# US 11,999,802 B2 · Compositions and methods for selective protein degradation

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- Number: US 11,999,802 B2
- Document ID: US11999802B2
- Status: Granted
- Kind: B2
- Application number: 16/757,026
- Filed: 2018-10-18
- Published: 2020-10-29
- Granted: 2024-06-04
- Earliest claimed: 2017-10-18
- Assignees: Novartis AG
- Applicants: Novartis AG
- Inventors: James E. Bradner; Andrei Golosov; Carleton Proctor Goold; Carla Patricia Pinto Guimaraes; Marc Horst Peter Hild; Gregory Motz; Nathan Thomas Ross; Jonathan M. Solomon; Rohan Eric John Beckwith; Seth Carbonneau
- Web page: https://patentgrep.com/patent/US11999802B2

## CPC

- A61K 31/138: Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine (atenolol A61K31/165; pindolol A61K31/404; timolol A61K31/5377)
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- A61K 31/454: containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
- A61K 31/4545: containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
- A61K 31/46: 8-Azabicyclo [3.2.1] octane; Derivatives thereof, e.g. atropine, cocaine
- A61K 31/55: having seven-membered rings, e.g. azelastine, pentylenetetrazole
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- 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 40/4211: {CD19 or B4}
- A61K 40/4215: {Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30}
- C07D 209/48: with oxygen atoms in positions 1 and 3, e.g. phthalimide
- C07D 213/81: Amides; Imides
- C07D 401/14: containing three or more hetero rings
- C07D 451/02: containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
- C07K 14/435: from animals; from humans
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/18: against material from animals or humans
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2815: {against CD8}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2875: {against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 (against NGF C07K16/22, against TNF C07K16/241)}
- C07K 16/2887: {against CD20}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C12N 9/003: {Dihydrofolate reductase [DHFR] (1.5.1.3)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/86: Viral vectors
- C12Y 105/01003: Dihydrofolate reductase (1.5.1.3)
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- 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
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- C07K 2319/50: containing protease site
- C07K 2319/81: containing a Zn-finger domain for DNA binding
- C07K 2319/95: containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- 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)
- 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)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention provides compositions including a fusion polypeptide and methods for making a fusion polypeptide that includes a COF1/CRBN-binding polypeptide, COF2/CRBN-binding polypeptide, or COF3/CRBN-binding polypeptide and a heterologous polypeptide of interest.

## Claims

### Claim 1

A fusion polypeptide comprising:
(i) a CRBN (cereblon)-binding polypeptide, and
(ii) a chimeric antigen receptor (CAR) that comprises, in a N-terminal to C-terminal direction, an antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains, wherein the CRBN-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 1.

### Claim 2 (depends on 1)

The fusion polypeptide of claim 1, wherein the CRBN-binding polypeptide is fused to the CAR.

### Claim 3 (depends on 1)

The fusion polypeptide of claim 1, wherein the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 20% of the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

### Claim 4 (depends on 1)

The fusion polypeptide of claim 1, wherein the degradation or ubiquitination of the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 70% of the degradation or ubiquitination of the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

### Claim 5 (depends on 1)

The fusion polypeptide of claim 1, wherein the fusion polypeptide further comprises a degradation domain, wherein the degradation domain is separated from the CRBN-binding polypeptide and the CAR by a heterologous protease cleavage site.

### Claim 6 (depends on 5)

The fusion polypeptide of claim 5, wherein the fusion polypeptide comprises, from N-terminus to C-terminus:
i) the degradation domain, the heterologous protease cleavage site, the CAR, and the CRBN-binding polypeptide;
ii) the degradation domain, the heterologous protease cleavage site, the CRBN-binding polypeptide, and the CAR;
iii) the CRBN-binding polypeptide, the CAR, the heterologous protease cleavage site, and the degradation domain; or
iv) the CAR, the CRBN-binding polypeptide, the heterologous protease cleavage site, and the degradation domain.

### Claim 7 (depends on 1)

A composition comprising the fusion polypeptide of claim 1, and a pharmaceutically acceptable carrier, excipient or stabilizer.

### Claim 8 (depends on 1)

The fusion polypeptide of claim 1, wherein the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 10% of the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

### Claim 9 (depends on 1)

The fusion polypeptide of claim 1, wherein the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 5% of the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

### Claim 10 (depends on 1)

The fusion polypeptide of claim 1, wherein the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 1% of the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

### Claim 11 (depends on 1)

The fusion polypeptide of claim 1, wherein the degradation or ubiquitination of the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 50% of the degradation or ubiquitination of the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

### Claim 12 (depends on 1)

The fusion polypeptide of claim 1, wherein the degradation or ubiquitination of the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 10% of the degradation or ubiquitination of the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

### Claim 13 (depends on 1)

The fusion polypeptide of claim 1, wherein the degradation or ubiquitination of the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 1% of the degradation or ubiquitination of the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

### Claim 14 (depends on 1)

The fusion polypeptide of claim 1, wherein the CRBN-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

### Claim 15 (depends on 1)

The fusion polypeptide of claim 1, wherein the CRBN-binding polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or 3.

### Claim 16 (depends on 5)

The fusion polypeptide of claim 5, wherein the degradation domain is an estrogen receptor (ER) domain.

### Claim 17 (depends on 5)

The fusion polypeptide of claim 5, wherein
the degradation domain is an ER domain and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 46 or 48.

### Claim 18 (depends on 1)

The fusion polypeptide of claim 1, wherein the CRBN-binding polypeptide is linked to the CAR by a peptide bond.

### Claim 19 (depends on 1)

The fusion polypeptide of claim 1, wherein the CRBN-binding polypeptide is linked to the CAR by a bond other than a peptide bond.

### Claim 20 (depends on 1)

The fusion polypeptide of claim 1, wherein the CAR is linked directly to the CRBN-binding polypeptide.

### Claim 21 (depends on 1)

The fusion polypeptide of claim 1, wherein the CAR is linked indirectly to the CRBN-binding polypeptide.

### Claim 22 (depends on 1)

The fusion polypeptide of claim 1, wherein the CRBN-binding polypeptide is operatively linked to the CAR via a linker, a glycine serine linker, or a linker comprising the amino acid sequence of SEQ ID NO: 28.

### Claim 23 (depends on 5)

The fusion polypeptide of claim 5, wherein the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, Granzyme B, Factor XA, Enterokinase, genenase, sortase, precission protease, thrombin, TEV protease, and elastase 1.

### Claim 24 (depends on 5)

The fusion polypeptide of claim 5, wherein the heterologous protease cleavage site comprises a sequence having a cleavage motif selected from the group consisting of Arg-X-Lys/Arg-Arg consensus motif (X can be any amino acid; SEQ ID NO: 52), Arg-X-X-X-Lys/Arg-Arg consensus motif (X can be any amino acid; SEQ ID NO: 53), Arg-Arg-X consensus motif (SEQ ID NO: 54), Ile-Glu-Pro-Asp-X consensus motif (SEQ ID NO: 55), Ile-Glu/Asp-Gly-Arg (SEQ ID NO: 56), Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 57), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO: 58), Leu-Pro-X-Thr-Gly/Ala consensus motif (SEQ ID NO: 59), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO: 60), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 61), Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 62), and [Ala-Gly-Ser-Val]X (X can be any amino acid; SEQ ID NO: 63).

### Claim 25 (depends on 5)

The fusion polypeptide of claim 5, wherein the heterologous protease cleavage site is cleaved by furin.

### Claim 26 (depends on 25)

The fusion polypeptide of claim 25, wherein the heterologous protease cleavage site comprises a furin cleavage site selected from the group consisting of RTKR (SEQ ID NO: 123); GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125); GTGAEDPRPSRKRR (SEQ ID NO: 127); LQWLEQQVAKRRTKR (SEQ ID NO: 129); GTGAEDPRPSRKRRSLGG (SEQ ID NO: 131); GTGAEDPRPSRKRRSLG (SEQ ID NO: 133); SLNLTESHNSRKKR (SEQ ID NO: 135); CKINGYPKRGRKRR (SEQ ID NO: 137); and SARNRQKR (SEQ ID NO: 34).

### Claim 27 (depends on 5)

The fusion polypeptide of claim 5, wherein the heterologous protease cleavage site is cleaved by a mammalian extracellular protease selected from the group consisting of Factor XA, Enterokinase, genenase, sortase, precission protease, thrombin, TEV protease, and elastase 1.

### Claim 28 (depends on 27)

The fusion polypeptide of claim 27, wherein the heterologous protease cleavage site comprises an amino acid sequence selected from the group consisting of Ile-Glu/Asp-Gly-Arg (SEQ ID NO: 56), Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 57), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO: 58), Leu-Pro-X-Thr-Gly/Ala consensus motif (SEQ ID NO: 59), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO: 60), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 61), Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 62), and [Ala-Gly-Ser-Val]-X (X can be any amino acid; SEQ ID NO: 63.

### Claim 29 (depends on 5)

The fusion polypeptide of claim 5, wherein the degradation domain is an FKB protein (FKBP) domain.

### Claim 30 (depends on 5)

The fusion polypeptide of claim 5, wherein the degradation domain is a dihydrofolate reductase (DHFR) domain.

### Claim 31 (depends on 5)

The fusion polypeptide of claim 5, wherein the degradation domain is an FKBP domain and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 50.

### Claim 32 (depends on 5)

The fusion polypeptide of claim 5, wherein the degradation domain is a DHFR domain and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 51.

### Claim 33 (depends on 1)

The fusion polypeptide of claim 1, wherein the CRBN-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 3.