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KR-20260066188-A - cd4 muteins and methods of using the same

KR20260066188AKR 20260066188 AKR20260066188 AKR 20260066188AKR-20260066188-A

Abstract

The present invention relates to a protein containing CD4 domain 1 and CD4 domain 2 (CD4 D1D2), wherein CD4 D1D2 contains one or more mutations compared to wild-type human CD4 D1D2, and to methods of using this protein to treat human immunodeficiency virus (HIV) infection in subjects.

Inventors

  • 파잔, 마이클
  • 가드너, 매튜
  • 페처, 이나
  • 앨퍼트, 마이클
  • 베일리, 찰스

Assignees

  • 더 스크립스 리서치 인스티튜트
  • 이뮨 인코포레이티드

Dates

Publication Date
20260512
Application Date
20190321
Priority Date
20180321

Claims (18)

  1. As a protein comprising human CD4 domain 1 and human domain 2 (CD4 D1D2) protein variants, The CD4 domain 1 (D1) comprises at least one permutation, and the CD4 domain 2 (D2) comprises at least one permutation, The above substitutions are each (a) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with a non-charged amino acid; (b) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; (c) Substitution of a non-charged amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; or (d) a substitution of glycine or an amino acid having a buried hydrophobic side chain in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 by a hydrophobic amino acid having a larger volume than the substituted amino acid; Each of the above substitutions is located at a position not identical to that of the wild-type human CD4 D1D2 represented by sequence number 1 within the non-human primate CD4 D1D2, and The above CD4 D1D2 variant is a protein comprising at least one substitution selected from the following group: (a) K1N, K2E, K2N, K2T, L5Y, L5I, L5E, L5W, L5V, L5F, L5T, K7E, K8E, T15N, T15E, T17N, T17E, S23N, S23T, S23K, S23Y, S23A, A55V, A55I, A55P, A55L, A55M, A55F, A55Y, A55W, A55T, I70E, I70L, I70V, K72S, K75E, K75Q, Q94E, L96V, L96Q, L96T, L96I, L96Y, Q110E, Q110H, L116F, L116W, V128I, V128L, R134G, R134T, K142R, K142G, K142S, V146I, V146L, V146F, V146W, N164D, N164H, N164R, N164E, K167T, K167R, K167L, V168I, V168L, and V176I; (b) K1N, K2E, K2N, K2T, L5Y, L5I, L5E, L5W, L5V, L5F, L5T, K7E, K8E, T15N, T15E, T17N, T17E, S23N, S23T, S23K, S23Y, S23A, A55V, A55I, A55P, A55L, A55M, A55F, A55Y, A55W, A55T, I70E, I70L, I70V, K72S, K75E, K75Q, Q94E, L96V, L96Q, L96T, L96I, L96Y, Q110E, Q110H, R134G, R134T, K142R, K142G, K142S, N164D, N164H, N164R, N164E, K167T, K167R, and K167L; (c) G6A, S23N, A55V, K72S, K75E, K75Q, Q94E, Q110E, V128L, R134G, V146I, N164D, K167T, and V168L; (d) K1N, K2N, K2T, K2E, K7E, K21N, K22T, K46T, R59Q, K75Q, K75E, R131T, R131Q, R134T, R134G, K142, K142G, K142S, K166N, K166Q, K166H, K166E, K167T, K171Q, K171S, K171N, and K171E; (e) A102V; or (f) G140A, K1N, K2N, K2E, K2T, G6A, K7E, K8E, T15N, T17E, K21N, K22T, S23N, S23T, A55V, I70E, K72S, K75E, Q94E, A102V, Q110E, L116F, L116W, V128L, R134G, G140A, V146I, V146L, V146F, V146W, N164D, K166N, K167T, V168L, and V176I.
  2. As a protein comprising human CD4 domain 1 and human domain 2 (CD4 D1D2) protein variants, The CD4 domain 1 (D1) comprises at least one permutation, and the CD4 domain 2 (D2) comprises at least one permutation, The above substitutions are each (a) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with a non-charged amino acid; (b) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; (c) Substitution of a non-charged amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; or (d) a substitution of glycine or an amino acid having a buried hydrophobic side chain in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 by a hydrophobic amino acid having a larger volume than the substituted amino acid; Each of the above substitutions is located at a position not identical to that of the wild-type human CD4 D1D2 represented by sequence number 1 within the non-human primate CD4 D1D2, and The above CD4 D1D2 protein variant is a protein comprising at least one of the K2E, G6A, K7E, K8E, T17N, T17E, S23N, S23T, A55V, A55I, I70E, K72S, K75E, K75Q, Q94E, Q110E, L116F, V128L, R134G, K142R, V146I, V146F, N164D, K167T, V168L, and V176I substitutions.
  3. As a protein comprising human CD4 domain 1 and human domain 2 (CD4 D1D2) protein variants, The CD4 domain 1 (D1) comprises at least one permutation, and the CD4 domain 2 (D2) comprises at least one permutation, The above substitutions are each (a) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with a non-charged amino acid; (b) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; (c) Substitution of a non-charged amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; or (d) a substitution of glycine or an amino acid having a buried hydrophobic side chain in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 by a hydrophobic amino acid having a larger volume than the substituted amino acid; Each of the above substitutions is in a position different from that of wild-type human CD4 D1D2 (sequence number 1) in non-human primate CD4 D1D2, and The above CD4 D1D2 protein variant is a protein comprising at least one of the G6A, S23N, A55V, K72S, K75E, K75Q, Q94E, Q110E, V128L, R134G, V146I, N164D, K167T, and V168L substitutions.
  4. In Paragraph 3, The above CD4 D1D2 protein variant is a protein comprising at least one of the K72S, K75E, K75Q, Q94E, Q110E, V128L, R134G, V146I, N164D, K167T, and V168L substitutions.
  5. As a protein comprising human CD4 domain 1 and human domain 2 (CD4 D1D2) protein variants, The CD4 domain 1 (D1) comprises at least one permutation, and the CD4 domain 2 (D2) comprises at least one permutation, The above substitutions are each (a) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with a non-charged amino acid; (b) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; (c) Substitution of a non-charged amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; or (d) a substitution of glycine or an amino acid having a buried hydrophobic side chain in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 by a hydrophobic amino acid having a larger volume than the substituted amino acid; Each of the above substitutions is located at a position not identical to that of the wild-type human CD4 D1D2 represented by sequence number 1 within the non-human primate CD4 D1D2, and The above CD4 D1D2 protein variant has the following substitutions: (1) A55V and K72S; (2) A55V and K75E; (3) A55V and K75Q; (4) A55V and Q94E; (5) A55V and Q110E; (6) A55V and L116F; (7) A55V and V128L; (8) A55V and R134G; (9) A55V and V146I; (10) A55V and N164D; (11) A55V and K167T; (12) A55V and V168L; (13) S23N and K72S; (14) S23N and K75E; (15) S23N and K75Q; (16) S23N and Q94E; (17) S23N and Q110E; (18) S23N and L116F; (19) S23N and V128I; (20) S23N and R134G; (21) S23N and V146I; (22) S23N and N164D; (23) S23N and K167T; or (24) Proteins including S23N and V168L.
  6. In any one of paragraphs 1 to 5, The above CD4 D1D2 protein variant is a protein comprising substitutions of G6A, S23N, A55V, V128L, R134G, and V168L.
  7. As a protein comprising human CD4 domain 1 and human CD4 domain 2 (CD4 D1D2) protein variants, At least one of the above substitutions is in the CD4 domain 1 (D1), and at least one of the above substitutions is in the CD4 domain 2 (D2), and Each of the above substitutions is (a) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with a non-charged amino acid; (b) Substitution of a basic amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; (c) Substitution of a non-charged amino acid in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 with an acidic amino acid; or (d) a substitution of glycine or an amino acid having a buried hydrophobic side chain in wild-type human CD4 D1D2 represented by SEQ ID NO. 1 by a hydrophobic amino acid having a larger volume than the substituted amino acid; Each of the above substitutions is located at a position not identical to that of the wild-type human CD4 D1D2 represented by sequence number 1 within the non-human primate CD4 D1D2, and The above CD4 D1D2 protein variant is a protein comprising SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, or SEQ ID NO. 44.
  8. In any one of paragraphs 1 through 7, The above CD4 D1D2 protein variant is a protein that does not contain the substitution of I76 by proline (I76P).
  9. In any one of paragraphs 1 through 7, The above CD4 D1D2 protein variant is a protein that does not contain the substitution of F98 by valine (F98V).
  10. In any one of paragraphs 1 through 7, It further includes a primate lentivirus envelope glycoprotein binding moiety, The binding moiety above is a protein comprising at least one sulfotyrosine.
  11. In Article 10, The binding moiety is a protein comprising the amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, or SEQ ID NO. 7.
  12. In any one of paragraphs 1 through 7, A protein containing an immunoglobulin Fc domain.
  13. Isolated nucleic acid comprising a nucleic acid sequence encoding a protein described in claims 1 to 7.
  14. An expression vector comprising the nucleic acid disclosed in claim 13.
  15. In Article 14, The above expression vector is an expression vector that is a viral vector.
  16. In Article 15, The above-mentioned viral vector is an expression vector, which is an adeno-associated virus (AAV) vector.
  17. A host cell comprising the expression vector described in claim 14.
  18. A pharmaceutical composition for treating HIV infection comprising a protein described in any one of claims 1 to 7.

Description

CD4 protein variants and methods of using the same Cross-reference regarding related applications This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 62/645,903 (filed March 21, 2018). The entire disclosure of the priority application is incorporated herein by reference in its entirety for all purposes. Statement of Government Support The present invention was made with government support under grant numbers AI091476, AI129868, and AI126623 awarded by the National Institutes of Health. The government holds certain rights to the present invention. The present invention generally relates to proteins containing CD4 and their use in HIV treatment. A polypeptide derived from CD4, which is a transmembrane receptor for human immunodeficiency virus (HIV) expressed on allowable host CD4+ T cells, can be utilized to inhibit HIV infection. By binding to viral particles, this CD4-derived polypeptide can mediate to block or "neutralize" HIV infection, thereby delaying the recognition of CD4 in allowable host cells. Soluble forms of CD4 (sCD4) were first proposed in the 1980s as an approach to suppressing HIV infection (Hussey, R. E. et al. (1988) NATURE, 331(6151): 78-81; Deen, K. C. et al. (1988) NATURE, 331(6151): 82-4; Smith, D. H. et al. (1987) SCIENCE, 238(4834): 1704-7; Fisher, R. A. et al. (1988) NATURE, 331(6151): 76-8; Watanabe, M. et al. (1989) NATURE, 337(6204): 267-70; Traunecker, A. et al. (1988) NATURE, 331(6151): 84-6). However, the short half-life of sCD4 in plasma has dampened enthusiasm for their use as recombinant protein therapeutic drugs for the treatment of HIV infection (Watanabe et al. (1989), supra; Schooley, R. T. et al. (1990) ANN. INTERN. MED., 112(4): 247-53). In addition, a significant portion of HIV strains isolated directly from patients (i.e., the first isolates) are resistant to neutralization by sCD4 (Ashkenazi, A. et al. (1991) Proc. Natl. Acad. Sci. USA, 88(16): 7056-60; Daar, E. S. et al. (1990) Proc. Natl. Acad. Sci. USA, 87(17): 6574-8; O'Brien, W. A. et al. (1992) J. Virol., 66(5): 3125-30). Previous improvements to the half-life of sCD4 were achieved by adding the immunoglobulin (Ig) fragment crystallizable (Fc) (Capon, DJ et al. (1989) NATURE, 337 (6207): 525-31; Chamow, SM et al. (1992) INT. J. CANCER SUPPL., 7: 69-72; Byrn, RA et al. (1990) NATURE, 344 (6267): 667-70). The form of sCD4 containing immunoglobulin Fc was named CD4-Ig. Despite these approaches, the half-life of human CD4-Ig still fell short of the half-life expected for conventional antibodies (Hodges, TL et al. (1991) ANTIMICROB. AGENTS CHEMOTHER., 35 (12): 2580-6; Kahn, JO et al. (1990) ANN. INTERN. MED., 112 (4): 254-61; Chamow, SM et al. (1994) BIOCONJUG. CHEM., 5 (2): 133-40). The suboptimal pharmacokinetic properties of CD4-Ig have limited its utility in therapeutic settings. Although development has been made to date, there is still a continuous need for new and effective treatments to treat and manage HIV and AIDS. The present invention is partially based on the discovery of proteins containing both human CD4 domain 1 and CD4 domain 2 (CD4 D1D2) having greater stability and/or activity than proteins containing naturally occurring human CD4 D1D2. In certain embodiments, the proteins exhibit higher agglutination temperatures and/or longer plasma half-lives when measured, for example, by dynamic light scattering (DLS) in thermal scan analysis, compared to proteins containing naturally occurring human CD4 D1D2. Additionally, it is considered that the proteins described herein may be suitable for the treatment of HIV in human subjects. In one embodiment, the present invention comprises a protein comprising a human CD4 domain 1 and domain 2 (CD4 D1D2) protein variant, wherein the CD4 D1D2 protein variant is at least 90% identical to wild-type human CD4 D1D2 (SEQ No. 1), and a. Substitution of basic amino acids in wild-type human CD4 D1D2 (Sequence No. 1) by non-charged amino acids; b. Substitution of basic amino acids in wild-type human CD4 D1D2 (Sequence No. 1) by acidic amino acids; c. Substitution of a non-charged amino acid in wild-type human CD4 D1D2 (Sequence No. 1) by an acidic amino acid; or d. comprising at least one substitution of glycine or an amino acid having a buried hydrophobic side chain in wild-type human CD4 D1D2 (SEQ No. 1) by a hydrophobic amino acid having a volume greater than that of the substituted amino acid; The substitution provides a protein at a position in non-human primate CD4 D1D2 that is not identical to that of wild-type human CD4 D1D2 (sequence number 1). In another aspect, the present invention comprises a protein comprising human CD4 domain 1 and domain 2 (CD4 D1D2) protein variants, wherein the CD4 D1D2 protein variant is at least 90% identical to wild-type human CD4 D1D2 (SEQ No. 1), and K1, K2, V4, L5, G6, K7, K8, D10, T11, V12, L14, T15, T17, A18, S19, Q20, K21, K22, S23, I24, Q25, H27, N30, N32, I34, K35, I36, L37, N39, G41