# US 11,992,505 B2 · In vitro production of red blood cells with proteins comprising sortase recognition motifs > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,992,505 B2 - Document ID: US11992505B2 - Status: Granted - Kind: B2 - Application number: 16/572,425 - Filed: 2019-09-16 - Published: 2020-03-05 - Granted: 2024-05-28 - Earliest claimed: 2013-05-10 - Assignees: Whitehead Institute for Biomedical Research - Applicants: Whitehead Institute for Biomedical Research - Inventors: Harvey Lodish; Hidde L. Ploegh; Hsiang-Ying Lee; Jiahai Shi; Lenka Hoffman; Novalia Pishesha - Web page: https://patentgrep.com/patent/US11992505B2 ## CPC - A61K 35/18: Erythrocytes (haemoglobin A61K38/42) - A01K 67/0275: Genetically modified vertebrates, e.g. transgenic - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C07K 14/47: from mammals - C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} - C07K 14/70582: {CD71} - C12N 5/0641: {Erythrocytes} - A01K 2217/072: maintaining or altering function, i.e. knock in - A01K 2227/105: Murine - A01K 2267/03: Animal model, e.g. for test or diseases - C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL] - C12N 2501/14: Erythropoietin [EPO] - C12N 2501/2303: Interleukin-3 (IL-3) - C12N 2501/2306: Interleukin-6 (IL-6) - C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand) - C12N 2501/999: Small molecules not provided for elsewhere - C12N 2506/11: from blood or immune system cells - C12N 2510/02: Cells for production - 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) - A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS - 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) ## Abstract Methods for the in vitro production of enucleated red blood cells and the enucleated red blood cells thus prepared are provided. Such enucleated red blood cells may express a sortaggable surface protein, which allows for surface modification in the presence of a sortase. Also described herein are surface modified enucleated red blood cells, e.g., conjugated with an agent of interest such as a peptide, a detectable label, or a chemotherapeutic agent, and uses thereof in delivering the agent to a subject. ## Claims ### Claim 1 A genetically engineered enucleated red blood cell, which comprises on the surface a first sortaggable surface fusion protein comprising (a) a first type I red blood cell transmembrane protein or a first type II red blood cell transmembrane protein; (b) a first peptide; and (c) a first therapeutic protein; wherein: (a) when the N-terminus of the first sortaggable surface fusion protein is exposed to the extracellular space, the first peptide is either N-terminal oligoglycine or N-terminal oligoalanine fused to the first type I red blood cell transmembrane protein and is exposed to the extracellular space, and the first peptide is fused to the first therapeutic protein via a first sortase recognition motif, or (b) when the C-terminus of the first sortaggable surface fusion protein is exposed to the extracellular space, the first peptide is a first sortase recognition motif which is fused to the C-terminus of the first type II red blood cell transmembrane protein and is exposed to the extracellular space, and the first peptide is fused to the first therapeutic protein via an oligoglycine or oligoalanine; and the first sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 1), LPXT (SEQ ID NO: 50), or LPXTA (SEQ ID NO: 2), wherein X is any amino acid residue; and wherein the genetically engineered enucleated red blood cell has a circulatory survival rate of greater than seven days. ### Claim 2 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first sortaggable surface fusion protein comprises the first type I red blood cell transmembrane protein. ### Claim 3 (depends on 2) The genetically engineered enucleated red blood cell of claim 2, wherein the first type I red blood cell transmembrane protein is glycophorin A (GPA). ### Claim 4 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first sortaggable surface fusion protein comprises the first type II red blood cell transmembrane protein. ### Claim 5 (depends on 4) The genetically engineered enucleated red blood cell of claim 4, wherein the type II red blood cell transmembrane protein is Kell or CD71. ### Claim 6 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 1), wherein X is any amino acid residue. ### Claim 7 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first therapeutic protein comprises a protein drug, an antigen, an enzyme, or a clotting or coagulation factor. ### Claim 8 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first therapeutic protein comprises a cytokine. ### Claim 9 (depends on 8) The genetically engineered enucleated red blood cell of claim 8, wherein the cytokine is an interferon, an interleukin, a colony stimulating factor, a leukemia inhibitory factor, or oncostatin M. ### Claim 10 (depends on 8) The genetically engineered enucleated red blood cell of claim 8, wherein the cytokine is a colony stimulating factor selected from the group consisting of G-CSF, GM-CSF, and M-CSF. ### Claim 11 (depends on 8) The genetically engineered enucleated red blood cell of claim 8, wherein the cytokine is an interleukin selected from the group consisting of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, and IL-15. ### Claim 12 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first therapeutic protein is fused to a detectable label. ### Claim 13 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first therapeutic protein is fused to a chemotherapeutic agent. ### Claim 14 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first therapeutic protein is fused to a lipid, a carbohydrate, a nucleic acid, a binding agent, a click-chemistry handle, or a small molecule. ### Claim 15 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first therapeutic protein comprises an enzyme. ### Claim 16 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first therapeutic protein comprises an antibody or a fragment thereof. ### Claim 17 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, wherein the first therapeutic protein is an antigen. ### Claim 18 (depends on 1) The genetically engineered enucleated red blood cell of claim 1, which further comprises on the surface a second sortaggable surface fusion protein, wherein the second sortaggable surface fusion protein comprises (a) a second type I red blood cell transmembrane protein or a second type II red blood cell transmembrane protein; (b) a second peptide; and (c) a second therapeutic protein; wherein: (a) when the N-terminus of the second sortaggable surface fusion protein is exposed to the extracellular space, the second peptide is either N-terminal oligoglycine or N-terminal oligoalanine fused to the second type I red blood cell transmembrane protein and is exposed to the extracellular space, and the second peptide is fused to the second therapeutic protein via a second sortase recognition motif, or (b) when the C-terminus of the second sortaggable surface fusion protein is exposed to the extracellular space, the second peptide is a second sortase recognition motif which is fused to the C-terminus of the second type II red blood cell transmembrane protein and is exposed to the extracellular space, and the second peptide is fused to the second therapeutic protein via an oligoglycine or oligoalanine; the second sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 1), LPXT (SEQ ID NO: 50), or LPXTA (SEQ ID NO: 2), wherein X is any amino acid residue; and the first sortase recognition motif differs from the second sortase recognition motif. ### Claim 19 (depends on 18) The genetically engineered enucleated red blood cell of claim 18, wherein the first peptide is different from the second peptide. ### Claim 20 (depends on 19) The genetically engineered enucleated red blood cell of claim 19, wherein the C-terminus of the first sortaggable surface protein is exposed to the extracellular space; wherein the first sortase recognition motif is the amino acid sequence LPXTA (SEQ ID NO: 2), wherein X is any amino acid residue; wherein the C-terminus of the second sortaggable surface protein is exposed to the extracellular space; and wherein the second sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 1). ### Claim 21 (depends on 20) The genetically engineered enucleated red blood cell of claim 20, wherein the C-terminus of the second sortaggable surface fusion protein is exposed to the extracellular space, and wherein the second type II red blood cell transmembrane protein is Kell. ### Claim 22 (depends on 18) The genetically engineered enucleated red blood cell of claim 18, wherein the first and the second therapeutic proteins are each fused to a functional moiety, and the functional moieties are different. ### Claim 23 (depends on 16) The genetically engineered enucleated red blood cell of claim 16, wherein the antibody is a single domain antibody. ### Claim 24 (depends on 17) The genetically engineered enucleated red blood cell of claim 17, wherein the antigen is a vaccine antigen, a cancer antigen, or an antigen derived from a pathogen.