# US 11,993,652 B2 · Tagged chimeric effector molecules and receptors thereof

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- Number: US 11,993,652 B2
- Document ID: US11993652B2
- Status: Granted
- Kind: B2
- Application number: 17/329,115
- Filed: 2021-05-24
- Published: 2022-06-02
- Granted: 2024-05-28
- Earliest claimed: 2013-12-20
- Assignees: Fred Hutchinson Cancer Center
- Applicants: Fred Hutchinson Cancer Center
- Inventors: Stanley R. Riddell; Lingfeng Liu
- Web page: https://patentgrep.com/patent/US11993652B2

## CPC

- C07K 16/2803: {against the immunoglobulin superfamily}
- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- A61K 38/1774: {Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)}
- A61K 38/2013: {IL-2}
- A61K 38/2086: {IL-13 to IL-16}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4211: {CD19 or B4}
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/2818: {against CD28 or CD152}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C12N 5/0636: {T lymphocytes}
- C12N 5/0637: {Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg}
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- G01N 33/56972: {White blood cells}
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/64: comprising a combination of variable region and constant region components
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/22: containing a Strep-tag
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/95: containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2510/00: Genetically modified cells
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present disclosure relates to tagged chimeric effector molecules and receptor molecules thereof for genetically engineering a host cell, wherein the recombinant host cell can be identified, isolated, sorted, induced to proliferate, tracked or eliminated. For example, a T cell may be recombinantly modified for use in adoptive immunotherapy.

## Claims

### Claim 1

A single chain fusion protein comprising an extracellular component and an intracellular component connected by a transmembrane domain,
wherein the extracellular component comprises a scFv, a tag cassette having the amino acid sequence of SEQ ID NO.:1 or SEQ ID NO.:2, and a connector region comprising a hinge,
wherein the intracellular component comprises an effector domain,
and wherein:
(a) the tag cassette is comprised in the scFv; and/or
(b) the tag cassette is located amino-terminal to the scFv.

### Claim 2 (depends on 1)

The single chain fusion protein of claim 1, wherein the scFv comprises a first variable domain linked to a second variable domain by the amino acid sequence of SEQ ID NO.:45.

### Claim 3 (depends on 1)

The single chain fusion protein of claim 1, wherein the tag cassette is located amino-terminal to the scFv.

### Claim 4 (depends on 1)

The single chain fusion protein of claim 1, wherein the tag cassette is comprised in the scFv.

### Claim 5 (depends on 1)

The single chain fusion protein of claim 1, further comprising one or more tag cassettes located carboxy-terminal to the scFv.

### Claim 6 (depends on 3)

The single chain fusion protein of claim 3, further comprising one or more tag cassettes located carboxy-terminal to the scFv.

### Claim 7 (depends on 4)

The single chain fusion protein of claim 4, further comprising one or more tag cassettes located carboxy-terminal to the scFv.

### Claim 8 (depends on 1)

The single chain fusion protein of claim 1, wherein the scFv comprises a variable region linker, wherein the variable region linker comprises the tag cassette.

### Claim 9 (depends on 1)

The single chain fusion protein of claim 1, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO.:54.

### Claim 10 (depends on 1)

The single chain fusion protein of claim 1, comprising a plurality of tag cassettes, each of the plurality of tag cassettes independently having the amino acid sequence of SEQ ID NO.:1 or 2.

### Claim 11 (depends on 1)

The single chain fusion protein of claim 1, comprising a plurality of tag cassettes, wherein the plurality of tag cassettes comprises at least two different tag cassettes.

### Claim 12 (depends on 1)

The single chain fusion protein of claim 1, comprising:
one or more tag cassettes located amino-terminal to the scFv and one or more tag cassettes comprised in the scFv;
(ii) one or more tag cassettes located amino-terminal to the scFv and one or more tag cassettes comprised in the connector region; or (iii) one or more tag cassettes comprised in the scFv and one or more tag cassettes comprised in the connector region.

### Claim 13 (depends on 1)

The single chain fusion protein of claim 1, further comprising one or more tag cassettes:
(i) immediately amino-terminal to the connector region; and/or
(ii) interposed between and connecting linker modules.

### Claim 14 (depends on 1)

The single chain fusion protein of claim 1, wherein the scFv recognizes a target and the target comprises a tumor antigen, a B-cell target, a TNF receptor superfamily member, a Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF-β superfamily member, a Wnt-related molecule, a T-cell target, a dendritic cell target, an NK cell target, a monocyte/macrophage cell target, an angiogenesis target, a receptor protein, a cancer cell, a cell associated with an autoimmune disease or disorder, a cell associated with an inflammatory disease or disorder, an infectious organism or cell, a pathogen-specific molecule expressed on infected cells, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor or pathogen associated peptide bound to HLA, hTERT peptide bound to HLA, tyrosinase peptide bound to HLA, WT-1 peptide bound to HLA, LTβR, LRP5, MUC1, OSMRβ, TCRα, TCRβ, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, WT-1, Robo1, α-fetoprotein (AFP), Frizzled, OX40, or CD79b.

### Claim 15 (depends on 1)

The single chain fusion protein of claim 1, wherein the effector domain or an effector portion thereof is a 4-1BB (CD137), CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof.

### Claim 16 (depends on 1)

The single chain fusion protein of claim 1, wherein the effector domain or effector portion thereof comprises CD3ζ and one or more of 4-1BB (CD137), CD27, CD28, and OX40 (CD134).

### Claim 17 (depends on 1)

The single chain fusion protein of claim 1, wherein the fusion protein comprises from amino-terminus to carboxy-terminus:
(a) the tag cassette, the scFv, the connector region comprising (i) the hinge and (ii) one or more tag cassettes, the hydrophobic portion, and the intracellular component comprising the effector domain; or (b) the scFv, comprising a variable region linker disposed between a first variable region and a second variable region and containing the tag cassette, the connector region comprising (i) the hinge and (ii) one or more tag cassettes, the hydrophobic portion, and the intracellular component comprising the effector domain.

### Claim 18

A nucleic acid molecule encoding a single chain fusion protein, wherein the single chain fusion protein comprises an extracellular component and an intracellular component connected by a transmembrane domain,
wherein the extracellular component comprises a scFv, a tag cassette having the amino acid sequence of SEQ ID NO.:1 or SEQ ID NO.:2, and a connector region comprising a hinge,
wherein the intracellular component comprises an effector domain,
and wherein:
(a) the tag cassette is comprised in the scFv; and/or
(b) the tag cassette is located amino-terminal to the scFv.

### Claim 19

A vector comprising a nucleic acid molecule encoding a single chain fusion protein, wherein the single chain fusion protein comprises an extracellular component and an intracellular component connected by a transmembrane domain,
wherein the extracellular component comprises a scFv, a tag cassette having the amino acid sequence of SEQ ID NO.:1 or SEQ ID NO.:2, and a connector region comprising a hinge,
wherein the intracellular component comprises an effector domain,
and wherein:
(a) the tag cassette is comprised in the scFv; and/or
(b) the tag cassette is located amino-terminal to the scFv.

### Claim 20

A host cell comprising a nucleic acid molecule encoding a single chain fusion protein, wherein the single chain fusion protein comprises an extracellular component and an intracellular component connected by a transmembrane domain,
wherein the extracellular component comprises a scFv, a tag cassette having the amino acid sequence of SEQ ID NO.:1 or SEQ ID NO.:2, and a connector region comprising a hinge,
wherein the intracellular component comprises an effector domain,
and wherein:
(a) the tag cassette is comprised in the scFv; and/or
(b) the tag cassette is located amino-terminal to the scFv.

### Claim 21 (depends on 20)

The host cell of claim 20, wherein the host cell is a T cell.

### Claim 22 (depends on 21)

The host cell of claim 21, wherein the host cell is a virus-specific T cell, a tumor antigen specific cytotoxic T cell, a naïve T cell, a memory stem T cell, a central or effector memory T cell, a CD4+ CD25+ regulatory T cell, a mature T cell, a CD8+ T cell, or a CD4+ T cell.

### Claim 23 (depends on 1)

A method comprising contacting a cell comprising the single chain fusion protein of claim 1 with a binding domain specific for the tag cassette.

### Claim 24 (depends on 20)

A method of treating a disease in a subject, the method comprising administering to the subject a host cell of claim 20, wherein the disease is associated with expression of a target recognized by the scFv.

### Claim 25 (depends on 24)

The method of claim 24, wherein the disease is a viral, bacterial, cancer, inflammatory, immune, or aging-associated disease.