# US 10,865,403 B2 · Engineered primate cystine/cysteine degrading enzymes for therapeutic uses

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,865,403 B2
- Document ID: US10865403B2
- Status: Granted
- Kind: B2
- Application number: 15/977,246
- Filed: 2018-05-11
- Published: 2018-11-15
- Granted: 2020-12-15
- Earliest claimed: 2017-05-12
- Assignees: Board of Regents, The University of Texas System
- Applicants: Board of Regents, The University of Texas System
- Inventors: George Georgiou; Everett Stone; Shira Cramer
- Web page: https://patentgrep.com/patent/US10865403B2

## CPC

- C12N 9/88: Lyases (4.)
- A61B 17/225: for extracorporeal shock wave lithotripsy [ESWL], e.g. by using ultrasonic waves
- 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 31/7076: containing purines, e.g. adenosine, adenylic acid
- A61K 38/16: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof {(enzyme inhibitors A61K38/005)}
- A61K 38/38: Albumins
- A61K 38/51: Lyases (4)
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/61: the organic macromolecular compound being a polysaccharide or a derivative thereof
- A61P 35/00: Antineoplastic agents
- C07K 14/001: {by chemical synthesis}
- C07K 14/76: Albumins
- C12Y 404/01001: Cystathionine gamma-lyase (4.4.1.1)
- 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 2319/00: Fusion polypeptide
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D)
- 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
- 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)
- C12Y: ENZYMES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Methods and compositions related to the engineering of a protein with L-cyst(e)ine degrading enzyme activity are described. For example, disclosed are modified cystathionine-γ-lyases comprising one or more amino acid substitutions and capable of degrading L-cyst(e)ine. Furthermore, compositions and methods are provided for the treatment of cancer and cystinuria using the disclosed modified enzymes or nucleic acids encoding said enzymes.

## Claims

### Claim 1

An isolated, modified primate cystathionine-γ-lyase (CGL) enzyme having the following substitutions relative to a native human CGL amino acid sequence (SEQ ID NO: 1), wherein the modified enzyme has both cystinase and cysteinase activity, and wherein said primate is selected from the group consisting of Homo sapiens, Pongo abelii, Pan troglodytes, Pan paniscus, and Macaca fascicularis; said substitutions comprising: a glutamic acid at position 55, a threonine at position 59, and a valine at position 339.

### Claim 2 (depends on 1)

The isolated, modified CGL enzyme of claim 1, further comprising an aspartic acid at position 336.

### Claim 3 (depends on 1)

The isolated, modified CGL enzyme of claim 1, wherein the isolated, modified CGL enzyme is a modified Pongo abelii CGL enzyme, wherein said substitutions comprise H55E, V59T, and E339V.

### Claim 4 (depends on 3)

The isolated, modified CGL enzyme of claim 3, further comprising a T336D substitution.

### Claim 5 (depends on 1)

The isolated, modified CGL enzyme of claim 1, wherein the isolated, modified CGL enzyme is a modified Homo sapiens CGL enzyme, a modified Macaca fascicularis CGL enzyme, a modified Pan troglodytes CGL enzyme, or a modified Pan paniscus CGL enzyme, wherein said substitutions comprise H55E, E59T, and E339V.

### Claim 6 (depends on 5)

The isolated, modified CGL enzyme of claim 5, further comprising a T336D substitution.

### Claim 7 (depends on 1)

The isolated, modified CGL enzyme of claim 1, further comprising a heterologous peptide segment or a polysaccharide.

### Claim 8 (depends on 7)

The isolated, modified CGL enzyme of claim 7, wherein the heterologous peptide segment is an XTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide.

### Claim 9 (depends on 7)

The isolated, modified CGL enzyme of claim 7, wherein the polysaccharide comprises polysialic acid polymers.

### Claim 10 (depends on 1)

The isolated, modified CGL enzyme of claim 1, wherein the enzyme is coupled to a polyethylene glycol (PEG).

### Claim 11 (depends on 10)

The isolated, modified CGL enzyme of claim 10, wherein the enzyme is coupled to the PEG via one or more lysine residues.

### Claim 12 (depends on 1)

A therapeutic formulation comprising an enzyme of claim 1, in a pharmaceutically acceptable carrier.

### Claim 13 (depends on 12)

A method of treating a tumor cell or subject having a tumor cell comprising administering to the tumor cell or the subject a therapeutically effective amount of the formulation of claim 12.

### Claim 14 (depends on 13)

The method of claim 13, wherein the subject is maintained on a L-cystine and/or L-cysteine restricted diet.

### Claim 15 (depends on 13)

The method of claim 13, wherein the subject is maintained on a normal diet.

### Claim 16 (depends on 13)

The method of claim 13, wherein the tumor cell has been identified as having a decreased expression level of a cystathionine-β-synthase or cystathionine-γ-lyase gene product relative to a non-tumor cell.

### Claim 17 (depends on 13)

The method of claim 13, wherein the tumor cell has been identified as having an increased expression level of a xCT(−) cystine/glutamate antiporter gene product relative to a non-tumor cell.

### Claim 18 (depends on 13)

The method of claim 13, wherein the tumor cell is a prostate cancer cells, a small cell lung cancer cell, or a hematological malignancy cell.

### Claim 19 (depends on 18)

The method of claim 18, wherein the hematologic malignancy cell is a chronic myelogenous leukemia cell, chronic lymphocytic leukemia cell, acute myelogenous leukemia cell, acute lymphocytic leukemia cell, acute monocytic leukemia cell, diffuse large B-cell lymphoma, myelodysplastic syndrome, chronic myelomonocytic leukemia, primary myelofibrosis, Hodgkin's lymphoma cell, non-Hodgkin's lymphoma cell, or multiple myeloma cell.

### Claim 20 (depends on 13)

The method of claim 13, wherein the subject is a human patient.

### Claim 21 (depends on 13)

The method of claim 13, wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intralesionally, intramuscularly, intravesicularlly, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a catheter.

### Claim 22 (depends on 13)

The method of claim 13, wherein the formulation is administered to a nutrient medium of the tumor cell.

### Claim 23 (depends on 22)

The method of claim 22, wherein the nutrient medium is blood, lymphatic fluid, or spinal fluid.

### Claim 24 (depends on 13)

The method of claim 13, further comprising administering at least a second anticancer therapy to the subject.

### Claim 25 (depends on 24)

The method of claim 24, wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy.

### Claim 26 (depends on 24)

The method of claim 24, wherein the secondary anticancer therapy is fludarabine.

### Claim 27 (depends on 12)

A method of treating a subject having or at risk of developing cystinuria comprising administering to the subject a therapeutically effective amount of a formulation of claim 12.

### Claim 28 (depends on 27)

The method of claim 27, wherein the subject is maintained on a L-cystine and/or L-cysteine restricted diet.

### Claim 29 (depends on 27)

The method of claim 27, wherein the subject is maintained on a methionine-restricted diet.

### Claim 30 (depends on 27)

The method of claim 27, wherein the subject is maintained on a normal diet.

### Claim 31 (depends on 27)

The method of claim 27, wherein the subject is a human patient.

### Claim 32 (depends on 27)

The method of claim 27, wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intramuscularly, intravascularly, subcutaneously, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a catheter.

### Claim 33 (depends on 27)

The method of claim 27, wherein the subject has previously been treated for cystinuria and the enzyme is administered to prevent the recurrence of cystinuria.

### Claim 34 (depends on 27)

The method of claim 27, further comprising administering at least a second cystinuria therapy to the subject.

### Claim 35 (depends on 34)

The method of claim 34, wherein the second cystinuria therapy is a surgical therapy or a shock wave therapy.

### Claim 36

An isolated, modified human cystathionine-γ-lyase (CGL) enzyme having the following substitutions relative to a native human CGL amino acid sequence (SEQ ID NO: 1), wherein the modified enzyme has both cystinase and cysteinase activity, said substitutions comprising: a glutamic acid at position 55, a threonine at position 59, and a valine at position 339.