# US 10,870,874 B2 · Binding fusion proteins, binding fusion protein-drug conjugates, XTEN-drug conjugates and methods of making and using same

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- Number: US 10,870,874 B2
- Document ID: US10870874B2
- Status: Granted
- Kind: B2
- Application number: 15/955,046
- Filed: 2018-04-17
- Published: 2018-12-06
- Granted: 2020-12-22
- Earliest claimed: 2010-04-02
- Assignees: Amunix Pharmaceuticals, Inc.
- Applicants: Amunix Pharmaceuticals, Inc.
- Inventors: Volker Schellenberger; Joshua Silverman; Chia-wei Wang; Benjamin Spink; Willem P. C. Stemmer; Nathan C. Geething
- Web page: https://patentgrep.com/patent/US10870874B2

## CPC

- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 11/08: Bronchodilators
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/70521: {CD28, CD152}
- C07K 14/7155: {for interleukins [IL]}
- C07K 14/8125: {Alpha-1-antitrypsin}
- C07K 16/18: against material from animals or humans
- C07K 16/241: {Tumor Necrosis Factors}
- C07K 16/244: {Interleukins [IL]}
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 16/32: against translation products of oncogenes
- C07K 16/40: against enzymes
- C12N 15/625: {containing a sequence coding for a signal sequence}
- 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 2317/31: multispecific
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C07K 2319/70: containing domain for protein-protein interaction
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

The present invention relates to binding fusion protein compositions comprising targeting moieties linked to extended recombinant polypeptide (XTEN), binding fusion protein-drug conjugate compositions, and XTEN-drug conjugate compositions, isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of using such compositions in treatment of diseases, disorders, and conditions.

## Claims

### Claim 1

A binding fusion protein comprising:
a first targeting moiety comprising a single-chain variable fragment (scFv) with specific binding affinity to CD3; and
a first extended recombinant polypeptide (XTEN), wherein the first XTEN comprises a motif selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

### Claim 2 (depends on 1)

The binding fusion protein of claim 1, wherein the first XTEN comprises an amino acid sequence which has at least 90% sequence identity to a sequence selected from the group consisting of AE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQ ID NO: 54) and AE864 (SEQ ID NO: 59).

### Claim 3 (depends on 2)

The binding fusion protein of claim 2, wherein the first XTEN comprises an amino acid sequence selected from the group consisting of AE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQ ID NO: 54) and AE864 (SEQ ID NO: 59).

### Claim 4 (depends on 1)

The binding fusion protein of claim 1, wherein the first targeting moiety has specific binding affinity for an epsilon subunit of CD3 (CDR).

### Claim 5 (depends on 1)

The binding fusion protein of claim 1, wherein the first XTEN is positioned N-terminal of the first targeting moiety.

### Claim 6 (depends on 1)

The binding fusion protein of claim 1, wherein the first XTEN is positioned C-terminal of the first targeting moiety.

### Claim 7 (depends on 1)

The binding fusion protein of claim 1, further comprising a second targeting moiety that differs from the first targeting moiety.

### Claim 8 (depends on 1)

The binding fusion protein of claim 1, wherein the binding fusion protein is configured according to formula I:
(XTEN)x-TM-(XTEN)y I
wherein x is either 0 or 1; y is either 0 or 1, wherein x+y≥1, and wherein TM is the first targeting moiety.

### Claim 9 (depends on 8)

The binding fusion protein of claim 8, wherein x is 1 and y is 1.

### Claim 10 (depends on 7)

The binding fusion protein of claim 7, further comprising a second XTEN, wherein the second XTEN comprises a motif selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

### Claim 11 (depends on 10)

The binding fusion protein of claim 10, wherein the second XTEN comprises an amino acid sequence which has at least 90% sequence identity to a sequence selected from the group consisting of AE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQ ID NO: 54) and AE864 (SEQ ID NO: 59).

### Claim 12 (depends on 11)

The binding fusion protein of claim 11, wherein the second XTEN comprises an amino acid sequence selected from the group consisting of AE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQ ID NO: 54) and AE864 (SEQ ID NO: 59).

### Claim 13 (depends on 10)

The binding fusion protein of claim 10, wherein the first XTEN is positioned at the N-terminus of the binding fusion protein and the second XTEN is positioned at the C-terminus of binding fusion protein.

### Claim 14 (depends on 1)

A pharmaceutical composition comprising the binding fusion protein of claim 1, and at least one pharmaceutically acceptable carrier.

### Claim 15 (depends on 14)

The pharmaceutical composition of claim 14, wherein the first XTEN comprises an amino acid sequence which has at least 90% sequence identity to a sequence selected from the group consisting of AE144 (SEQ ID NO: 41), AE288 (SEQ ID NO: 47), AE576 (SEQ ID NO: 54) and AE864 (SEQ ID NO: 59).

### Claim 16 (depends on 14)

A kit comprising:
packaging material;
at least a first container comprising the pharmaceutical composition of claim 14;
a label identifying the pharmaceutical composition and providing storage and handling conditions; and
a sheet of instructions for the reconstitution and/or administration of the pharmaceutical composition to a subject.

### Claim 17 (depends on 14)

A method of treating a human disease, the method comprising administering a therapeutically effective dose of the pharmaceutical composition of claim 14 to direct cytotoxic activity of T-cells towards tumor cells.

### Claim 18 (depends on 17)

The method of claim 17, wherein the human disease is selected from the group consisting of breast carcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma, colorectal carcinoma, liver carcinoma, ovarian carcinoma, thecoma, arrhenoblastoma, cervical carcinoma, endometrial carcinoma, fibrosarcoma, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinoma, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinoma, thyroid carcinoma, Wilm's tumor, renal cell carcinoma, and prostate carcinoma.

### Claim 19 (depends on 7)

The isolated binding fusion protein of claim 7, wherein the second targeting moiety comprises a second single-chain variable fragment (scFv).