JPWO2020051493A5

JPWO2020051493A5 -

Info

Publication number: JPWO2020051493A5
Application number: JP2021512773A
Authority: JP
Publication date: 2022-09-14

Claims

Genetically modified hematopoietic cells having elevated intracellular lactate concentrations compared to homotypic native hematopoietic cells ,
(i) a lactate-modulating polypeptide; and
(ii) a chimeric receptor polypeptide
wherein the chimeric receptor polypeptide comprises:
(a) an extracellular target binding domain;
(b) a transmembrane domain, and
(c) a cytoplasmic signaling domain
genetically modified hematopoietic cells .

2. The genetically modified hematopoietic cell of Claim 1 , wherein said lactate regulatory polypeptide is monocarboxylic acid transporter (MCT), lactate dehydrogenase A (LDHA) , or pyruvate dehydrogenase kinase 1 ( PDKl).

3. The genetically modified hematopoietic cell of claim 2 , wherein said lactate regulating polypeptide is a monocarboxylic acid transporter (MCT), which is MCT1, MCT2, or MCT4.

Said chimeric receptor polypeptide is :
(1) is an antibody binding T cell receptor (ACTR) polypeptide wherein (a) is an extracellular Fc binding domain ; or
(2) is a chimeric receptor antigen (CAR) polypeptide, wherein (a) is an extracellular antigen-binding domain;
The genetically modified hematopoietic cell according to any one of claims 1-3 .

Said chimeric receptor polypeptide is :
(i) further comprising at least one co-stimulatory signaling domain ;
(ii) does not contain a co-stimulatory signaling domain; and
(iii) said cytoplasmic signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM);
5. The genetically modified hematopoietic cell of any one of claims 1-4 , which is a CAR polypeptide comprising one or more of the characteristics of .

The CAR polypeptide further comprises a hinge domain located at the C-terminus of (a) and the N-terminus of (b) ; and/or the chimeric receptor polypeptide further comprises a signal peptide at its N-terminus. The genetically modified hematopoietic cell of any one of claims 1-5 .

The chimeric receptor polypeptide is the CAR polypeptide, wherein the extracellular target binding domain of (a) is an antigen binding domain, and the antigen binding domain is specific for a tumor antigen, a pathogen antigen, or an autoantigen. 7. The genetically modified hematopoietic cell of any one of claims 1 to 6 , which is a single chain antibody fragment (scFv) that binds to effective immune cells.

8. The genetically modified hematopoietic cell of Claim 7 , wherein said antigen binding domain binds said tumor antigen associated with a hematological or solid tumor .

said tumor antigen associated with said hematological tumor is selected from the group consisting of CD19, CD20, CD22, kappa chain, CD30, CD123, CD33, LeY, CD138, CD5, BCMA, CD7, CD40, and IL-1RAP ; or
Said tumor antigens associated with said solid tumors are GD2, GPC3, FOLR, HER2, EphA2, EFGRVIII, IL13RA2, VEGFR2, ROR1, NKG2D, EpCAM, CEA, mesothelin, MUC1, CLDN18.2, CD171, CD133, PSCA, cMET , EGFR, PSMA, FAP, CD70, MUC16, L1-CAM, and CAIX,
9. The genetically modified hematopoietic cell of claim 8 .

said transmembrane domain is from the group consisting of CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16A, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, and FGFR2B 10. The genetically modified hematopoietic cell of any one of claims 1-9, which is of a selected membrane protein.

Said CAR polypeptide comprises said at least one co-stimulatory signaling domain , said co-stimulatory signaling domain being 4-1BB, CD28, CD28 _LL→GG variant, OX40, ICOS, CD27, GITR, ICOS, HVEM, TIM1 11. The genetically modified hematopoietic cell of any one of claims 5 to 10 , which is a co-stimulatory molecule selected from the group consisting of , LFA1, and CD2.

12. The genetically modified hematopoietic cell of any one of claims 5-11 , wherein said CAR polypeptide comprises two co-stimulatory signaling domains.

one of said costimulatory signaling domains is a CD28 costimulatory signaling domain ;
the other costimulatory domain is selected from the group consisting of a 4-1BB costimulatory signaling domain, an OX40 costimulatory signaling domain, a CD27 costimulatory signaling domain, and an ICOS costimulatory signaling domain ; or
The two co-stimulatory domains are:
(i) CD28 and 4-1BB, or
(ii) CD28 _LL→GG variant a and 4-1BB
13. The genetically modified hematopoietic cell of claim 12 , which is

14. The genetically modified hematopoietic cell of any one of claims 1 to 13 , wherein said cytoplasmic signaling domain of (c) is the cytoplasmic domain of CD3ζ or FcεR1γ.

The CAR polypeptide comprises (i) a CD28 co-stimulatory domain in combination with a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof, or (ii) a 4-1BB co-stimulatory domain with a CD8 transmembrane domain, CD8 hinge 8. The genetically modified hematopoietic cell of claim 7 , comprising domains, or combinations thereof.

8. The genetically modified hematopoietic cell of Claim 7 , wherein said CAR polypeptide comprises the amino acid sequence of SEQ ID NO:97 or 98.

17. The genetically modified hematopoietic cell of any one of claims 1-16 , wherein said hematopoietic cell is a hematopoietic stem cell or an immune cell.

18. The genetically modified hematopoietic cell of Claim 17, wherein said hematopoietic cell is said immune cell that is a natural killer cell, macrophage, neutrophil, eosinophil, or T cell.

said hematopoietic cells are immune cells, said immune cells having inhibited expression of endogenous T cell receptors, endogenous major histocompatibility complex, endogenous beta-2-microglobulin, or a combination thereof 19. The genetically modified hematopoietic cell of claim 18 , which is a T cell that has been replenished or depleted.

Said hematopoietic cell comprises a nucleic acid or set of nucleic acids, said nucleic acid or said set of nucleic acids collectively:
20. The genetic modification of any one of claims 1-19 , comprising (A) a first nucleotide sequence encoding said lactate regulator, and (B) a second nucleotide sequence encoding said chimeric receptor polypeptide. hematopoietic cells.

21. The genetically modified hematopoietic cell of Claim 20 , wherein said hematopoietic cell comprises said nucleic acid, said nucleic acid comprising both said first nucleotide sequence and said second nucleotide sequence.

Said nucleic acid further comprises a third nucleotide sequence located between said first nucleotide sequence and said second nucleotide sequence, said third nucleotide sequence being a ribosome skip site, an internal ribosome entry site (IRES), or 22. The genetically modified hematopoietic cell of claim 21 , encoding a second promoter.

23. The genetically modified hematopoietic cell of claim 22 , wherein said third nucleotide sequence encodes a ribosome skip site, said ribosome skip site being a P2A peptide.

24. The genetically modified hematopoietic cell of any one of claims 20-23 , wherein said nucleic acid or said nucleic acid set is comprised within a vector or set of vectors that is a viral vector .

25. A pharmaceutical composition comprising the genetically modified hematopoietic cells of any one of claims 1-24 and a pharmaceutically acceptable carrier.

25. A population of genetically modified hematopoietic cells for use in inhibiting target cells expressing a target antigen in a subject, wherein said genetically modified hematopoietic cells are the genetically modified hematopoietic cells of any one of claims 1-24 . including _
said genetically modified hematopoietic cells express a CAR polypeptide, said CAR polypeptide comprising an extracellular antigen binding domain specific for a target antigen; and
said target antigen is an immune cell specific for a tumor antigen, a pathogen antigen, or an autoantigen;
A population of said genetically modified hematopoietic cells.

27. A population of genetically modified hematopoietic cells for use according to claim 26, wherein at least some of the cells expressing the target antigen are placed in a low glucose environment.

28. A population of genetically modified hematopoietic cells for use according to claim 26 or 27, wherein said genetically modified hematopoietic cells are autologous cells.

29. Genetically modified hematopoietic cells for use according to any one of claims 26 to 28 , wherein said genetically modified hematopoietic cells are ex vivo activated, expanded or both. a group of

30. A population of genetically modified hematopoietic cells for use according to any one of claims 26 to 29 , wherein said subject is a human patient suffering from cancer and said target antigen is a tumor antigen. .

Said cancer is cancer of B cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelial 31. Genetically modified hematopoiesis for use according to claim 30 , selected from the group consisting of cancer, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer. a population of cells .

32. A gene for use according to claim 31 , wherein said cancer of B-cell origin is selected from the group consisting of B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma. A population of engineered hematopoietic cells .

Collectively the following:
(A) a first nucleotide sequence encoding the chimeric receptor polypeptide of any one of claims 1 and 4-16 ; and (B) the lactic acid of any one of claims 1-3. A nucleic acid or set of nucleic acids comprising a second nucleotide sequence that encodes a regulatory polypeptide .

34. The nucleic acid or nucleic acid set of claim 33, wherein said nucleic acid or said nucleic acid set is an RNA molecule or set of RNA molecules.

said nucleic acid comprises both said first nucleotide sequence and said second nucleotide sequence, and said nucleic acid further comprises a third nucleotide sequence located between said first nucleotide sequence and said second nucleotide sequence; 35. The nucleic acid or nucleic acid set of claim 33 or 34 , wherein said third nucleotide sequence encodes a ribosome skip site, an internal ribosome entry site (IRES), or a second promoter.

36. The nucleic acid or nucleic acid set of claim 35 , wherein said ribosome skip site is a P2A peptide.

37. The nucleic acid or nucleic acid set of any one of claims 33-36 , wherein said nucleic acid or said nucleic acid set is comprised within a vector or set of vectors.

38. The nucleic acid or nucleic acid set of claim 37, wherein said vector or said set of vectors comprises one or more viral vectors.