MODIFIED HEMATOPOIETIC STEM/PROGENITOR AND NON-T EFFECTOR CELLS, AND USES THEREOF
Hematopoietic stem/progenitor cells (HSPC) and/or non-T effector cells are genetically modified to express (i) an extracellular component including a ligand binding domain that binds a cellular marker preferentially expressed on an unwanted cell; and (ii) an intracellular component comprising an effector domain. Among other uses, the modified cells can be administered to patients to target unwanted cancer cells without the need for immunological matching before administration.
1 . A method of preparing genetically engineered natural killer (NK) cells for administration to a subject, comprising:
selecting an umbilical cord blood or placental blood sample without immunological matching to the subject,
lysing red blood cells in the sample;
enriching the sample for CD34+ hematopoietic stem and progenitor cells (HSPCs);
culturing the CD34+ enriched HSPCs in a culture medium comprising interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), Flt-3 ligand (FLT-3L), stem cell factor (SCF) and a solid phase coated with Delta1 ext-IgG and recombinant human fibronectin or fragments thereof to form an expanded HSPC sample;
wherein the HSPCs have been genetically modified to express a molecule comprising an extracellular component having a ligand binding domain that binds a cellular marker on an unwanted cell, linked to a transmembrane domain linked to an intracellular component having an effector domain; and
culturing the genetically-modified expanded HSPCs in a culture medium comprising IL-2 and IL-15, thereby preparing the genetically engineered NK cells for administration to the subject without immunological matching.
2 . The method of claim 1 , wherein the ligand binding domain binds to CD19, ROR1, Her2, PSMA, PSCA, mesothelin, WT1, or CD20; and the intracellular component comprises an effector domain selected from CD3ζ, CD28, and 4-1BB.
3 . The method of claim 2 , wherein the ligand binding domain binds to CD19.
4 . The method of claim 3 , wherein the ligand binding domain is a scFv comprising the CDRs of monoclonal antibody FMC63.
5 . The method of claim 2 , wherein the ligand binding domain binds to ROR1.
6 . The method of claim 5 , wherein the ligand binding domain is a scFv comprising the CDRs of monoclonal antibody 2A2, R11 or R12.
7 . The method of claim 2 , wherein the extracellular component further comprises a spacer region comprising a portion of a hinge region of a human antibody between the ligand binding domain and the transmembrane domain.
8 . The method of claim 7 , wherein the spacer region comprises a Fc domain and a human IgG4 heavy chain hinge.
9 . The method of claim 2 , wherein the transmembrane domain is a CD28 transmembrane domain or a CD4 transmembrane domain.
10 . The method of claim 1 , wherein the genetically engineered NK cells are cryopreserved in the presence of a cryoprotective agent.
11 . The method of claim 1 , further comprising formulating the genetically engineered NK cells for infusion to the subject.