# US 12,178,835 B2 · Allograft tolerance without the need for systemic immune suppression

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- Number: US 12,178,835 B2
- Document ID: US12178835B2
- Status: Granted
- Kind: B2
- Application number: 18/175,409
- Filed: 2023-02-27
- Published: 2023-12-28
- Granted: 2024-12-31
- Earliest claimed: 2017-06-12
- Assignees: Sinai Health System
- Applicants: Sinai Health System
- Inventors: Andras Nagy; Jeffrey Harding; Kristina Nagy
- Web page: https://patentgrep.com/patent/US12178835B2

## CPC

- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- 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 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 25/16: Anti-Parkinson drugs
- A61P 27/02: Ophthalmic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 14/47: from mammals
- C07K 14/521: {Chemokines}
- C07K 14/70503: {Immunoglobulin superfamily}
- C07K 14/70532: {B7 molecules, e.g. CD80, CD86}
- C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)}
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 15/09: Recombinant DNA-technology
- A61K 38/1709: {from mammals}
- A61K 38/1774: {Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)}
- A61K 38/1841: {Transforming growth factor [TGF]}
- C12N 2510/00: Genetically modified cells
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A cell genetically modified to comprise at least one mechanism for providing a local immunosuppression at a transplant site when transplanted in an allogeneic host is, and methods for making and using the same is provided. The cell comprises a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting, and whose function is one or more of: to mitigate antigen presenting cell activation and function; to mitigate graft attacking leukocyte activity or cytolytic function; to mitigate macrophage cytolytic function and phagocytosis of allograft cells; to induce apoptosis in graft attacking leukocytes; to mitigate local inflammatory proteins; and to protect against leukocyte-mediated apoptosis.

## Claims

### Claim 1

A cell genetically modified to comprise transgenes comprising:
human Fas ligand (FASLG), CD200, C-C motif chemokine ligand 21 (CCL21), serpin family B member 9 (SERPINB9), milk fat globule-EFG factor 8 (MFGE8), Programmed cell death-ligand 1 (PD-L1), major histocompatibility complex, class I, G (HLA-G), and CD47; or
murine Fas ligand (FasL), Cd200, C-C motif chemokine ligand 21B (Ccl21b), Serine Protease Inhibitor 6 (Spi6), Mfge8, Pd-11, histocompatibility 2, M region locus 3 (H2-M3), and Cd47; and
wherein the human FASLG transgene encodes a protein having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 10,
the human CD200 transgene encodes a protein having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 6,
the human CCL21 transgene encodes a protein having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 2,
the human SERPINB9 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 8,
the human MFGE8 transgene encodes a protein having at least 95% identity to sequence as set forth in SEQ ID NO: 14,
the human PD-L1 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 12,
the human HLA-G transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 16, and
the human CD47 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 4; or
the murine FasI transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 9,
the murine Cd200 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 5,
the murine Ccl21b transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 1,
the murine Spi6 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 7,
the murine Mfge8 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 13,
the murine Pd-11 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 11,
the murine H2-M3 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 15, and
the murine Cd47 transgene encodes a protein having at least 95% sequence identity to sequence as set forth in SEQ ID NO: 3.

### Claim 2 (depends on 1)

The cell of claim 1, wherein the cell is an allogeneic cell in reference to a subject.

### Claim 3 (depends on 1)

The cell of claim 1, wherein the cell is a human cell or a murine cell.

### Claim 4 (depends on 1)

The cell of claim 1, wherein the cell is a stem cell.

### Claim 5 (depends on 1)

The cell of claim 1, where the cell further comprises a heterologous sequence encoding a therapeutic agent.

### Claim 6 (depends on 5)

The cell of claim 5, wherein the therapeutic agent comprises a wild-type version of a gene that is mutated within a subject.

### Claim 7 (depends on 5)

The cell of claim 5, wherein the therapeutic agent comprises an enzyme, an antibody, a growth factor, or a cytokine.

### Claim 8 (depends on 1)

The cell of claim 1, wherein expression of the transgenes is under the control of one or more constitutive promoters.

### Claim 9 (depends on 8)

The cell of claim 8, wherein the one or more constitutive promoters comprise a cytomegalovirus (CMV) immediate-early enhancer/chicken β-actin (CAG) promoter, a cytomegalovirus (CMV) promoter, a human elongation factor-1 alpha (EF1a) promoter, a 3-phosphoglycerate kinase (PGK) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a Simian Virus 40 (SV40) promoter, a thymidine kinase (tk) promoter of herpes simplex virus (HSV), mouse mammary tumor virus (MMTV) promoter, a long terminal repeat (LTR) promoter of human immunodeficiency virus (HIV), a promoter of Moloney virus, an Epstein barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.

### Claim 10 (depends on 1)

The cell of claim 1, wherein the cell further comprises one or more of the following transgenes: transforming growth factor beta (TGF-β), 5′-nucleotidase ecto (Cd73), ectonucleoside triphosphate diphosphohydrolase 1 (Cd39), lymphocyte activating 3 (Lag3), interleukin 1 receptor type 2 (Il1r2), atypical chemokine receptor 2 (Ackr2), tumor necrosis factor receptor superfamily, member 22 (Tnfrsf22), tumor necrosis factor receptor superfamily, member 23 (Tnfrsf23), tumor necrosis factor receptor superfamily, member 10 (Tnfrsf10), defender against cell death 1 (Dad1), or a dominant negative form of the IFNγ receptor (IFNγR1 d39).

### Claim 11 (depends on 1)

The cell of claim 1, wherein the cell further comprises a genetic modification of one or more cell division locus/loci (CDL).

### Claim 12 (depends on 11)

The cell of claim 11, wherein the genetic modification of the one or more cell division locus/loci (CDL) comprises insertion of an ablation link (ALINK) system or an exogenous activator of regulation of cell division locus/loci (EARC) system.

### Claim 13 (depends on 11)

The cell of claim 11, wherein the CDL comprises cyclin dependent kinase 1 (CDK1), DNA topoisomerase II alpha (TOP2A), centromere protein A (CENPA), baculoviral IAP repeat containing 5 (BIRC5), or eukaryotic translation elongation factor 2 (EEF2).

### Claim 14 (depends on 1)

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