# US 10,865,242 B2 · Method and compositions for cellular immunotherapy

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- Number: US 10,865,242 B2
- Document ID: US10865242B2
- Status: Granted
- Kind: B2
- Application number: 15/302,403
- Filed: 2015-04-08
- Published: 2018-01-11
- Granted: 2020-12-15
- Earliest claimed: 2014-04-10
- Assignees: Seattle Children's Hospital
- Applicants: Seattle Children's Hospital
- Inventors: Michael C. Jensen
- Web page: https://patentgrep.com/patent/US10865242B2

## CPC

- C07K 16/2803: {against the immunoglobulin superfamily}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 38/1774: {Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)}
- A61K 38/179: {for growth factors; for growth regulators}
- A61K 38/1793: {for cytokines; for lymphokines; for interferons}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- 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 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 13/12: of the kidneys
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- 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 14/71: for growth factors; for growth regulators
- C07K 14/7151: {for tumor necrosis factor [TNF], for lymphotoxin [LT]}
- C07K 16/2818: {against CD28 or CD152}
- C07K 16/32: against translation products of oncogenes
- C12N 5/0636: {T lymphocytes}
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N 15/85: for animal cells
- C12Y 207/10001: Receptor protein-tyrosine kinase (2.7.10.1)
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/572: {cytotoxic response}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/524: CH2 domain
- C07K 2317/526: CH3 domain
- C07K 2317/53: Hinge
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2319/02: containing a signal sequence
- 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 15/62: DNA sequences coding for fusion proteins
- C12N 2510/00: Genetically modified cells
- C12N 2800/90: Vectors containing a transposable element
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- 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)
- C12Y: ENZYMES
- 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 invention provides nucleic acids, vectors, host cells, methods and compositions to confer and/or augment immune responses mediated by cellular immunotherapy, such as by adoptively transferring CD8+ central memory T cells or combinations of central memory T cells with CD4+ T cells that are genetically modified to express a chimeric receptor. In some alternatives the genetically modified host cell comprises a nucleic acid comprising a polynucleotide coding for a ligand binding domain, a polynucleotide comprising a customized spacer region, a polynucleotide comprising a transmembrane domain, and a polynucleotide comprising an intracellular signaling domain. In some alternatives, the ligand binding domains binds to CD171.

## Claims

### Claim 1

A nucleic acid encoding a chimeric receptor comprising:
a) a polynucleotide encoding a ligand binding domain comprising the nucleotide sequence of residues 2150 to 2875 set forth in SEQ ID NO:54, wherein the ligand binding domain specifically binds to or interacts with CD171;
b) a polynucleotide encoding a polypeptide spacer comprising the amino acid sequence set forth in SEQ ID NO:01, and wherein the spacer has a length greater than or equal to 12 and less than or equal to 119 consecutive amino acids;
c) a polynucleotide encoding a transmembrane domain; and
d) a polynucleotide encoding an intracellular signaling domain;
wherein the chimeric receptor comprises an increased in vivo activity for a survival rate of a subject having a neuroblastoma and for neuroblastoma regression in the subject compared to an anti-CD171 chimeric receptor comprising a spacer having a length greater than 119 consecutive amino acids.

### Claim 2 (depends on 1)

The nucleic acid of claim 1, wherein the spacer comprises a portion of a hinge region of a human antibody.

### Claim 3 (depends on 2)

The nucleic acid of claim 2, wherein the intracellular signaling domain comprises all of or a portion of CD3 zeta in combination with a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3, and a combination thereof.

### Claim 4 (depends on 3)

The nucleic acid of claim 3, wherein the intracellular signaling domain comprises a portion of CD3 zeta and a portion of 4-1BB.

### Claim 5 (depends on 4)

The nucleic acid of claim 4, further comprising a polynucleotide encoding a marker sequence.

### Claim 6 (depends on 1)

An engineered cell comprising the nucleic acid of claim 1.

### Claim 7 (depends on 6)

The engineered cell of claim 6, wherein the engineered cell is a CD8+ cytotoxic T lymphocyte cell selected from the group consisting of a naïve CD8+ T cell, a central memory CD8+ T cell, an effector memory CD8+ T cell, and a bulk CD8+ T cell.

### Claim 8 (depends on 7)

The engineered cell of claim 7, wherein the CD8+ cytotoxic T lymphocyte cell is a central memory T cell positive for CD45RO, CD62L, and CD8.

### Claim 9 (depends on 6)

The engineered cell of claim 6, wherein the engineered cell is a CD4+ helper T lymphocyte cell selected from the group consisting of a naïve CD4+ T cell, a central memory CD4+ T cell, an effector memory CD4+ T cell, and a bulk CD4+ T cell.

### Claim 10 (depends on 9)

The engineered cell of claim 9, wherein the CD4+ helper T lymphocyte cell is a naïve CD4+ T cell positive for CD45RA, CD62L, CD4, and negative for CD45RO.

### Claim 11 (depends on 6)

The engineered cell of claim 6, wherein the engineered cell is a precursor T cell.

### Claim 12 (depends on 6)

The engineered cell of claim 6, wherein the engineered cell is a hematopoietic stem cell.

### Claim 13

A nucleic acid encoding a chimeric receptor comprising:
a) a polynucleotide encoding a ligand binding domain, wherein the ligand binding domain specifically binds to or interacts with CD171;
b) a polynucleotide encoding a polypeptide spacer comprising the amino acid sequence set forth in SEQ ID NO:01;
c) a polynucleotide encoding a transmembrane domain; and
d) a polynucleotide encoding an intracellular signaling domain;
wherein the chimeric receptor comprises the sequence set forth in SEQ ID NO:55 or SEQ ID NO:56; and
wherein the chimeric receptor comprises an increased in vivo activity for a survival rate of a subject having a neuroblastoma and for neuroblastoma regression in the subject compared to an anti-CD171 chimeric receptor comprising a spacer having a length greater than 119 consecutive amino acids.

### Claim 14 (depends on 1)

The nucleic acid of claim 1, wherein the nucleic acid comprises at least 95% sequence identity to the sequence as set forth in SEQ ID NO:55 or SEQ ID NO:56.

### Claim 15 (depends on 1)

The nucleic acid of claim 1, wherein the spacer has a length greater than or equal to 12 and less than or equal to 15 consecutive amino acids.

### Claim 16 (depends on 1)

The nucleic acid of claim 1, wherein the spacer comprises the amino acid sequence set forth in SEQ ID NO:21.

### Claim 17 (depends on 1)

The nucleic acid of claim 1, wherein the spacer comprises the amino acid sequence set forth in SEQ ID NO:59.