Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

Abstract

For the monitoring of chimeric antigen receptor (CAR) T-cell therapies, antigen-based CAR detection methods are usually applied. However, for each target-antigen, a separate detection system is required. Furthermore, when monitored CAR T-cells in the blood of patients treated with bispecific antibodies or T-cell engagers (bsAbs/BiTEs) recognize the same antigen, these methods produce false-positive results in clinical diagnostics. Anti-CAR-linker monoclonal antibodies (mAbs) targeting the linker sequence between the variable domains of the antigen binding CAR fragment promise a universal and unbiased CAR detection. To test this, we analyzed clinical specimens of all BCMA- and CD19-targeting CAR T-cell products currently approved for clinical use. We found a highly specific and sensitive CAR detection using anti-CAR-linker mAb in blood cells from patients treated with Ide-cel, Tisa-cel, Axi-cel, Brexu-cel, and Liso-cel. For Ide-cel and Tisa-cel, the sensitivity was significantly lower compared to that for antigen-based CAR detection assays. Strikingly, the specificity of anti-CAR linker mAb was not affected by the simultaneous presence of bispecific blinatumomab or teclistamab for Axi-cel, Brexu-cel, Liso-cel, or Ide-cel, respectively. Cilta-cel (containing a monomeric G₄S-CAR linker) could not be detected by anti-CAR linker mAb. In conclusion, anti-CAR-linker mAbs are highly specific and useful for CAR T-cell monitoring but are not universally applicable.

Keywords: BiTE; CAR; CAR T-cell monitoring; anti-CAR-linker mAbs; bispecific antibody; combination treatments; detection; diagnostic.

Figure 1

Schematical representation of CAR T-cell detection with the classical ligand-based assay and anti-CAR linker mAb. (A) A schematical representation of CD19 and BCMA tumor antigens and their targeting CARs are shown. The recombinantly produced and biotinylated ligands and APC-conjugated anti-biotin Abs are used to visualize CAR expression on the surface of T-cells. (B) The general structure of a chimeric antigen receptor (CAR) is depicted as an illustration. The single-chain variable fragment (scFv) of CARs contains either a Whitlow linker (GSTSGSGKPGSGEGSTKG) or a (G₄S)₃ linker (GGGGSGGGGSGGGGS), targeted by anti-Whitlow mAb or by anti-G₄S monoclonal antibody (mAb), respectively. Biotinylated CAR-linker mABs and APC-conjugated anti-biotin Abs are used for the universal visualization of CAR expression on the surface of T-cells.

Figure 2

Anti-CAR linker mAbs allows for specific and sensitive CAR T monitoring in patients treated with any clinically approved CAR T therapies except Cilta-cel. (A) The structures of BCMA- and CD19-specific CARs are depicted in the illustration. CD19 antigen-specific CARs: Brexu-cel, Liso-cel, Axi-cel, and Tisa-cel; BCMA-specific CARs: Ide-cel and Cilta-cel. (B) Flow cytometry-based CAR detection analyses are shown, performed with biotinylated anti-CAR linker mAbs and APC-conjugated anti-biotin Abs to all approved BCMA and CD19 CAR T-cell products using isolated lymphocyte specimens from patients’ blood (n = 3). (C) Flow cytometry-based CAR detection analyses are shown, performed with Alexa 647-conjugated anti-CAR linker mAbs to all approved BCMA and CD19 CAR T-cell products using isolated lymphocyte specimens from patients’ blood (n = 3). The CAR-positive cells were determined from the CD3-positive cells and significances calculated based on BCMA- and CD19-antigen-based CAR detection reagent (Miltenyi = 100%). Statistical analysis was performed using Student’s t-test. * p ≤ 0.05%; ** p ≤ 0.01%; *** p ≤ 0.001%.

Figure 3

Anti-CAR linker mAbs allows for specific CAR T monitoring in patients pretreated with BiTE/bsAb, targeting the same antigen as the CAR. (A) The structure of the blinatumomab (CD19×CD3 BiTE) and teclistamab (BCMA×CD3 bsAb) are illustrated. (B) Shown is a schematic overview of CAR T-cell detection strategies based on the CD19 or BCMA CAR detection reagent (Miltenyi) that leads to a false positive staining in patients pretreated with BiTE/bsAb targeting the same antigen as the CAR. The structure of CD19- or BCMA-specific CAR and CD3 on the cell surface of a T-cell is depicted as an illustration. (C) Shown is a schematic overview of CAR T-cell detection strategies based on anti-CAR linker mAbs. The specificity of the CAR detection is not affected by BiTE/bsAb targeting the same antigen as the CAR. (D) Detection of CAR-positive cells in CD3-positive blood T-lymphocytes from patients, treated either with Brexu-cel, Liso-cel, and Axi-cel (anti-CD19 CAR T-cells) simultaneously in the presence or absence of blinatumomab BiTE (CD19×CD3) or treated either with Ide-cel (anti-BCMA CAR T-cells) simultaneously in the presence or absence of teclistamab bsAb (BCMA×CD3). Representative clinical blood specimens of a Lymphoma or MM patient are shown as dot plots. CAR-negative and CAR-positive cells are visualized using blue and green dots, respectively. Green and red checkmarks denote for specific or artificial CAR T-cell detection, respectively. Shown are the CD3-positive cells. Note that CD19- and BCMA-based CAR detection reagents (Miltenyi) interfere with blinatumomab and teclistamab, respectively, and bind to CD3 on the surface of all T-cells, irrespective of whether they carry a CAR or not. This leads to false positive results (denoted by dots shown in orange or red). The problem is solved using anti-CAR linker mAbs targeting the artificial linker sequence between the variable heavy- and light-chain domains of the scFv.