CAR-T secreting a bispecific T-cell adapter and its application in the treatment of solid tumors
A bispecific, adapter technology, applied in receptors/cell surface antigens/cell surface determinants, antineoplastic drugs, animal cells, etc.
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Embodiment 1
[0072] Example 1 Design and Production of Bispecific T Cell Adapter Constructs
[0073] Three novel bispecific T cell adapter molecules (Table 1) were designed to enhance the functional efficacy of CAR-T cells against solid tumors. Functional efficacy includes, but is not limited to, increased cytotoxicity of the CAR-T cells of the present invention.
[0074] a. NKG2D-bispecific T cell adapter : Human NKG2D linked to anti-human CD3 ScFv (SP34) via a G4S linker.
[0075] b. MN14-bispecific T cell adapter : Anti-human CEACAM5 (CEA) ScFv (MN14op) linked to anti-human CD3 ScFv (SP34) via a G4S linker.
[0076] c. T84.66 - bispecific T cell adapter : Anti-human CEACAM5 (CEA) ScFv (T84.66) was linked to anti-human CD3 ScFv (SP34) through a G4S linker.
[0077] Table 1: Amino acid sequences of bispecific T cell adapter molecules
[0078]
Embodiment 2
[0079] Example 2 Modification of Primary Human T Cells Using Bicistronic Transposons to Deliver Bispecific T Cell Adapters and Chimeric Antigen Receptors (CARs)
[0080] 1. Design of CAR to co-express bispecific T cell adapter molecules
[0081] The CAR complex in this example consists of a target cell ligand-binding ScFv domain, a CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. The CAR complex and the bispecific T cell adapter molecule are separated by a P2A domain ( figure 2 ). A bicistronic CAR construct was designed to carry a bispecific T cell adapter construct.
[0082] We designed and made the following CAR and CAR-P2A-bispecific T cell adapter constructs, as shown in Table 2, and the sequences of these CARs are shown in Table 3.
[0083] Table 2. CAR and CAR-P2A-bispecific T cell adapter constructs
[0084]
[0085] The amino acid sequence of P2A is: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 7). The amino acid...
Embodiment 3
[0093] Example 3 Identification and expression of CAR-T cells co-expressing bispecific T cell adapter molecules
[0094] Human primary T cells were isolated and purified from PBMCs of healthy donors and electroporated with a transposon carrying a CAR-P2A-bispecific T cell adapter construct. Transduced CAR-T cells were analyzed by flow cytometry, and their integration was detected by staining with AF488-linked anti-Fab'.
[0095] All CAR-T cells showed good levels of integration ( Figure 4A , Figure 4B , Figure 4C ). Flow cytometric analysis of transduced T cells represented by viability (left) and CAR expression (right). Numbers in each plot represent the frequency of parental cells expressing each parameter. Three novel constructs, LBC010, LBC021, and LBC022, were compared with LBC001 and LBC017 CAR-T cells that did not express the bispecific T cell adapter molecule. By FACS analysis, CAR-T cells with or without bispecific T cell adapters showed comparable expression...
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