# US 12,600,762 B2 · Masked cytokines and methods of use thereof

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- Number: US 12,600,762 B2
- Document ID: US12600762B2
- Status: Granted
- Kind: B2
- Application number: 17/787,741
- Filed: 2021-01-11
- Published: 2023-04-06
- Granted: 2026-04-14
- Earliest claimed: 2020-01-11
- Assignees: AskGene Pharma Inc.
- Applicants: Askgene Pharma, Inc.
- Inventors: Yuefeng Lu; Chunxiao Yu; Jian-Feng Lu; Liqin Liu
- Web page: https://patentgrep.com/patent/US12600762B2

## CPC

- C07K 14/7155: {for interleukins [IL]}
- C07K 14/54: Interleukins [IL]
- C07K 14/5418: {IL-7}
- C07K 14/5443: {IL-15}
- C07K 14/55: IL-2
- C07K 16/244: {Interleukins [IL]}
- C07K 16/2818: {against CD28 or CD152}
- 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 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2319/00: Fusion polypeptide
- 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)

## Abstract

Provided herein are prodrugs and methods of making and using thereof for stimulating the immune system, or treating cancer, autoimmune or an infectious disease.

## Claims

### Claim 1

A prodrug comprising a cytokine moiety, a masking moiety, and a carrier moiety, wherein:
a) the masking moiety binds to the cytokine moiety and inhibits a biological activity of the cytokine, wherein the masking moiety comprises a single chain variable fragment (scFv) against the cytokine moiety or comprises an extracellular domain (ECD) of a receptor for the cytokine moiety;
b) the carrier moiety comprises an antigen-binding moiety, wherein the antigen-binding moiety binds to an antigen expressed on the surface of a target immune cell; and
c) the masking moiety is linked directly or indirectly to the carrier moiety;
wherein the target immune cell expresses on its surface a cytokine receptor for the cytokine moiety such that when the prodrug binds to the antigen on the target immune cell through the antigen-binding moiety, the cytokine moiety binds to the cytokine receptor on the target immune cell and exerts the biological activity on the target immune cell,
wherein the cytokine moiety comprises an IL-2 polypeptide, an IL-7 polypeptide, or an IL-21 polypeptide.

### Claim 2 (depends on 1)

The prodrug of claim 1, wherein the receptor for the cytokine moiety comprises two or more subunits.

### Claim 3 (depends on 1)

The prodrug of claim 1, wherein the antigen is selected from Programmed Cell Death 1 (PD-1), Programmed Cell Death Ligand 1 (PD-L1), Cytotoxic T-lymphocyte Associated Protein 4 (CTLA-4), T-cell Immunoreceptor with Ig and ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin Domain containing-3 (TIM-3), Lymphocyte Activation Gene 3 (LAG-3), Cluster of Differentiation 25 (CD25), Cluster of Differentiation 16a (CD16a), Cluster of Differentiation 16b (CD16b), Natural Killer Group 2 Member D (NKG2D), Natural Killer Cell P44-related protein (NKP44), Natural Killer Cell Proliferation-associated Gene P30 (NKP30), Cluster of Differentiation 19 (CD19), Cluster of Differentiation 20 (CD20), Cluster of Differentiation 30 (CD30), Cluster of Differentiation 38 (CD38), B-cell Maturation Antigen (BCMA), and signal regulatory protein alpha (SIRP alpha).

### Claim 4 (depends on 1)

The prodrug of claim 1, wherein the cytokine moiety comprises an IL-2 polypeptide.

### Claim 5 (depends on 1)

The prodrug of claim 1, wherein the immune cell is a T cell, the antigen is PD-1, and the cytokine moiety comprises an IL-2.

### Claim 6 (depends on 1)

The prodrug of claim 1, wherein
i) the cytokine moiety comprises an IL-7 polypeptide and the masking moiety comprises an extracellular domain (ECD) of IL-7 receptor α (IL-7Rα ECD),
ii) the cytokine moiety comprises an IL-21 polypeptide and the masking moiety comprises an ECD of IL-21 receptor α (IL-21Rα ECD),
iii) the cytokine moiety comprises an IL-2 polypeptide and the masking moiety comprises an ECD of IL-2 receptor β (IL-2Rβ ECD),
iv) the cytokine moiety comprises an IL-21 polypeptide and the masking moiety comprises an scFv against IL-21, or
v) the cytokine moiety comprises an IL-2 polypeptide and the masking moiety comprises an scFv against IL-2.

### Claim 7 (depends on 6)

The prodrug of claim 6, wherein the cytokine moiety comprises an IL-2 polypeptide comprising SEQ ID NO: 6 or SEQ ID NO: 62.

### Claim 8 (depends on 6)

The prodrug of claim 6, wherein the cytokine moiety comprises an IL-2 polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 6 or SEQ ID NO: 62.

### Claim 9 (depends on 6)

The prodrug of claim 6, wherein the cytokine moiety comprises an IL-2 polypeptide and the masking moiety comprises an IL-2Rβ ECD, wherein the IL-2-Rβ ECD comprises SEQ ID NO: 11, or an amino acid sequence that is at least 95% identical thereto.

### Claim 10 (depends on 6)

The prodrug of claim 6, wherein the cytokine moiety comprises an IL-2 polypeptide and the masking moiety comprises an scFv against IL-2, and wherein the scFv comprises:
a) a heavy chain complementarity-determining region 1 (HCDR1), a heavy chain complementarity-determining region 2 (HCDR2), a heavy chain complementarity-determining region 3 (HCDR3), a light chain complementarity-determining region 1 (LCDR1), a light chain complementarity-determining region 2 (LCDR2), and a light chain complementarity-determining region 3 (LCDR3) of an scFv sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23,
b) the heavy and light chain variable domains of an scFv sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, or
c) an HCDR1, an HCDR2, and an HCDR3 of a heavy chain variable domain sequence set forth in SEQ ID NO: 60 and a LCDR1, a LCDR2 and a LCDR3 of a light chain variable domain sequence set forth in SEQ ID NO: 61.

### Claim 11 (depends on 6)

The prodrug of claim 6, wherein the cytokine moiety comprises an IL-2 polypeptide and the masking moiety comprises an scFv against IL-2, wherein the scFv comprises:
a) SEQ ID NO: 22 or SEQ ID NO: 23, or an amino acid sequence that is at least 95% identical thereto; or
b) SEQ ID NOs: 60 and 61.

### Claim 12 (depends on 6)

The prodrug of claim 6, wherein the cytokine moiety comprises an IL-21 polypeptide comprising any one of SEQ ID NOs: 1-5, or an amino acid sequence that is at least 95% identical thereto.

### Claim 13 (depends on 6)

The prodrug of claim 6, wherein the cytokine moiety comprises an IL-21 polypeptide and the masking moiety comprises an IL-21Ra ECD, or an IL-21Ry ECD, wherein the masking moiety comprises any one of SEQ ID NOs: 12, 13, and 63-73, or an amino acid sequence that is at least 95% identical thereto.

### Claim 14 (depends on 6)

The prodrug of claim 6, wherein the cytokine moiety comprises an IL-21 polypeptide and the masking moiety comprises an scFv against human IL-21, wherein the scFv comprises:
a) an HCDR1, an HCDR2, an HCDR3, a LCDR1, a LCDR2, and a LCDR3 of an scFv sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21,
b) the heavy and light chain variable domains of an scFv sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21, or
c) SEQ ID NO: 20 or SEQ ID NO: 21, or an amino acid sequence that is at least 95% identical thereto.

### Claim 15 (depends on 1)

The prodrug of claim 1, wherein the masking moiety comprises an ECD of a receptor α or γ of the cytokine.

### Claim 16 (depends on 1)

The prodrug of claim 1, wherein the prodrug comprises an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety comprises two light chains each comprising SEQ ID NO: 44, and two heavy chains comprising respectively:
SEQ ID NOs: 24 and 25,
SEQ ID NOs: 35 and 36,
SEQ ID NOs: 39 and 41,
SEQ ID NOs: 42 and 43, or
SEQ ID NOs: 42 and 74.

### Claim 17 (depends on 1)

The prodrug of claim 1, wherein the prodrug comprises an anti-CTLA4 antibody moiety, wherein the anti-CTLA4 antibody moiety comprises two light chains each comprising SEQ ID NO: 50, and two heavy chains comprising respectively:
SEQ ID NOs: 51 and 54,
SEQ ID NOs: 51 and 55,
SEQ ID NOs: 51 and 56,
SEQ ID NOs: 53 and 58, or
SEQ ID NOs: 53 and 59.

### Claim 18 (depends on 1)

A pharmaceutical composition comprising the prodrug of claim 1 and a pharmaceutically acceptable excipient.

### Claim 19 (depends on 1)

The prodrug of claim 1, wherein the immune cell is a T cell, the antigen is PD-1, and the cytokine moiety comprises an IL-21 polypeptide.