GENETIC MODIFIED PLURI- OR MULTIPOTENT STEM CELLS AND USES THEREOF
The invention concerns pluri- or multipotent stem cells (SCs), e.g. human pluri- or multipotent stem cells (hSCs) engineered to express a multispecific antibody and which further express, on their surface, a human immune cell co-stimulatory ligand or an active fragment thereof.
1 .- 14 . (canceled)
15 . A method of preventing acute myeloid leukemia (AML) in a subject in need thereof, comprising administering an isolated human mesenchymal stem cell (hMSC) to said subject to prevent AML, wherein said hMSC comprises a nucleic acid sequence encoding a bispecific antibody comprising:
i) a first antigen binding domain that specifically binds to CD33, the first antigen binding domain comprising a variable light (V L ) chain and a variable heavy (V H ) chain; and
ii) a second antigen binding domain that specifically binds to CD3, the second antigen binding domain comprising a V L chain and a V H chain,
wherein said hMSC expresses and secretes said bispecific antibody, and does not express a T cell co-stimulatory ligand or an active fragment thereof.
16 . The method of claim 15 , wherein said bispecific antibody is a single chain antibody.
17 . The method of claim 15 , wherein said bispecific antibody comprises a linker
i) comprising the amino acid sequence G 4 S (SEQ ID NO:33); or
ii) consisting of the amino acid sequence (G 4 S) 3 (SEQ ID NO:34).
18 . The method of claim 15 , wherein:
i) the VH of the first antigen binding domain comprises a V H CDR1, a V H CDR2 and a VH CDR3, wherein
a) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5,
b) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:9, SEQ ID NO:13, and SEQ ID NO:17,
c) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:18,
d) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:11, SEQ ID NO:15, and SEQ ID NO:19, or
e) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:12, SEQ ID NO:16, and SEQ ID NO:20; and
ii) the V L of the first antigen binding domain comprises a V L CDR1, a V L CDR2 and a V L CDR3, wherein
a) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8,
b) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:21, SEQ ID NO:25, and SEQ ID NO:29,
c) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:30,
d) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:31, or
e) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:24, SEQ ID NO:28, and SEQ ID NO:32.
19 . A vector encoding a bispecific antibody comprising:
i) a first antigen binding domain that specifically binds to CD33, the first antigen binding domain comprising a variable light (V L ) chain and a variable heavy (V H ) chain; and
ii) a second antigen binding domain that specifically binds to CD3, the second antigen binding domain comprising a V L chain and a V H chain.
20 . The vector of claim 19 , wherein said vector is a lentiviral vector or a retroviral vector.
21 . The vector of claim 19 , wherein said vector comprises a spleen focus forming virus (SFFV)-derived internal promoter.
22 . The vector of claim 19 , wherein said bispecific antibody is a single chain antibody.
23 . The vector of claim 19 , wherein said bispecific antibody comprises a linker
i) comprising the amino acid sequence G 4 S (SEQ ID NO:33); or
ii) consisting of the amino acid sequence (G 4 S) 3 (SEQ ID NO:34).
24 . The vector of claim 19 , wherein:
i) the V H of the first antigen binding domain comprises a V H CDR1, a V H CDR2 and a V H CDR3, wherein
a) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5,
b) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:9, SEQ ID NO: 13, and SEQ ID NO:17,
c) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:18,
d) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:11, SEQ ID NO:15, and SEQ ID NO:19, or
e) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:12, SEQ ID NO:16, and SEQ ID NO:20; and
ii) the V L of the first antigen binding domain comprises a V L CDR1, a V L CDR2 and a V L CDR3, wherein
a) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8,
b) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:21, SEQ ID NO:25, and SEQ ID NO:29,
c) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:30,
d) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:31, or
e) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:24, SEQ ID NO:28, and SEQ ID NO:32.
25 . A method of producing a bispecific antibody, said method comprising:
i) transfecting a human mesenchymal stem cell (hMSC) with a vector encoding a bispecific antibody, wherein said bispecific antibody comprises:
a) a first antigen binding domain that specifically binds to CD33, the first antigen binding domain comprising a variable light (V L ) chain and a variable heavy (V H ) chain, and
b) a second antigen binding domain that specifically binds to CD3, the second antigen binding domain comprising a V L chain and a V H chain, wherein said hMSC expresses and secretes said bispecific antibody; and
ii) purifying said bispecific antibody from cell culture.
26 . The method of claim 25 , wherein said vector is a lentiviral vector or a retroviral vector.
27 . The method of claim 25 , wherein said vector comprises a spleen focus forming virus (SFFV)-derived internal promoter.
28 . The method of claim 25 , wherein said bispecific antibody is a single chain antibody.
29 . The method of claim 25 , wherein said bispecific antibody comprises a linker
i) comprising the amino acid sequence G 4 S (SEQ ID NO:33); or
ii) consisting of the amino acid sequence (G 4 S) 3 (SEQ ID NO:34).
30 . The method of claim 25 , wherein:
i) the V H of the first antigen binding domain comprises a V H CDR1, a V H CDR2 and a V H CDR3, wherein
a) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5,
b) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:9, SEQ ID NO:13, and SEQ ID NO:17,
c) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:18,
d) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:11, SEQ ID NO:15, and SEQ ID NO:19, or
e) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:12, SEQ ID NO:16, and SEQ ID NO:20; and
ii) the V L of the first antigen binding domain comprises a V L CDR1, a V L CDR2 and a V L CDR3, wherein
a) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8,
b) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:21, SEQ ID NO:25, and SEQ ID NO:29,
c) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:30,
d) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:31, or
e) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:24, SEQ ID NO:28, and SEQ ID NO:32.
31 . A method of inducing or enhancing T cell proliferation in a subject in need thereof, comprising administering a human mesenchymal stem cell (hMSC), wherein said hMSC comprises:
i) a nucleic acid sequence encoding a bispecific antibody comprising:
a) a first antigen binding domain that specifically binds to CD33, the first antigen binding domain comprising a variable light (V L ) chain and a variable heavy (V H ) chain, and
b) a second antigen binding domain that specifically binds to CD3, the second antigen binding domain comprising a V L chain and a V H chain; and
ii) a nucleic acid sequence encoding a T cell co-stimulatory ligand or an active fragment thereof, wherein said T cell co-stimulatory ligand is selected from the group consisting of CD28 ligands CD80 (B7-1) and CD86 (B7-2), CD137 ligand (CD137L, 4-1BB ligand), Ox40 ligand OX40L (CD252), CD27 ligand CD70 (CD27L), Inducible T-cell Costimulator (ICOS) ligand ICOSL (CD275), lymphocyte function-associated antigen (LFA) 1 ligand intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2 (CD102), and ICAM-3 (CD50), and 2B4 (CD44) ligand SLAMF2 (CD48), and
wherein said hMSC expresses and secretes said bispecific antibody and functionally expresses said T cell co-stimulatory ligand or active fragment thereof.
32 . The method of claim 31 , wherein said bispecific antibody is a single chain antibody.
33 . The method of claim 31 , wherein said bispecific antibody comprises a linker
i) comprising the amino acid sequence G 4 S (SEQ ID NO:33); or
ii) consisting of the amino acid sequence (G 4 S) 3 (SEQ ID NO:34).
34 . The method of claim 31 , wherein:
i) the V H of the first antigen binding domain comprises a V H CDR1, a V H CDR2 and a V H CDR3, wherein
a) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5,
b) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:9, SEQ ID NO:13, and SEQ ID NO:17,
c) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:18,
d) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:11, SEQ ID NO:15, and SEQ ID NO:19, or
e) said V H CDR1, V H CDR2 and V H CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:12, SEQ ID NO:16, and SEQ ID NO:20; and
ii) the V L of the first antigen binding domain comprises a V L CDR1, a V L CDR2 and a V L CDR3, wherein
a) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8,
b) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:21, SEQ ID NO:25, and SEQ ID NO:29,
c) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:30,
d) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:31, or
e) said V L CDR1, V L CDR2 and V L CDR3 of the first antigen binding domain respectively comprise SEQ ID NO:24, SEQ ID NO:28, and SEQ ID NO:32.
35 . The method of claim 31 , wherein said T cell co-stimulatory ligand is 4-1BB ligand, B7-1, or B7-2.
36 . The method of claim 31 , wherein said T cell proliferation is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, or 500% after administration of said hMSC.