# US 11,111,287 B2 · Chimeric proteins and methods of immunotherapy

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- Number: US 11,111,287 B2
- Document ID: US11111287B2
- Status: Granted
- Kind: B2
- Application number: 16/414,721
- Filed: 2019-05-16
- Published: 2019-11-14
- Granted: 2021-09-07
- Earliest claimed: 2016-01-11
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Lei S. Qi; Bing C. Wang
- Web page: https://patentgrep.com/patent/US11111287B2

## CPC

- C07K 14/70521: {CD28, CD152}
- 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 40/36: Immune checkpoint inhibitors
- A61K 40/4211: {CD19 or B4}
- A61P 35/00: Antineoplastic agents
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/1086: {Preparation or screening of expression libraries, e.g. reporter assays}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/01: containing a localisation/targetting motif
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/09: containing a nuclear localisation signal
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present disclosure provides systems for immune cell regulation and methods of immunotherapy. Systems of the present disclosure for immune cell regulation comprise a chimeric receptor polypeptide, a chimeric adaptor polypeptide, a gene modulating polypeptide (GMP), and a cleavage moiety.

## Claims

### Claim 1

A system capable of inducing death of a target cell, comprising:
(a) a chimeric transmembrane receptor polypeptide (receptor) comprising a ligand binding domain, an immune cell signaling domain, and a gene modulating polypeptide (GMP), the GMP comprising an actuator moiety linked to a cleavage recognition site, wherein the actuator moiety modulates expression and/or activity of an immune regulatory protein of the lymphocyte, and wherein the immune regulatory protein enhances lymphocyte cytotoxicity and/or reduces a side effect of lymphocyte activation; and
(b) a chimeric adaptor polypeptide (adaptor) comprising a receptor binding moiety linked to a cleavage moiety, wherein the cleavage moiety is capable of cleaving the cleavage recognition site on the receptor when the receptor binding moiety of the adaptor binds the immune cell signaling domain of the receptor in response to binding of the ligand binding domain of the receptor to a ligand present on the target cell, and wherein the adaptor does not bind a ligand present on the target cell,
wherein the receptor is activatable upon binding to the ligand present on the target cell to recruit the adaptor to the receptor, and wherein the recruited adaptor releases the actuator moiety from the GMP of the receptor by action of the cleavage moiety at the cleavage recognition site to induce death of the target cell.

### Claim 2 (depends on 1)

The system of claim 1, wherein the ligand binding domain comprises a single-chain variable fragment (scFv).

### Claim 3 (depends on 1)

The system of claim 1, wherein the ligand binding domain binds CD19 or a fragment thereof.

### Claim 4 (depends on 1)

The system of claim 1, wherein the immune cell signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

### Claim 5 (depends on 1)

The system of claim 1, wherein the actuator moiety comprises a CRISPR-associated polypeptide (Cas), a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a recombinase, a flippase, a transposase, or an Argonaute protein.

### Claim 6 (depends on 1)

The system of claim 1, wherein the actuator moiety comprises a Cas protein that forms a complex with a guide RNA (gRNA).

### Claim 7 (depends on 6)

The system of claim 6, wherein the Cas protein is Cas9, Cpf1, or C2c2.

### Claim 8 (depends on 6)

The system of claim 6, wherein the Cas protein substantially lacks DNA cleavage activity.

### Claim 9 (depends on 6)

The system of claim 6, wherein the Cas protein is linked to a transcriptional repressor or a transcriptional activator.

### Claim 10 (depends on 1)

The system of claim 1, wherein the cleavage recognition site comprises a polypeptide sequence and the cleavage moiety comprises a protease.

### Claim 11 (depends on 1)

The system of claim 1, wherein the cleavage moiety is a TEV protease, and wherein the cleavage recognition site is a TEV protease cleavage site.

### Claim 12 (depends on 1)

A lymphocyte expressing the system of claim 1.

### Claim 13 (depends on 1)

A population of lymphocyte cells, individual lymphocyte cells expressing the system of claim 1, wherein said population of lymphocyte cells is characterized in that: upon exposing said population of lymphocyte cells to a target cell population in an in vitro cell death assay, the population of lymphocyte cells induces death of at least 45% of the target cells within about 2 days when the ratio of the number of lymphocyte cells in said population of lymphocyte cells to the number of target cells in the target cell population is about 1:2 or less.