Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells

Human peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from Guangdong General Hospital, after obtaining informed consent and for research use only. Pan T cells were enriched from the PBMCs using a “Pan T cell isolation kit” (Miltenyi Biotec, Germany). CD8+ T cells were positively isolated from the Pan T cells using “CD8 microbeads” (Miltenyi Biotec, Germany); the unlabeled cells that passed through the column were collected, representing the CD4+ T cells.

CD19.28z, Meso.28z, HER2.28z, and PSCA.28z CARs were constructed by linking sequences from a signal peptide derived from GM-CSF (GenBank; AAA58735.1, aa 1–19) to the corresponding antigen-specific single-chain variable fragment (scFv); these CARs do not have a spacer domain. Anti-CD19 scFv derived from FMC63 monoclonal antibody, anti-Mesothelin scFv derived from SS1 monoclonal antibody, anti-HER2 scFv derived from FRP5 monoclonal antibody, and PSCA scFv derived from humanized 1G8 monoclonal antibody. The CARs were codon-optimized and chemically synthesized using Genscript. A spacer containing a hinge domain and a CH3 domain derived from human IgG4 (GenBank; AAC82527.1, aa 98–329) was included in PSCA-H.28z CAR; an additional IgD hinge spacer was included in MUC1-H.28z CAR. The scFv of anti-MUC1 CAR derived from HMFG2 monoclonal antibody. The resulting products were sub-cloned into a Pwpxld-based lentiviral backbone plasmid encoding the transmembrane and intracellular domains of CD28 (aa 153–220) and the intracellular domain of CD3-ζ (aa 52–163). CD19-H.28z CAR and Meso-H.28z CAR were constructed by overlapping PCR, adding the hinge domain and the CH3 of IgG4 to the 3′ end of the scFv.

Pwpxld (encoding the CARs or GFP), Pspax2 (expressing the three required lentiviral proteins), and PMD.2G (encoding the lentiviral envelope protein) were transfected into HEK293T cells with PEI reagent (Life Technologies). The lentiviral supernatants were collected 48 and 72 h after transfection and were passed through a 0.4 μm filter. Human Pan T cells, CD8+ T cells, or CD4 T cells were stimulated with microbeads loaded with anti-human CD3, anti-human CD2, and anti-human CD28 antibodies (Miltenyi Biotec, Germany) in a 3:1 bead:cell ratio for 72 h and cultured in RPMI 1640, supplemented with 10% FBS, 10 mM of HEPES, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 2 mM of l-glutamine, r-human IL-2 (300 IU/ml, PeproTech, CT, USA), and r-human IL-15 (5 ng/ml, PeproTech, CT, USA). On day 3 after cell activation, the activated T cells were transduced with lentiviral supernatants and Polybrene 8 μg/ml (Takara) once; then, the cells were washed with PBS 3 times to completely remove any residual lentiviral supernatants. The cells were then resuspended in complete medium to achieve the expansion. The microbeads were removed on day 5. Fresh medium was added every 2 days to maintain an appropriate cell density ranging from 5 × 10⁵ to 1 × 10⁶ cells/ml.

All of the samples were analyzed with an LSR Fortessa or C6 (BD Bioscience), and the data were analyzed using FlowJo software. CAR T cells were detected by GFP, and T cell phenotypes were evaluated via CD3 PE-cy7 (clone OKT3, eBioscience), CD3 BV421 (clone UCHT1, BD), CD8a PE (clone HT8a, eBioscience), CD8a PE-cf594 (clone RPA-T8, BD), and CD4 APC (clone OKT4, eBioscience). All FACS plots representing the CAR T cell phenotypes were gated on CD3 and GFP double positive cells.

For the 19.28z T and 19-H.28z T cells, Nalm6 cell lysates were used as a chemoattractant in the lower chamber, while for the Meso.28z T and Meso-H.28z T cells, A549 cell lysates were used as a chemoattractant in the lower chamber. The T cells were cultured in an insert coated with Matrigel for 24 h, and the cells that transmigrated to the lower chamber were counted by flow cytometry. The percentage of migration was calculated as follows: (CAR T cells migrating through the Matrigel chamber membrane/total CAR T cells in insert membrane before assay begin) × 100.

NSI mice (NOD-scid-IL2Rg⁻/⁻mice, Guangzhou Institutes of Biomedicine and Health (GIBH)), aged 6–12 weeks were used to construct the xenograft tumor mouse models. All of the animal studies were carried out in accordance with instructional guidelines from the China Council on Animal Care and under protocols approved by the guidelines of the Ethics Committee of Animal Experiments at GIBH. Equivalent numbers of male and female mice were used. The NALM6-GL leukemia lines were intravenously inoculated into 2 × 10⁵ cells, and the A549-GL carcinoma lines were inoculated into 5 × 10⁵ cells subcutaneously (flank). The mice then received an adoptive transfer of CAR T cells intravenously 3–14 days later, as indicated in the individual experiments. To control the differences in transduction efficiency, non-transduced T cells were supplemented to ensure that both the number of CAR+ T cells, and the total number of T cells remained constant across all CAR T cell groups. The leukemia burden was evaluated using a cooled CCD camera system (IVIS 100 Series Imaging System, Xenogen, Alameda, CA, USA). The mice were injected intraperitoneally with d-luciferin firefly potassium salt at 75 mg/kg and then imaged 5 min later with an exposure time of 30 s. Quantification of the total and average emissions was performed using Living Image software (Xenogen).

All graphs report the mean ± SEM. All statistical analyses were performed with Prism software version 6.0 (GraphPad). The statistical significance of all data was calculated using an unpaired Student’s t test with the Bonferroni correction for multiple comparisons, where applicable. P < 0.05 was considered significant and is designated with an asterisk in all figures.

To characterize the functionality of hinges in CAR vectors, we constructed a series of lentiviral vectors for CAR production that contained the same transmembrane and intracellular domains (CD28 costimulatory receptor in tandem with CD3ζ) but different scFv-binding and hinge domains, tagged with GFP (Fig. 1a). We then transduced these CAR vectors into human primary T cells and monitored the growth of the CAR T cells from day 6 post-transfection. Interestingly, we found that the percentages of CAR T cells were greater when the CAR vectors contained a hinge domain, regardless of the scFv. For example, the percentages of 19-H.28z and Meso-H.28z T cells increased from 3.0 to 40.7% (Fig. 1b; Additional file 1: Figure S1) and from 34 to 74.3% (Fig. 1c; Additional file 1: Figure S1), respectively. In addition, the percentages of PSCA-H.28z T cells increased from 4.7 to 31.1% (Fig. 1d; Additional file 1: Figure S1) and from 1.6 to 58.2% (Fig. 1i). Similarly, the percentages of MUC1-H.28z T cells also increased from 6.9 to 17.3%, although the MUC1-H.28z CAR was designed with an IgD hinge, which indicates that the ability to increase the CAR T cell percentage is not restricted to IgG4-CH3 hinges (Fig. 1i). The absolute number of CAR T cells also significantly increased (Fig. 1e–g). In contrast, the percentages of CAR T cells without a hinge domain tended to be stable throughout the in vitro expansion process (Fig. 1b–d; Additional file 1: Figure S1), and their total cell numbers were less than those of CAR T cells containing a hinge domain (Fig. 1e–g). Taken together, these results demonstrate that the incorporation of a hinge domain can promote the growth of CAR T cells in vitro.

To identify which T cell subsets were most responsive to the incorporation of a hinge domain, we monitored the ratios of CD4+ and CD8+ CAR T cells in culture and found that the percentages of CD4+ 19-H.28z T and CD4+ Meso-H.28z T cells increased from 3.13 to 29.6% (Fig. 2a, left; Additional file 2: Figure S2) and from 19.9 to 46.5% (Fig. 2a, middle), respectively. In addition, the percentages of CD4+ PSCA-H.28z T cells increased from 3.06 to 34.3% (Fig. 2a, right) and from 0.92 to 46% (Fig. 2b, left). The CD4+ MUC1-H.28z T cells also increased from 4.14 to 14.9% (Fig. 2b, right). However, the influence of the hinge domain on the percentages of CD8+ CAR T cells was less pronounced, as the percentages of CD8+ 19-H.28z T and CD8+ Meso-H.28z T cells increased from 0.4 to 10.5% (Fig. 2a, left; Additional file 2: Figure S2) and from 14.1 to 23.8% (Fig. 2a, middle), respectively. In addition, the CD8+ PSCA-H.28z T cells increased from 0.92 to 6.34% (Fig. 2a, right) and from 0.67 to 12.6% (Fig. 2b, left). The CD8+ MUC1-H.28z T cells also increased from 2.85 to 4.61% (Fig. 2b, right). The percentage of CAR T cells without a hinge domain tended to be stable throughout the in vitro culture period (Additional file 3: Figure S3). Interestingly, when we isolated CD4+ T and CD8+ T cells and cultured them separately in vitro, the percentages of CAR T cells with or without a hinge domain all tended to be stable in both CD4+ T and CD8+ T cells, consistent with the GFP control T cells (Fig. 2c). This suggests that the use of a hinge to enhance CD4+ CAR T cell expansion requires the co-participation of CD8+ CAR T cells. In summary, hinge incorporation mainly promotes CD4+ CAR T cell expansion during the in vitro culture period.

To study whether the incorporation of a hinge domain affects the cytotoxicity of CAR T cells, we compared the killing capacities of anti-CD19 and anti-mesothelin CARs with and without a hinge. Both 19.28z T and 19-H.28z T cells efficiently lysed the NALM6-GL (Fig. 3a), indicating that the killing capacities of these two CARs were similar. Similarly, there were no significant differences between the lysis capacities of Meso.28z T and Meso-H.28z CAR T cells (Fig. 3b). For cytokine production, both 19-H.28z T and Meso-H.28z T cells produced similar levels of IL2 and IFN-γ compared with their hinge-free counterparts (Fig. 3c–f). Next, we compared the migratory capacity of GFP T, 19.28z T, and 19-H.28z T cells, using NALM6 cell lysate as a chemoattractant in the lower chamber of the transwell plate. Interestingly, we found that the 19-H.28z T cells transmigrated the Matrigel more efficiently than the 19.28z T cells (Fig. 3g). Similar results were also obtained in the Meso-H.28z T cells (Fig. 3h), suggesting that hinge incorporation enhanced the migratory and invasion capabilities of CAR T cells.

Subsequently, we evaluated the in vivo antitumor capacities of CAR T cells with or without the hinge domain in cell-line-derived xenograft-bearing mice. Immune deficient NSI mice were intravenously injected with 2 × 10⁵ NALM6-GL cells, followed by the infusion of a single dose of 2 × 10⁶ GFP, 19.28z, or 19-H.28z T cells on day 7 (Fig. 4a) [32‐36]. Bioluminescence imaging (BLI) showed a reduced tumor burden in the mice infused with 19.28z T and 19-H.28z T cells compared with those infused with GFP T cells on day 14. However, both the 19.28z T and 19-H.28z T-cell-infused mice relapsed on day 22 (Fig. 4c). The survival times of the 19.28z T and 19-H.28z T cell groups also showed no significant difference (Fig. 4b). In summary, the introduction of a hinge into anti-CD19 specific CARs did not enhance their in vivo antitumor capacities.

To study whether a hinge can affect the antitumor capacities of CARs that are specific for solid tumors, a solid-tumor mouse model was established, in which A549-GL cells were subcutaneously injected into NSI mice. Mice bearing an established tumor were treated I.V. with two doses of Meso.28z, Meso-H.28z, or GFP T cells, with the first dose on day 7 and the second on day 10. The tumor diameters were measured every 6 days. On day 49, the mice were sacrificed (Fig. 5a). Interestingly, tumor growth in both the Meso.28z T and Meso-H.28z T cell groups was delayed compared with the blank and GFP T cell group, but the delay was greater in the Meso-H.28z T group (Fig. 5b). Results consistent with these were also obtained from weight measurement and photographic inspection of the tumors (Fig. 5c–d). These results demonstrated that the incorporation of a hinge domain enhanced the antitumor capacities of anti-mesothelin CARs.