# US 2017/0281682 A1 · Chimeric receptors and uses thereof in immune therapy

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- Number: US 2017/0281682 A1
- Document ID: US20170281682A1
- Status: Published application
- Kind: A1
- Application number: 15/509,133
- Filed: 2015-09-09
- Published: 2017-10-05
- Earliest claimed: 2014-09-09
- Assignees: Unum Therapeutics Inc.
- Applicants: Unum Therapeutics Inc.
- Inventors: Charles Wilson; Kathleen McGinness
- Web page: https://patentgrep.com/patent/US20170281682A1

## CPC

- C07K 14/70535: {Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)}
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- 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 40/4205: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4}
- A61K 40/4211: {CD19 or B4}
- A61K 40/4221: {CD20}
- A61K 40/4258: {Gangliosides, e.g. GM2, GD2 or GD3}
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/04: Immunostimulants
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/70517: {CD8}
- C07K 16/3084: {against tumour-associated gangliosides}
- C07K 16/32: against translation products of oncogenes
- C12N 5/0636: {T lymphocytes}
- C12N 15/86: Viral vectors
- A61K 2039/505: {comprising antibodies}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/70: containing domain for protein-protein interaction
- C12N 2510/00: Genetically modified cells
- C12N 2740/10043: viral genome or elements thereof as genetic vector
- 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

Disclosed herein are chimeric receptors comprising an extracellular domain with affinity and specific for the Fc portion of an immunoglobulin molecule (Ig), an Fc-binding domain; a transmembrane domain; at least one co-stimulatory signaling domain; and a cytoplasmic signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). Also provided herein are nucleic acids encoding such chimeric receptors and immune cells expressing the chimeric receptors. Such immune cells can be used to enhance antibody-dependent cell-mediated cytotoxicity and/or to enhance antibody-based immunotherapy, such as cancer immunotherapy.

## Claims

### Claim 1

A chimeric receptor, comprising:
(a) an Fc binding domain;
(b) a transmembrane domain;
(c) at least one co-stimulatory signaling domain; and
(d) a cytoplasmic signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); wherein either (c) or (d) is located at the C-terminus of the chimeric receptor; and wherein
(i) if (a) is an extracellular ligand-binding domain of CD16A, (d) does not comprise an ITAM domain of an Fc receptor, and
(ii) the chimeric receptor is not a receptor comprising, from N-terminus to C-terminus, an extracellular ligand-binding domain of F158 CD16A or V158 CD16A, a hinge and transmembrane domain of CD8a, a co-stimulatory signaling domain of 4-1BB, and a cytoplasmic signaling domain of CD3ζ.

### Claim 2 (depends on 1)

The chimeric receptor of claim 1, wherein (d) is located at the C-terminus of the chimeric receptor.

### Claim 3 (depends on 1)

The chimeric receptor of claim 1, further comprising (e) a hinge domain, which is located at the C-terminus of (a) and the N-terminus of (b).

### Claim 4 (depends on 1)

The chimeric receptor of claim 1, wherein the chimeric receptor further comprises a signal peptide at its N-terminus.

### Claim 5 (depends on 1)

The chimeric receptor of claim 1, wherein the Fc binding domain of (a) is selected from the group consisting of:
(i) an extracellular ligand-binding domain of an Fc-receptor, which optionally is an Fc-gamma receptor, an Fc-alpha receptor, or an Fc-episilon receptor,
(ii) an antibody fragment that binds the Fc portion of an immunoglobulin,
(iii) a naturally-occurring protein that binds the Fc portion of an immunoglobulin or an Fc-binding fragment thereof, and
(iv) a synthetic polypeptide that binds the Fc portion of an immunoglobulin.

### Claim 6 (depends on 5)

The chimeric receptor of claim 5, wherein the Fc binding domain is (i), which is an extracellular ligand-binding domain of CD16A, CD32A, or CD64A.

### Claim 7 (depends on 6)

The chimeric receptor of claim 6, wherein the Fc binding domain is an extracellular ligand-binding domain of CD32A or CD64A.

### Claim 8 (depends on 5)

The chimeric receptor of claim 5, wherein the Fc binding domain is (ii), which is a single chain variable fragment (ScFv), a domain antibody, or a nanobody.

### Claim 9 (depends on 5)

The chimeric receptor of claim 5, wherein the Fc binding domain is (iii), which is Protein A or Protein G.

### Claim 10 (depends on 5)

The chimeric receptor of claim 5, wherein the Fc binding domain is (iv), which is a Kunitz peptide, a SMIP, an avimer, an affibody, a DARPin, or an anticalin.

### Claim 11 (depends on 1)

The chimeric receptor of claim 1, wherein the transmembrane domain of (b) is of a single-pass membrane protein.

### Claim 12 (depends on 11)

The chimeric receptor of claim 11, wherein the transmembrane domain is of a membrane protein selected from the group consisting of CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16A, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, and FGFR2B.

### Claim 13 (depends on 11)

The chimeric receptor of claim 11, wherein the transmembrane domain is of a membrane protein selected from the group consisting of CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16A, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, and FGFR2B.

### Claim 14 (depends on 1)

The chimeric receptor of claim 1, wherein the transmembrane domain of (b) is a non-naturally occurring hydrophobic protein segment.

### Claim 15 (depends on 1)

The chimeric receptor of claim 1, wherein the at least one co-stimulatory signaling domain of (c) is of a co-stimulatory molecule selected from the group consisting of 4-1BB, CD28, CD28LL→GG variant, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, and CD2.

### Claim 16 (depends on 1)

The chimeric receptor of claim 1, wherein the at least one co-stimulatory signaling domain of (c) is of a co-stimulatory molecule selected from the group consisting of CD28, CD28LL→GG variant, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, and CD2.

### Claim 17 (depends on 1)

The chimeric receptor of claim 1, wherein the chimeric receptor comprises two co-stimulatory signaling domains.

### Claim 18 (depends on 17)

The chimeric receptor of claim 17, wherein the two co-stimulatory domains are:
(i) CD28 and 4-1BB; or
(ii) CD28LL→GG variant and 4-1BB.

### Claim 19 (depends on 1)

The chimeric receptor of claim 1, wherein the cytoplasmic signaling domain of (d) is a cytoplasmic domain of CD3 or FcεR1γ.

### Claim 20 (depends on 3)

The chimeric receptor of claim 3, wherein the hinge domain of (e) is of CD8α or IgG.

### Claim 21 (depends on 3)

The chimeric receptor of claim 3, wherein the hinge domain is a non-naturally occurring peptide.

### Claim 22 (depends on 21)

The chimeric receptor of claim 21, wherein the hinge domain is an extended recombinant polypeptide (XTEN) or a (Gly4Ser)n polypeptide, in which n is an integer of 3-12, inclusive.

### Claim 23 (depends on 3)

The chimeric receptor of claim 3, wherein the chimeric receptor comprises components (a)-(e) as shown in Table 3.

### Claim 24 (depends on 23)

The chimeric receptor of claim 23, wherein the chimeric receptor comprises the amino acid sequence selected from SEQ ID NOs:2-11.

### Claim 25 (depends on 3)

The chimeric receptor of claim 3, wherein the chimeric receptor comprises components (a)-(e) as shown in Table 4.

### Claim 26 (depends on 25)

The chimeric receptor of claim 25, wherein the chimeric receptor comprises the amino acid sequence selected from SEQ ID NOs:12-17.

### Claim 27 (depends on 3)

The chimeric receptor of claim 3, wherein the chimeric receptor comprises components (a)-(e) as shown in Table 5.

### Claim 28 (depends on 27)

The chimeric receptor of claim 27, wherein the chimeric receptor comprises the amino acid sequence selected from SEQ ID NOs:18-30, and 32-56.

### Claim 29 (depends on 1)

A nucleic acid comprising a nucleotide sequence encoding a chimeric receptor of claim 1.

### Claim 30 (depends on 29)

The nucleic acid of claim 29, wherein the nucleic acid is an RNA molecule.

### Claim 31 (depends on 29)

A vector comprising the nucleic acid of claim 29.

### Claim 32 (depends on 31)

The vector of claim 31, wherein the vector is an expression vector.

### Claim 33 (depends on 32)

The vector of claim 32, wherein the vector is a viral vector.

### Claim 34 (depends on 33)

The vector of claim 33, wherein the viral vector is a lentiviral vector or a retroviral vector.

### Claim 35 (depends on 29)

A host cell, comprising a nucleic acid of claim 29.

### Claim 36 (depends on 35)

The host cell of claim 35, wherein the host cell is an immune cell.

### Claim 37 (depends on 36)

The host cell of claim 36, wherein the immune cell is a natural killer cell, macrophage, neutrophil, eosinophil, or T cell.

### Claim 38 (depends on 35)

The host cell of claim 35, wherein the host cell is a T cell in which the expression of the endogenous T cell receptor has been inhibited or eliminated.

### Claim 39 (depends on 35)

A pharmaceutical composition, comprising (a) or a host cell of claim 35 and (b) a pharmaceutically acceptable carrier.

### Claim 40 (depends on 39)

The pharmaceutical composition of claim 39, wherein the composition further comprises an Fc-containing therapeutic agent.

### Claim 41 (depends on 40)

The pharmaceutical composition of claim 40, wherein the Fc-containing therapeutic agent is an Fc fusion protein.

### Claim 42 (depends on 40)

The pharmaceutical composition of claim 40, wherein the Fc-containing therapeutic agent is a therapeutic antibody or an Fc fusion protein.

### Claim 43 (depends on 42)

The pharmaceutical composition of claim 42, wherein the Fc-containing therapeutic agent is a therapeutic antibody selected from the group consisting of Adalimumab, Ado-Trastuzumab emtansine, Alemtuzumab, Basiliximab, Bevacizumab, Belimumab, Brentuximab, Canakinumab, Cetuximab, Certolizumab, Daclizumab, Denosumab, Dinutuximab, Eculizumab, Efalizumab, Epratuzumab, Gemtuzumab, Golimumab, Ibritumomab, Infliximab, Ipilimumab, Labetuzumab, Muromonab, Natalizumab, Obinutuzumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Pertuzumab, Ramucirumab, Ranibizumab, Rituximab, Tocilizumab, Trastuzumab, Tositumomab. Ustekinumab, and Vedolizumab.

### Claim 44 (depends on 35)

A kit, comprising:
a first pharmaceutical composition that comprises (i) a host cell of claim 35 and (ii) a pharmaceutically acceptable carrier; and
a second pharmaceutical composition that comprises an Fc-containing therapeutic agent and a pharmaceutically acceptable carrier.

### Claim 45 (depends on 44)

The kit of claim 44, wherein the Fc-containing therapeutic agent is an Fc fusion protein.

### Claim 46 (depends on 44)

The kit of claim 44, wherein the Fc-containing therapeutic agent is a therapeutic antibody.

### Claim 47 (depends on 46)

The kit of claim 46, wherein the therapeutic antibody is selected from the group consisting of Adalimumab, Ado-Trastuzumab emtansine, Alemtuzumab, Basiliximab, Bevacizumab, Belimumab, Brentuximab, Canakinumab, Cetuximab, Certolizumab, Daclizumab, Denosumab, Dinutuximab, Eculizumab, Efalizumab, Epratuzumab, Gemtuzumab, Golimumab, Ibritumomab, Infliximab, Ipilimumab, Labetuzumab, Muromonab, Natalizumab, Obinutuzumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Pertuzumab, Ramucirumab, Ranibizumab, Rituximab, Tocilizumab, Trastuzumab, Tositumomab. Ustekinumab, and Vedolizumab.

### Claim 48 (depends on 1)

A method for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) or for enhancing efficacy of an antibody-based immunotherapy in a subject, comprising administering to the subject an effective amount of host immune cells that express a chimeric receptor of claim 1.

### Claim 49 (depends on 48)

The method of claim 48, wherein the host immune cells are natural killer cells, macrophages, neutrophils, eosinophils, T cells, or a combination thereof.

### Claim 50 (depends on 48)

The method of claim 48, wherein the host immune cells are autologous.

### Claim 51 (depends on 48)

The method of claim 48, wherein the host immune cells are allogeneic.

### Claim 52 (depends on 48)

The method of claim 48, wherein the host immune cells are activated, expanded, or both ex vivo.

### Claim 53 (depends on 48)

method of claim 48, wherein the subject has been treated or is being treating with an Fc-containing therapeutic agent.

### Claim 54 (depends on 53)

The method of claim 53, wherein the Fc-containing therapeutic agent is a therapeutic antibody or a Fc fusion protein.

### Claim 55 (depends on 54)

The method of claim 54, wherein the Fc-containing therapeutic agent is a therapeutic antibody selected from the group consisting of Adalimumab, Ado-Trastuzumab emtansine, Alemtuzumab, Basiliximab, Bevacizumab, Belimumab, Brentuximab, Canakinumab, Cetuximab, Certolizumab, Daclizumab, Denosumab, Dinutuximab, Eculizumab, Efalizumab, Epratuzumab, Gemtuzumab, Golimumab, Ibritumomab, Infliximab, Ipilimumab, Labetuzumab, Muromonab, Natalizumab, Obinutuzumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Pertuzumab, Ramucirumab, Ranibizumab, Rituximab, Toclizumab, Tositumomab, Trastuzumab, Ustekinumab, and Vedolizumab.

### Claim 56 (depends on 53)

The method of claim 53, wherein the subject is a human patient suffering from a cancer and the Fc-containing therapeutic agent is for treating the cancer.

### Claim 57 (depends on 56)

The method of claim 56, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia.

### Claim 58 (depends on 57)

The method of claim 57, wherein the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, and thyroid cancer.

### Claim 59 (depends on 58)

The method of claim 58, wherein the cancer of b-cell original is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

### Claim 60 (depends on 1)

A method for preparing immune cells expressing a chimeric receptor, comprising:
(i) providing a population of immune cells;
(ii) introducing into the immune cells a nucleic acid encoding a chimeric receptor of claim 1; and
(iii) culturing the immune cells under conditions allowing for expression of the chimeric receptor.

### Claim 61 (depends on 60)

The method of claim 60, wherein the population of immune cells [[are]] is derived from peripheral blood mononuclear cells (PBMC).

### Claim 62 (depends on 60)

The method of claim 60, wherein the immune cells are natural killer cells, macrophages, neutrophils, eosinophils, T cells, or a combination thereof.

### Claim 63 (depends on 60)

The method of claim 60, wherein the immune cells are derived from a human patient.

### Claim 64 (depends on 63)

The method of claim 63, wherein the human patient is a cancer patient.

### Claim 65 (depends on 60)

The method of claim 60, wherein the nucleic acid is a viral vector.

### Claim 66 (depends on 65)

The method of claim 65, wherein the viral vector is a lentiviral vector or a retroviral vector.

### Claim 67 (depends on 60)

The method of claim 60, wherein the nucleic acid is an RNA molecule.

### Claim 68 (depends on 60)

The method of claim 60, wherein the vector is introduced into the immune cells by lentiviral transduction, retroviral transduction, DNA electroporation, or RNA electroporation.

### Claim 69 (depends on 60)

The method of claim 60, further comprising (iv) activating the immune cells expressing the chimeric receptor.

### Claim 70 (depends on 69)

The method of claim 69, wherein the immune cells comprise T cells, which are activated in the presence of one or more of anti-CD3 antibody, anti-CD28 antibody, IL-2, and phytohemoagglutinin.

### Claim 71 (depends on 70)

The method of claim 70, wherein the immune cells are T cells in which the endogenous T cell receptors are inhibited or eliminated.

### Claim 72 (depends on 69)

The method of claim 69, wherein the immune cells comprise natural killer cells, which are activated in the presence of one or more of 4-1BB ligand, anti-4-1BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-12, IL-21 and K562 cells.