Genetic modified pluri- or multipotent stem cells and uses thereof

Abstract

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.