# US 11,440,948 B2 · Modified non-natural NKG2D ligands that selectively deliver attached heterologous molecules to non-natural NKG2D receptors on car-cells

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- Number: US 11,440,948 B2
- Document ID: US11440948B2
- Status: Granted
- Kind: B2
- Application number: 16/774,567
- Filed: 2020-01-28
- Published: 2020-07-30
- Granted: 2022-09-13
- Earliest claimed: 2019-01-28
- Assignees: Xyphos Biosciences Inc.; The J. David Gladstone Institutes
- Applicants: Xyphos Biosciences Inc.; The J. David Gladstone Institutes, A Testamentary Trust Established Under the Will of J. David Gladstone
- Inventors: Kaman Kim; Nigel Killeen; Eytan Herzig; Warner Greene
- Web page: https://patentgrep.com/patent/US11440948B2

## CPC

- C07K 14/7056: {Lectin superfamily, e.g. CD23, CD72}
- 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 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4205: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4}
- A61K 40/4224: {Molecules with a "CD" designation not provided for elsewhere}
- A61K 40/46: Viral antigens
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- C07K 14/16: HIV-1 {; HIV-2}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/1145: {Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2887: {against CD20}
- C07K 16/32: against translation products of oncogenes
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2317/71: Decreased effector function due to an Fc-modification
- 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 2319/00: Fusion polypeptide
- 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
- 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

## Abstract

This application relates generally to the production of modified, non-natural α1-α2 domains of NKG2D ligands with attached polypeptides having specific target-binding properties, for example, antibodies or variable fragments of antibodies, that are selectively delivered to Chimeric Antigen Receptors (CARs) comprised of modified, non-natural NKG2D receptors on engineered mammalian cells. The targeting of surface-expressed molecules includes those of virus-infected cells that can then be attacked and ablated by engineered cells of the immunity system expressing CARs cognate to the modified, non-natural α1-α2 domains of NKG2D ligands with attached polypeptides.

## Claims

### Claim 1

A modified, non-natural ligand for a modified, non-natural NKG2D (Natural Killer Group 2D) receptor,
wherein the ligand has an attached heterologous molecule that selectively binds a human immunodeficiency virus (HIV) protein present on the surface of a cell infected by HIV, wherein said heterologous molecule selectively binds to an epitope sequence that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 169 and SEQ ID NO: 170, and
wherein the modified ligand with its attached heterologous molecule can selectively bind to a modified, non-natural NKG2D receptor of a chimeric antigen receptor (CAR)-cell and cause the destruction of the HIV-infected cell.

### Claim 2 (depends on 1)

The modified, non-natural ligand of claim 1, wherein said modified, non-natural ligand comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-72.

### Claim 3 (depends on 1)

The modified, non-natural ligand of claim 1, wherein said modified, non-natural ligand binds to a non-natural NKG2D receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 54 and SEQ ID NO: 154.

### Claim 4 (depends on 1)

The modified, non-natural ligand of claim 1, wherein said modified, non-natural ligand is bound to a CAR-cell.

### Claim 5 (depends on 4)

The modified, non-natural ligand of claim 4, wherein said CAR-cell comprises a modified, non-natural NKG2D receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 54 and SEQ ID NO: 154, and
wherein a modified, non-natural ligand that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-72 to which is attached a heterologous molecule or atom that does not bind an HIV protein is further bound to said CAR-cell.

### Claim 6 (depends on 5)

The modified, non-natural ligand of claim 5, wherein said heterologous molecule or atom that does not bind an HIV protein modulates a function of the CAR-cell.

### Claim 7 (depends on 6)

The modified, non-natural ligand of claim 6, wherein said function is a function selected from the group consisting of proliferation, differentiation, ablation, imaging, antagonism of immunosuppression, homing, and cytolysis of a cell not infected by HIV.