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US-12617833-B2 - Tethered interleukin-15 and interleukin-21

US12617833B2US 12617833 B2US12617833 B2US 12617833B2US-12617833-B2

Abstract

Disclosed are nucleic acids and polypeptides which provide the co-expression of interleukin (IL)-21 and IL-15 by a host cell, each interleukin being bound to the cell membrane by a cell membrane anchor moiety. Also disclosed are related recombinant expression vectors, host cells, populations of cells, pharmaceutical compositions, and methods of treating or preventing cancer.

Inventors

  • Christian S. Hinrichs
  • Benjamin Y. Jin

Assignees

  • THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES

Dates

Publication Date
20260505
Application Date
20190207

Claims (20)

  1. 1 . A nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of Formula I: S 1 —N 1 -L 1 -C 1 a -L 2 -S 2 —N 2 -L 3 -C 2 b (Formula I), wherein: each of S 1 and S 2 is, independently, a signal sequence; one of N 1 and N 2 is an interleukin (IL)-21 amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2, and one of N 1 and N 2 is an IL-15 amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3; L 2 is a cleavable linker sequence comprising the amino acid sequence of GDVEX 1 NPGP (SEQ ID NO: 6), wherein X 1 of SEQ ID NO: 6 is any naturally occurring amino acid residue; each of C 1 and C 2 is, independently, a B7-1 transmembrane-intracellular amino acid sequence, a B7-2 transmembrane-intracellular amino acid sequence, a CD8α transmembrane-intracellular amino acid sequence, a B7-1 transmembrane amino acid sequence, a B7-2 transmembrane amino acid sequence, or a CD8α transmembrane amino acid sequence; and each one of a and b is 1, and each of L 1 and L 3 is, independently, (i) a polypeptide of Formula III: X 1 m X 2 n X 3 p X 4 q (Formula III), wherein: each of m, p, and q is, independently, 0 or 1; n is an integer from 20 to 65; X 2 is a plurality of amino acid residues, each of which is independently selected from glycine and serine; and each of X 1 , X 3 , and X 4 is, independently, any one naturally occurring amino acid residue; (ii) a polypeptide of Formula IV: X 5 r X 6 s X 7 t (Formula IV), wherein: s is 1; each of r and t is, independently, an integer from 20 to 25; each of X 5 and X 7 is, independently, a plurality of amino acid residues, each of which is, independently, selected from alanine, lysine, and glutamic acid; and X 6 is any one naturally occurring amino acid residue; or iii) 10 to 30 amino acid residues selected, independently, from glycine, serine, threonine, lysine, glutamic acid, and proline.
  2. 2 . The nucleic acid of claim 1 , wherein each of L 1 and L 3 is, independently, 10 to 65 amino acid residues.
  3. 3 . The nucleic acid of claim 1 , wherein L 2 is 20 to 30 amino acid residues.
  4. 4 . The nucleic acid of claim 1 , wherein L 2 is a (i) porcine teschovirus-1 2A (P2A) amino acid sequence, (ii) equine rhinitis A virus (E2A) amino acid sequence, (iii) thosea asigna virus 2A (T2A) amino acid sequence, (iv) foot-and-mouth disease virus (F2A) amino acid sequence, or (v) furin-cleavable-P2A amino acid sequence, wherein each one of the P2A, E2A, T2A, F2A, and furin-cleavable P2A amino acid sequence comprises the amino acid sequence of GDVEXINPGP (SEQ ID NO: 6), wherein X 1 of SEQ ID NO: 6 is any naturally occurring amino acid residue.
  5. 5 . The nucleic acid of claim 1 , wherein each of C 1 and C 2 is, independently, IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 4) or LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 5).
  6. 6 . The nucleic acid of claim 1 , wherein N 1 is an IL-21 amino acid sequence and N 2 is an IL-15 amino acid sequence.
  7. 7 . The nucleic acid of claim 1 , comprising a nucleotide sequence encoding an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 32-37.
  8. 8 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the nucleic acid of claim 1 .
  9. 9 . A host cell expressing the nucleic acid of claim 1 .
  10. 10 . A recombinant expression vector comprising the nucleic acid of claim 1 .
  11. 11 . A host cell comprising the recombinant expression vector of claim 10 .
  12. 12 . A population of cells comprising the host cell of claim 11 .
  13. 13 . The host cell of claim 11 , wherein the host cell comprises an antigen-specific receptor.
  14. 14 . The host cell of claim 13 , wherein the antigen-specific receptor is a chimeric antigen receptor (CAR).
  15. 15 . The host cell of claim 13 , wherein the antigen-specific receptor is an endogenous T cell receptor (TCR).
  16. 16 . The host cell of claim 13 , wherein the antigen-specific receptor is an exogenous TCR.
  17. 17 . The host cell of claim 13 , wherein the antigen-specific receptor has antigenic specificity for a cancer antigen.
  18. 18 . A method of treating a tumor in a mammal, the method comprising administering to the mammal the nucleic acid of claim 1 , in an amount effective to treat the tumor in the mammal.
  19. 19 . A method of enhancing the immune response of a mammal to a vaccine, the method comprising administering to the mammal (i) the vaccine and (ii) the nucleic acid of claim 1 in an amount effective to enhance the immune response of the mammal to the vaccine.
  20. 20 . One or more polypeptides encoded by the nucleic acid of claim 1 .

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This patent application is the U.S. national stage of PCT/US2019/016975, filed Feb. 7, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62/628,454, filed Feb. 9, 2018, which is incorporated by reference in its entirety herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant Numbers ZIABC011478 awarded by the National Institutes of Health, National Cancer Institute. The Government has certain rights in this invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 71,205 Byte ASCII (Text) file named “750024_ST25.TXT” dated Jul. 21, 2020. BACKGROUND OF THE INVENTION Adoptive cell therapy can be an effective treatment for cancer in some patients. However, obstacles to the overall success of adoptive cell therapy still exist. For example, the in vivo persistence, survival, and anti-tumor activity of the transferred T cells can, in some cases, decrease following adoptive transfer. Despite considerable research in the field of adoptive cell therapy, there still exists a need for improved methods and products for producing cells for adoptive cell therapy and treating and/or preventing cancer. BRIEF SUMMARY OF THE INVENTION An embodiment of the invention provides a nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of Formula I: S1—N1-L1-C1a-L2-S2—N2-L3-C2b   (Formula I), wherein: each of S1 and S2 is, independently, a signal sequence;one of N1 and N2 is an interleukin (IL)-21 amino acid sequence and one of N1 and N2 is an IL-15 amino acid sequence;each of L1, L2, and L3 is, independently, a linker sequence;each of C1 and C2 is, independently, a transmembrane-intracellular amino acid sequence or a transmembrane amino acid sequence; andeach of a and b is, independently, 0 or 1. Further embodiments of the invention provide related recombinant expression vectors, polypeptides, host cells, populations of cells, pharmaceutical compositions, and methods of treating or preventing cancer. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) FIG. 1A is a schematic illustrating the composition of a tethered IL-15 (TeIL-15) construct. Each TeIL-15 construct includes a signal sequence, IL-15 mature amino acid sequence, linker, and cell membrane anchor moiety (“anchor”). FIG. 1B is a schematic illustrating the composition of a tethered IL-21 (TeIL-21) construct. Each TeIL-21 construct includes a signal sequence, IL-21 mature amino acid sequence, linker, and cell membrane anchor moiety (“anchor”). FIG. 1C is a schematic illustrating the composition of tethered IL-21/tethered IL-15 (TeIL-21/15) construct. Each TeIL-21/15 construct includes a TeIL-21 construct of FIG. 1B, a cleavable linker, and a TeIL-15 construct of FIG. 1A. FIG. 2 is a graph showing the number of live cells measured at various time points (days) after IL-2 was withdrawn from the media seven days after transduction of the cells with a vector encoding the TeIL-15 Lr1, TeIL-15 Lr2, IL-15 RA, TeIL-15 Lr6, or IL-15S construct of Table 2. Untransduced (Un Tdx) cells served as a negative control. Untransduced cells cultured in the presence of exogenous IL-15 served as a positive control. FIG. 3 is a graph showing the tumor size (mm2) measured in tumor-bearing mice on the indicated number of days after infusion of untransduced (*) or transduced cells (n=5 in each group). Cells were transduced with (i) DMF5 TCR and TeIL-15 Lr1Ar2 (open squares), (ii) DMF5 TCR and TeIL-21 Lr8Ar1 (open triangles), (iii) DMF5 TCR and TeIL-21/15 E2A Ar1 (open diamonds), (iv) E7 TCR and TeIL-15 Lr1Ar2 (closed squares), (v) E7 TCR and TeIL-21 Lr8Ar1 (closed triangles), (vi) E7 TCR and TeIL-21/15 E2A Ar1 (closed diamonds), (vi) DMF5 TCR alone (open circles), or (vii) E7 TCR alone (closed circles). FIG. 4A is a graph showing the tumor size (mm2) measured in each of five tumor-bearing mice on the indicated number of days after infusion of untransduced T cells. FIG. 4B is a graph showing the tumor size (mm2) measured in each of five tumor-bearing mice on the indicated number of days after infusion of T cells transduced with the E7 TCR alone. FIG. 4C is a graph showing the tumor size (mm2) measured in each of five tumor-bearing mice on the indicated number of days after infusion of T cells transduced with the E7 TCR and TeIL-15. FIG. 4D is a graph showing the tumor size (mm2) measured in each of five tumor-bearing mice on the indicated number of days after infusion of T cells transduced with the E7 TCR and TeIL-21. FIG. 4E is a graph showing the tumor size (mm2) measured in each of five tumor-bearing mice on the indicated number of days after infusion of T cells transduced with the E7 TCR and TeIL-21/15. FIG. 4F is a graph showing the tum