# US 10,370,643 B2 · LILRB2 and notch-mediated expansion of hematopoietic precursor cells

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- Number: US 10,370,643 B2
- Document ID: US10370643B2
- Status: Granted
- Kind: B2
- Application number: 15/313,043
- Filed: 2015-05-21
- Published: 2017-09-14
- Granted: 2019-08-06
- Earliest claimed: 2014-05-22
- Assignees: Fred Hutchinson Cancer Research Center; Board of Regents, The University of Texas System
- Applicants: Fred Hutchinson Cancer Research Center; Board of Regents of the University of Texas System
- Inventors: Irwin D. Bernstein; Chengcheng Zhang; Mi Deng; Zhigang Lu; Junke Zheng
- Web page: https://patentgrep.com/patent/US10370643B2

## CPC

- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C12N 5/0662: {Stem cells}
- G01N 33/56966: {Animal cells}
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/113: Acidic fibroblast growth factor (aFGF, FGF-1)
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/17: Angiopoietin
- 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 2501/42: Notch; Delta; Jagged; Serrate
- C12N 2501/599: with CD designations not provided for elsewhere
- C12N 2501/91: Heparin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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 current disclosure describes methods of expanding precursor cells for hematopoietic transplantation in subjects. The methods culture precursor cells in media containing an immobilized high molecular weight LILRB2 agonist or an LILRB2 agonist in combination with a Notch agonist. The expanded cells can be used to treat a variety of hematopoietic disorders.

## Claims

### Claim 1

A method of expanding hematopoietic stem cells and hematopoietic progenitor cells ex vivo, comprising culturing said cells with (i) a Notch agonist and (ii) a leukocyte immunoglobulin-like receptor B2 (LILRB2) agonist immobilized on a first solid phase, wherein the immobilized LILRB2 agonist is an antibody to the LILRB2 receptor, or an antigen-binding fragment of said antibody.

### Claim 2 (depends on 1)

The method of claim 1, wherein the hematopoietic stem cells and hematopoietic progenitor cells are human cells.

### Claim 3 (depends on 2)

The method of claim 2, wherein the hematopoietic stem cells and hematopoietic progenitor cells are obtained from bone marrow, umbilical cord blood, placental blood, or Wharton's jelly.

### Claim 4 (depends on 1)

The method of claim 1, wherein the antibody to the LILRB2 receptor is a monoclonal antibody.

### Claim 5 (depends on 1)

The method of claim 1, wherein the antibody to the LILRB2 receptor is a polyclonal antibody.

### Claim 6 (depends on 1)

The method of claim 1, wherein the LILRB2 agonist is an Fv, Fab, Fab′, F(ab′)2, or single chain Fv fragment (scFv).

### Claim 7 (depends on 1)

The method of claim 1, wherein LILRB2 agonist binds to the Ig1 domain of LILRB2.

### Claim 8 (depends on 1)

The method of claim 1, wherein LILRB2 agonist binds to the Ig4 domain of LILRB2.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first solid phase is the surface of a tissue culture dish.

### Claim 10 (depends on 1)

The method of claim 1, wherein the Notch agonist is immobilized on the first solid phase.

### Claim 11 (depends on 1)

The method of claim 1, wherein the Notch agonist is immobilized on a second solid phase that is not the first solid phase.

### Claim 12 (depends on 1)

The method of claim 1, wherein the Notch agonist is an extracellular, Notch-interacting domain of a Delta protein.

### Claim 13 (depends on 1)

The method of claim 1, wherein the Notch agonist is Deltaext-IgG.

### Claim 14 (depends on 1)

The method of claim 1, wherein the Notch agonist is an antibody that specifically binds to Notch-1.

### Claim 15 (depends on 1)

The method of claim 1, wherein the Notch agonist is an antibody that specifically binds to Notch-2.

### Claim 16 (depends on 1)

The method of claim 1, wherein the culturing step is performed in the presence of a culture medium comprising stem cell factor (SCF), thrombopoietin (TPO), and Flt3-ligand.

### Claim 17 (depends on 1)

The method of claim 1, wherein the culturing step is performed in the presence of a culture medium comprising SCF, Flt3-ligand, interleukin-6 (IL-6), TPO, fibroblast growth factor-1 (FGF1), and interleukin-3 (IL-3).

### Claim 18 (depends on 17)

The method of claim 17, wherein the culture medium comprises SCF, Flt3-ligand, IL-6, TPO, FGF1, IL-3, and heparin.

### Claim 19 (depends on 1)

The method of claim 1, wherein the culturing step is performed in the presence of a culture medium comprising retronectin.