# US 2020/0399387 A1 · Humanized bcma antibody and bcma-car-t cells

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- Number: US 2020/0399387 A1
- Document ID: US20200399387A1
- Status: Published application
- Kind: A1
- Application number: 17/010,798
- Filed: 2020-09-02
- Published: 2020-12-24
- Earliest claimed: 2019-01-16
- Assignees: Caribou Biosciences, Inc.
- Applicants: Caribou Biosciences, Inc.
- Inventors: LIJUN Wu; Vita Golubovskaya
- Web page: https://patentgrep.com/patent/US20200399387A1

## CPC

- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- 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 40/4215: {Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30}
- A61P 35/00: Antineoplastic agents
- 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)}
- C12N 5/0636: {T lymphocytes}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- 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 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/54: {characterised by the route of administration}
- A61K 2039/804: {Blood cells [leukemia, lymphoma]}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/46: characterised by the cancer treated
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/70: characterized by effect upon binding to a cell or to an antigen
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- 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)

## Abstract

The present invention is directed to a humanized BCMA single-chain variable fragment (scFv), comprising VH having the amino acid sequence of SEQ ID NO: 3 and VL having the amino acid sequence of SEQ ID NO: 5. The present invention is also directed to a BCMA chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) of the present invention, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain. This humanized BCMA-CAR-T cells have specific killing activity against BCMA-positive tumor cells.

## Claims

### Claim at position 1

-11. (canceled)

### Claim 12

An anti-BCMA single-chain variable fragment (scFv), comprising:
a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 3; and
a light chain variable region VL) comprising the amino acid sequence of SEQ ID NO: 5.

### Claim 13 (depends on 12)

The scFv of claim 12, further comprising a linker in between the VH and the VL.

### Claim 14 (depends on 13)

The scFv of claim 13, wherein the linker comprises SEQ ID NO: 4.

### Claim 15 (depends on 13)

The scFv of claim 13, comprising the amino acid sequence of SEQ ID NO: 14.

### Claim 16

An anti-BCMA chimeric antigen receptor (CAR), comprising:
an anti-BCMA single-chain variable fragment (scFv) comprising
a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 3, and
a light chain variable region VL) comprising the amino acid sequence of SEQ ID NO: 5;
a transmembrane domain;
a co-stimulatory domain; and
an activating domain.

### Claim 17 (depends on 16)

The CAR of claim 16, wherein the transmembrane domain is a transmembrane domain selected from the group consisting of a T cell receptor α chain, a T cell receptor β chain, a CD3 zeta chain, a CD28, a CD3ε, a CD45, a CD4, a CD5, a CD8, a CD9, a CD16, a CD22, a CD33, a CD37, a CD64, a CD80, a CD86, a CD134, a CD137, an ICOS, a CD154, and a GITR.

### Claim 18 (depends on 17)

The CAR of claim 17, wherein the transmembrane domain comprises a transmembrane domain of a CD8.

### Claim 19 (depends on 16)

The CAR of claim 16, wherein the co-stimulatory domain is a co-stimulatory domain selected from the group consisting of a CD28, a 4-1BB, a GITR, an ICOS-1, a CD27, an OX-40, and a DAP10.

### Claim 20 (depends on 19)

The CAR of claim 19, wherein the co-stimulatory domain comprises a 4-1BB co-stimulatory domain.

### Claim 21 (depends on 16)

The CAR of claim 16, wherein the activating domain comprises a CD3 zeta activating domain.

### Claim 22 (depends on 16)

The CAR of claim 16, wherein the transmembrane domain comprises a transmembrane domain of a CD8, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain, and the activating domain comprises a CD3 zeta activating domain.

### Claim 23 (depends on 16)

The CAR of claim 16, comprising the amino acid sequence of SEQ ID NO: 17.

### Claim 24 (depends on 16)

A nucleic acid, having a 5′ end and a 3′ end, encoding the CAR of claim 16.

### Claim 25 (depends on 24)

The nucleic acid of claim 24, further comprising a leader sequence at the 5′ end.

### Claim 26 (depends on 25)

The nucleic acid of claim 25, wherein the leader sequence comprises the nucleic acid sequence of SEQ ID NO: 7.

### Claim 27 (depends on 24)

The nucleic acid of claim 24, further comprising a promoter at the 5′ end.

### Claim 28 (depends on 27)

The nucleic acid of claim 27, wherein the promoter comprises an EF1a promoter.

### Claim 29 (depends on 24)

A vector, comprising the nucleic acid of claim 24.

### Claim 30 (depends on 30)

The vector of claim 30, wherein the vector is a virus vector.

### Claim 31 (depends on 16)

A CAR-T cell, comprising the CAR of claim 16.

### Claim 32 (depends on 23)

A CAR-T cell, comprising the CAR of claim 23.

### Claim 33

A method of killing BCMA-positive cancer cells comprising:
contacting the BCMA-positive cancer cells with a CAR-T cell comprising an anti-BCMA chimeric antigen receptor (CAR) comprising
an anti-BCMA single-chain variable fragment (scFv) comprising
a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 3, and
a light chain variable region VL) comprising the amino acid sequence of SEQ ID NO: 5;
a transmembrane domain;
a co-stimulatory domain; and
an activating domain.

### Claim 34 (depends on 33)

The method of claim 33, wherein the contacting is in vitro.

### Claim 35 (depends on 33)

The method of claim 33, wherein the BCMA-positive cancer cells comprise multiple myeloma cancer cells.

### Claim 36 (depends on 35)

The method of claim 35, wherein the multiple myeloma cancer cells comprise human cells.

### Claim 37 (depends on 33)

The method of claim 33, wherein the contacting is intra-tumoral.

### Claim 38

A method of making an anti-BCMA CAR-T cell, comprising:
introducing into a T cell a nucleic acid encoding an anti-BCMA chimeric antigen receptor (CAR) comprising
an anti-BCMA single-chain variable fragment (scFv) comprising
a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 3, and
a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 5;
a transmembrane domain;
a co-stimulatory domain; and
an activating domain.

### Claim 39 (depends on 38)

The method of claim 38, wherein the nucleic acid is introduced into the T cell in vitro.

### Claim 40 (depends on 38)

The method of claim 38, wherein the nucleic acid is introduced into the T cell by a virus vector.

### Claim 41 (depends on 40)

The method of claim 40, wherein the virus vector is selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector, a Sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV vector.