# US 11,896,620 B2 · Engineered immune cell and use thereof

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- Number: US 11,896,620 B2
- Document ID: US11896620B2
- Status: Granted
- Kind: B2
- Application number: 17/908,109
- Filed: 2021-05-24
- Published: 2023-05-04
- Granted: 2024-02-13
- Earliest claimed: 2020-05-27
- Assignees: Bioheng Therapeutics Limited
- Applicants: Nanjing Bioheng Biotech Co., Ltd
- Inventors: Yun Xing; Zhonghui Yan; Ying Xiong; Rongrong Pu; Jiangtao Ren; Xiaohong He; Yanbin Wang; Lu Han
- Web page: https://patentgrep.com/patent/US11896620B2

## CPC

- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/2803: {against the immunoglobulin superfamily}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/30: characterised by the recombinant expression of specific molecules in the cells of the immune system
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/35: Cytokines
- A61K 40/4211: {CD19 or B4}
- A61K 40/4234: {Interleukins [IL]}
- A61K 40/4236: {Chemokines}
- A61P 35/00: Antineoplastic agents
- C12N 5/0636: {T lymphocytes}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/54: Pancreas
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/70: characterized by effect upon binding to a cell or to an antigen
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 15/86: Viral vectors
- C12N 2510/00: Genetically modified cells
- C12N 2740/13043: viral genome or elements thereof as genetic vector
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

An engineered immune cell, which expresses (i) a cell surface molecule that specifically recognizes a ligand, (ii) an exogenous interleukin, and (iii) an exogenous Flt3L, XCL2, and/or XCL1; the engineered immune cell can be used for treating cancer, infection, or autoimmune diseases; and compared with a traditional engineered immune cell, the engineered immune cell has significantly improved tumor killing activity.

## Claims

### Claim 1

An engineered immune cell, which expresses (i) a cell surface molecule that specifically recognizes a ligand, (ii) an exogenous IL-7, and (iii) an exogenous XCL2 and/or XCL1 gene.

### Claim 2 (depends on 1)

The engineered immune cell according to claim 1, wherein the cell surface molecule that specifically recognizes a ligand is a chimeric antigen receptor or a T cell receptor.

### Claim 3 (depends on 2)

The engineered immune cell according to claim 2, wherein the cell surface molecule that specifically recognizes a ligand is the chimeric antigen receptor containing a ligand binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

### Claim 4 (depends on 1)

The engineered immune cell according to claim 1, wherein an encoding gene of the IL-7 is represented by SEQ ID NO: 23 or 27, or the IL-7 is represented by SEQ ID NO: 24 or 28.

### Claim 5 (depends on 1)

The engineered immune cell according to claim 1, wherein the XCL1 gene is represented by SEQ ID NO: 25 or 29, or a polypeptide encoded by the XCL1 gene is represented by SEQ ID NO: 26 or 30.

### Claim 6 (depends on 1)

The engineered immune cell according to claim 1, wherein the XCL2 gene is represented by SEQ ID NO: 67, or a polypeptide encoded by the XCL2 gene is represented by SEQ ID NO: 68.

### Claim 7 (depends on 1)

The engineered immune cell according to claim 1, wherein the immune cell is selected from the group consisting of a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell and an NKT cell.

### Claim 8 (depends on 7)

The engineered immune cell according to claim 7, wherein the T cell is a CD4+/CD8+ T cell, a CD4+ helper T cell, a CD8+ T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γδ-T cell, or an αβ-T cell.

### Claim 9 (depends on 3)

The engineered immune cell according to claim 3, wherein the ligand binding domain is selected from the group consisting of scFv, Fab, single domain antibody, nanobody, antigen binding ligand, recombinant fibronectin domain, anticalin and DARPIN.

### Claim 10 (depends on 3)

The engineered immune cell according to claim 3, wherein the ligand binding domain binds to a target selected from the group consisting of: TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRCSD, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 1Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, S SEA-4, CD20, AFP, Folate receptor α, ERBB2 (Her2/neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-ab1, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRCSD, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1 a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos associated antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2 ETS fusion gene, NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal tract carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF β, APRIL, NKG2D and any combination thereof.

### Claim 11 (depends on 3)

The engineered immune cell according to claim 3, wherein the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 a, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

### Claim 12 (depends on 3)

The engineered immune cell according to claim 3, wherein the intracellular signaling domain is a signaling domain of a protein selected from the group consisting of: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66d.

### Claim 13 (depends on 3)

The engineered immune cell according to claim 3, wherein the co-stimulatory domain is one or more co-stimulatory signaling domains of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70.

### Claim 14 (depends on 1)

The engineered immune cell according to claim 1, wherein the immune cell further contains at least one inactive gene selected from the group consisting of CD52, GR, TCR α, TCR β, CD3 γ, CD3 δ, CD3 ε, CD247 ζ, HLA-I, HLA-II, B2M, PD1, CTLA-4, LAG3 and TIM3.

### Claim 15 (depends on 1)

The engineered immune cell according to claim 1, wherein expression of the IL-7, XCL2, and/or XCL1 gene is a conditional expression.

### Claim 16 (depends on 15)

The engineered immune cell according to claim 15, wherein the IL-7, XCL2, and/or XCL1 gene is operably linked to an inducible, repressible, or tissue-specific promoter so as to be conditionally expressed.

### Claim 17

A nucleic acid molecule, which contains: (i) a nucleic acid sequence encoding a cell surface molecule that specifically recognizes a ligand, (ii) a nucleic acid sequence encoding an IL-7, and (iii) a nucleic acid sequence encoding XCL2 and/or XCL1.

### Claim 18 (depends on 17)

The nucleic acid molecule according to claim 17, wherein the cell surface molecule that specifically recognizes a ligand is a chimeric antigen receptor.

### Claim 19 (depends on 17)

A vector, which contains the nucleic acid molecule according to claim 17.

### Claim 20 (depends on 19)

The vector according to claim 19, wherein the vector is selected from the group consisting of plasmid, retrovirus, lentivirus, adenovirus, vaccinia virus, Rous Sarcoma Virus (RSV), polyoma virus, and adeno-associated virus (AAV).

### Claim 21 (depends on 1)

A pharmaceutical composition, which contains the engineered immune cell according to claim 1.

### Claim 22 (depends on 21)

The pharmaceutical composition according to claim 21, wherein said composition is for treating cancers, infections or autoimmune diseases.