying drug candidates and for stimulating an effective immune response to the S protein. SUMMARY In some embodiments, the present disclosure provides engineered proteins, comprising an engineered coronavirus S protein ectodomain having at least 90% identity to: (a) positions 14-1208 of SEQ ID NO: 1 or 2; (b) positions 14-1160 of SEQ ID NO: 1 or 2; or (c) positions 319-1208 of SEQ ID NO: 1 or 2, said engineered protein comprising at least one mutation relative to the sequence of SEQ ID NO: 1 or 2, said at least one mutation comprising: (1) an engineered disulfide bond;(2) a cavity filling substitution;(3) a substitution that provides an electrostatic or polar interaction; and/or(4) a proline substitution. In further aspects, an engineering protein comprises at least one engineered disulfide bond and at least one cavity filling substitution. In still further aspects, an engineering protein comprises at least one engineered disulfide bond and at least one substitution that provides an electrostatic or polar interaction. In still further aspects, an engineering protein comprises at least one engineered disulfide bond and at least one proline substitution. In additional aspects, an engineering protein comprises at least one cavity filling substitution and at least one substitution that provides an electrostatic or polar interaction. In still further aspects, an engineering protein comprises at least one cavity filling substitution and at least one proline substitution. In still further aspects, an engineering protein comprises at least one substitution that provides an electrostatic or polar interaction and at least one proline substitution. In some embodiments, the present disclosure provides engineered proteins comprising an engineered coronavirus S protein ectodomain that comprises a sequence at least 90% identical to: (a) positions 14-1208 of SEQ ID NO: 1 or 2; (b) positions 14-1160 of SEQ ID NO: 1 or 2; or (c) positions 319-1208 of SEQ ID NO: 1 or 2; wherein the engineered protein comprises the following substitutions relative to the sequence of SEQ ID NO: 1 or 2: F817P, A892P, A899P, A942P, K986P, and V987P. In further aspects, the engineered coronavirus S protein has at least about 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity to: (a) positions 14-1208 of SEQ ID NO: 1 or 2; (b) positions 14-1160 of SEQ ID NO: 1 or 2; or (c) positions 319-1208 of SEQ ID NO: 1 or 2. In further embodiments, the present disclosure provides engineered proteins, comprising an engineered coronavirus S protein ectodomain having at least 90% identity to: (a) positions 14-1208 of SEQ ID NO: 1 or 2; (b) positions 14-1160 of SEQ ID NO: 1 or 2; or (c) positions 319-1208 of SEQ ID NO: 1 or 2, said at least one mutation comprising: (i) a substitution at a position corresponding to: T724, T752, T778, T961, I1013, H1058, S735, T859, I770, A1015, L727, S1021, Q901, S875, T912, H1088, L1141, V1040, L966, A766, T778, L938, V963, V911, N1108, V705, A893, N703, A672, A694, A1080, I1132, P862, T547, N978, S758, Q762, D1118, S659, S698, R1039, V722, A930, A903, Q913, S974, D979, P728, V951, V736, L858, S884, P