Car-t cell assay for specificity test of novel antigen binding moieties
The present invention generally relates to specificity assays using cell cultures, in particular to chimeric antigen receptor (CAR) expressing reporter T (CAR-T cell) assays to test antigen binding moieties in different formats. Furthermore, the present invention relates to the use of CAR-T cells, transfected/transduced with an engineered chimeric antigen receptor (CAR) comprising a target antigen binding moiety capable of specific binding to a target antigen, e.g., tumor associated antigens.
1. A method for assessing the specificity of an antigen binding moiety comprising the steps of:
a. providing an antigen binding moiety specific for a target antigen, wherein said antigen binding moiety is a Fab fragment;
b. generating a chimeric antigen receptor (CAR) expressing reporter T (CAR-T) cell by:
i. transferring the antigen binding moiety into a CAR vector system operationally coupled to a response element;
ii. transferring the CAR vector system into a reporter T cell comprising a reporter gene under the control of the response element;
c. contacting the reporter CAR-T cell with a target cell comprising the target antigen on the surface, in particular wherein the target cell is a cancer cell; and
d. determining T cell activation by determining the expression of the reporter gene to establish the specificity of the antigen binding moiety.
2. The method according to claim 1 , wherein the antigen binding moiety comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) and wherein coding polynucleotide sequences for the VH and VL domains of the antigen binding moiety are transferred to the CAR vector system.
3. The method according to claim 1 , wherein the CAR vector system encodes a CAR comprising a Fab or a crossFab fragment, an anchoring transmembrane domain and at least one intracellular signaling and/or co-signaling domain.
4. The method according to claim 3 , wherein binding of the target antigen to the reporter CAR-T cell leads to activation of the intracellular signaling and/or co-signaling domain.
5. The method according to claim 1 , wherein activation of the intracellular signaling domain leads to activation of the response element.
6. The method according to claim 1 , wherein activation of the response element leads to expression of the reporter gene.
7. The method according to claim 1 , wherein the reporter gene is coding for luminescent protein.
8. The method according to claim 1 , wherein the target antigen is a cell surface receptor.
9. The method according to claim 1 , wherein the target antigen is a peptide bound to a molecule of the human major histocompatibility complex (WIC).
10. The method according to claim 9 , wherein the antigen binding moiety is a T cell receptor like (TCRL) antigen binding moiety.
11. The method according claim 1 , wherein high level of expression of the reporter gene in the presence of the target cell and low level of expression of the reporter gene in the absence of the target cell is indicative for high specificity of the antigen binding moiety.
12. The method according to claim 1 , wherein high level of expression of the reporter gene in the presence of the target cell and low level of expression of the reporter gene in the absence of the target cell is indicative for high specificity of a T cell bispecific (TCB) antibody comprising the antigen binding moiety.
13. The method according to claim 1 , wherein the method is an in vitro method.
14. A method for generating a TCB antibody, wherein the TCB antibody format comprises a first antigen binding moiety specific for a target antigen and a second antigen binding moiety capable of specific binding to a T cell activating receptor, wherein the first antigen binding moiety is selected by a method comprising:
a. providing an antigen binding moiety specific for a target antigen;
b. generating a chimeric antigen receptor (CAR) expressing reporter T (CAR-T) cell by:
iii. transferring the antigen binding moiety into a CAR vector system operationally coupled to a response element;
iv. transferring the CAR vector system into a reporter T cell comprising a reporter gene under the control of the response element;
c. contacting the reporter CAR-T cell with a target cell comprising the target antigen on the surface, in particular wherein the target cell is a cancer cell; and
d. determining T cell activation by determining the expression of the reporter gene to establish the specificity of the antigen binding moiety.
15. The method of claim 14 , wherein the T cell activating receptor is CD3.
16. The method of claim 1 , wherein the antigen binding moiety comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) and wherein coding polynucleotide sequences for the VH and VL domains of the antigen binding moiety are transferred to the CAR vector system.
17. The method of claim 4 , wherein activation of the intracellular signaling domain leads to activation of the response element.
18. The method of claim 15 , wherein said first antigen binding moiety is a Fab fragment.
19. The method of claim 18 , wherein said antigen binding moiety is a crossover Fab fragment.
20. The method of claim 15 , wherein the second antigen binding moiety said TCB comprises a heavy chain complementarity determining region (HCDR1) of SEQ ID NO:88, an HCDR2 of SEQ ID NO:89 and an HCDR3 of SEQ ID NO:90, and light chain complementarity determining region (LCDR1) of SEQ ID NO:91, an LCDR2 of SEQ ID NO:92, and an LCDR3 of SEQ ID NO:93.
21. The method of claim 14 , wherein said TCB antibody is a monoclonal antibody.