# US 12,005,081 B2 · Chimeric receptors and methods of use thereof

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- Number: US 12,005,081 B2
- Document ID: US12005081B2
- Status: Granted
- Kind: B2
- Application number: 17/338,528
- Filed: 2021-06-03
- Published: 2021-09-30
- Granted: 2024-06-11
- Earliest claimed: 2019-04-30
- Assignees: Senti Biosciences, Inc.
- Applicants: Senti Biosciences, Inc.
- Inventors: Brian Scott Garrison; Jennifer Chien; Kathryn Armstrong Loving; Russell Morrison Gordley; Michelle Elizabeth Hung
- Web page: https://patentgrep.com/patent/US12005081B2

## CPC

- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4202: {Receptors, cell surface antigens or cell surface determinants}
- A61K 40/421: {Immunoglobulin superfamily}
- A61K 40/4214: {Receptors for cytokines}
- A61K 40/4217: {Receptors for interleukins [IL]}
- A61K 40/4254: {Adhesion molecules, e.g. NRCAM, EpCAM or cadherins}
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- 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 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2851: {against the lectin superfamily, e.g. CD23, CD72}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C12N 5/0636: {T lymphocytes}
- 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)
- A61K 2239/17: Hinge-spacer domain
- A61K 2239/28: Expressing multiple CARs, TCRs or antigens
- A61K 2239/29: Multispecific CARs
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- 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
- C07K 2319/43: containing a FLAG-tag
- C07K 2319/50: containing protease site
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are acute myeloid leukemia antigen targets for chimeric receptors and methods of using same.

## Claims

### Claim 1

A method of treating a subject having acute myeloid leukemia (AML), wherein the method comprises delivering to the subject an autologous or an allogeneic T cell or Natural Killer (NK) cell comprising:
(a) an anti-FLT3 chimeric antigen receptor (CAR) comprising a FLT3-binding domain and an anti-CD33 CAR comprising a CD33-binding domain, or a bivalent CAR comprising a FLT3-binding domain and a CD33-binding domain,
wherein the FLT3-binding domain comprises a heavy chain variable domain (VH) comprising a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 81, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 82, and a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 83 and a light chain variable domain (VL) comprising a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 84, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 85, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 86, and
wherein the CD33-binding domain comprises VH comprising a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 123, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 124, and a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 125 and a VL comprising a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 126, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 127, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 128; and
(b) an inhibitory chimeric receptor comprising an extracellular antigen-binding domain that binds to EMCN.

### Claim 2 (depends on 1)

The method of claim 1, wherein the T cell or the NK cell comprises the bivalent CAR.

### Claim 3 (depends on 1)

The method of claim 1, wherein the anti-FLT3 CAR and the anti-CD33 CAR, or the bivalent CAR comprises a CD3zeta-chain intracellular signaling domain and one or more additional intracellular signaling domains selected from the group consisting signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, and a 2B4 intracellular signaling domain.

### Claim 4 (depends on 3)

The method of claim 3, wherein the anti-FLT3 CAR and the anti-CD33 CAR, or the bivalent CAR comprises a transmembrane domain selected from the group consisting of: a CD8 transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain.

### Claim 5 (depends on 4)

The method of claim 4, wherein the anti-FLT3 CAR and the anti-CD33 CAR, or the bivalent CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain, and the spacer region has an amino acid sequence selected from the group consisting of the amino acid sequences of: SEQ ID NOs: 55-64.

### Claim 6 (depends on 1)

The method of claim 1, wherein the inhibitory chimeric receptor comprises a scFv, and wherein the scFv comprises an antigen binding domain obtained from an anti-EMCN antibody.

### Claim 7 (depends on 1)

The method of claim 1, wherein the FLT3-binding domain comprises an scFv and the CD33-binding domain comprises an scFv.

### Claim 8 (depends on 7)

The method of claim 7, wherein the T cell or the NK cell comprises the bivalent CAR and wherein the scFvs are separated from each other by a peptide linker, and wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 74.

### Claim 9 (depends on 8)

The method of claim 8, wherein each of the scFvs comprises a VH and a VL, the VH and the VL are separated by a peptide linker, and wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 27.

### Claim 10 (depends on 1)

The method of claim 1, wherein the T cell or the NK cell is allogeneic.

### Claim 11 (depends on 1)

The method of claim 1, wherein the delivering comprises intravenous (IV) administration.

### Claim 12 (depends on 1)

The method of claim 1, wherein the AML expresses CD33.

### Claim 13 (depends on 1)

The method of claim 1, wherein the AML expresses FLT3.

### Claim 14 (depends on 1)

The method of claim 1, wherein the AML expresses both CD33 and FLT3.

### Claim 15

A method of providing an anti-tumor immunity in a subject, wherein the anti-tumor immunity comprises targeting AML, the method comprising administering to the subject in need thereof a therapeutically effective dose of an autologous or an allogeneic T cell or NK cell comprising:
(a) an anti-FLT3 CAR comprising a FLT3-binding domain and an anti-CD33 CAR comprising a CD33-binding domain, or a bivalent CAR comprising a FLT3-binding domain and a CD33-binding domain,
wherein the FLT3-binding domain comprises a VH comprising a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 81, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 82, and a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 83 and a VL comprising a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 84, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 85, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 86, and
wherein the CD33-binding domain comprises a VH comprising a CDR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 123, a CDR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 124, and a CDR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 125 and a VL comprising a CDR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 126, a CDR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 127, and a CDR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 128; and
(b) an inhibitory chimeric receptor comprising an extracellular antigen-binding domain that binds to EMCN.

### Claim 16 (depends on 15)

The method of claim 15, wherein the T cell or the NK cell comprises the bivalent CAR.

### Claim 17 (depends on 15)

The method of claim 15, wherein the anti-FLT3 CAR and the anti-CD33 CAR, or the bivalent CAR comprises a CD3zeta-chain intracellular signaling domain and one or more additional intracellular signaling domains selected from the group consisting of: a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, and a 2B4 intracellular signaling domain.

### Claim 18 (depends on 17)

The method of claim 17, wherein the anti-FLT3 CAR and the anti-CD33 CAR, or the bivalent CAR comprises a transmembrane domain selected from the group consisting of: a CD8 transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain.

### Claim 19 (depends on 18)

The method of claim 18, wherein the anti-FLT3 CAR and the anti-CD33 CAR, or the bivalent CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain, and the spacer region has an amino acid sequence selected from the group consisting of the amino acid sequences of: SEQ ID NOs: 55-64.

### Claim 20 (depends on 15)

The method of claim 15, wherein the inhibitory chimeric receptor comprises a scFv, and wherein the scFv comprises an antigen binding domain obtained from an anti-EMCN antibody.

### Claim 21 (depends on 15)

The method of claim 15, wherein the FLT3-binding domain comprises an scFv and the CD33-binding domain comprises an scFv.

### Claim 22 (depends on 21)

The method of claim 21, wherein the T cell or the NK cell comprises the bivalent CAR and wherein the scFvs are separated from each other by a peptide linker, and wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 74.

### Claim 23 (depends on 22)

The method of claim 22, wherein each of the scFvs comprises a VH and a VL, the VH and the VL are separated by a peptide linker, and wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 27.

### Claim 24 (depends on 15)

The method of claim 15, wherein the T cell or the NK cell is allogeneic.

### Claim 25 (depends on 15)

The method of claim 15, wherein the administering comprises IV administration.

### Claim 26 (depends on 15)

The method of claim 15, wherein the AML expresses CD33.

### Claim 27 (depends on 15)

The method of claim 15, wherein the AML expresses FLT3.

### Claim 28 (depends on 15)

The method of claim 15, wherein the AML expresses both CD33 and FLT3.