First immunotherapeutic CAR-T cells against the immune checkpoint protein HLA-G

Abstract

Background: CAR-T cells immunotherapy is a breakthrough in the treatment of hematological malignancies such as acute lymphoblastic leukemia (ALL) and B-cell malignancies. However, CAR-T therapies face major hurdles such as the lack of tumor-specific antigen (TSA), and immunosuppressive tumor microenvironment sometimes caused by the tumorous expression of immune checkpoints (ICPs) such as HLA-G. Indeed, HLA-G is remarkable because it is both a potent ICP and a TSA. HLA-G tumor expression causes immune escape by impairing innate and adaptive immune responses and by inducing a suppressive microenvironment. Yet, to date, no immunotherapy targets it.

Methods: We have developed two anti-HLA-G third-generation CARs based on new anti-HLA-G monoclonal antibodies.

Results: Anti-HLA-G CAR-T cells were specific for immunosuppressive HLA-G isoforms. HLA-G-activated CAR-T cells polarized toward T helper 1, and became cytotoxic against HLA-G⁺ tumor cells. In vivo, anti-HLA-G CAR-T cells were able to control and eliminate HLA-G⁺ tumor cells. The interaction of tumor-HLA-G with interleukin (IL)T2-expressing T cells is known to result in effector T cell functional inhibition, but anti-HLA-G CAR-T cells were insensitive to this inhibition and still exerted their function even when expressing ILT2. Lastly, we show that anti-HLA-G CAR-T cells differentiated into long-term memory effector cells, and seemed not to lose function even after repeated stimulation by HLA-G-expressing tumor cells.

Conclusion: We report for the first time that HLA-G, which is both a TSA and an ICP, constitutes a valid target for CAR-T cell therapy to specifically target and eliminate both tumor cells and HLA-G⁺ suppressive cells.

Keywords: adoptive; antigens; biomarkers; carbohydrate; chimeric antigen; immunotherapy; receptors; tumor; tumor escape; tumor-associated.

Figure 1

Anti-HLA-G CAR design and expression vector. (A) Schematic representation of anti-HLA-G monoclonal antibodies specificities: LFTT1 monoclonal antibody (mAb) is specific for HLA-G1/β2m-associated isoform and 15E7 mAb is specific for HLA-G1/β2m-free isoform. (B) Schematic representation of the third-generation anti-HLA-G CAR protein and lentiviral vector backbones used to transduce human CD3⁺ T cells. (C) Lentiviral constructs details; SP: signal peptide from mouse Igκ. scFv: single chain fragment from either LFTT1 (CAR-LFTT1) or 15E7 (CAR-15E7). Hinges: classical IgG4 hinge, IgG4 hinge+CH3 domain of human IgG4 and IgG4 hinge+CH2-CH3 domain of human IgG4. TM CD28: transmembrane domain of human CD28. CD28, 4-1BB, CD3ζ: endodomains of respectively CD28, 4-1BB and CD3ζ human proteins. P2A: cleavage site. trCD19: truncated CD19. U3, R, U5: HIV-1 LTR regions. Φ: encapsidation signal. RRE, Rev Response Element. EF1α, full length elongation factor-1α promoter. WPRE, Woodchuck Post-transcriptionnal Response element mutated for HBx ATG codon start. (D) Validation of transduction and co-expression of CAR using the CAR^CH2-CH3 construct proteins and trCD19 reporter for both CAR-LFTT1 and CAR-15E7 T cells from primary human T cells. Transduced T cells were labeled with CD19 and IgG4 antibodies and analyzed by flow cytometry.

Figure 2

Anti-HLA-G CAR-T cells are specific and activated by different HLA-G isoforms. (A) Anti-HLA-G monoclonal antibodies specificity against HLA-G^–/+ cell lines. Representative dot plot of K562, K562-HLA-G1 and JEG-3 tumor cell lines labeling with the anti-HLA-G 15E7 and LFTT1 monoclonal antibodies. Upregulation of activation-associated markers CD69, CD25 and programmed cell death protein 1 (PD-1) were monitored on activated non-transduced T cells, CAR-15E7 and CAR-LFTT1 T cells following a 6-hour incubation with K562, K562-HLA-G1 or JEG-3 cell lines. (B) Representative dot plot of the upregulation of CD69 and CD25 on activated non-transduced T cells and CAR-T cells sets. (C) Upregulation of activation-associated markers CD69, CD25 and PD-1 was determined on CAR expressing T cells (CD8⁺/trCD19⁺) in comparison to CAR negative T cells (CD8⁺/trCD19^-) following incubation with K562, K562-HLA-G1 or JEG-3 cell lines (n=3, NT, non-transduced).

Figure 3

CAR-LFTT1 and CAR-15E7 are cytotoxic against HLA-G1 tumor cells. (A) K562, K562-HLA-G1 or JEG-3 tumor target cells were exposed to activated non-transduced T (NT) cells, CAR-15E7 or CAR-LFTT1 T cells sets at different E:T ratio. Target cells were labeled with CFSE and % of tumor cell lysis represent the percentage of CFSE⁺ cells labeled with live/dead at 24 hours posteffector exposure (n=6). Significance was determined using a Mann-Whitney U test of unpaired t-test (*p<0,05; **p<0002) (n=6). (B) CD107a degranulation of CAR-LFTT1, CAR-15E7 sets or activated NT human T cells was monitored on CD8⁺/trCD19⁺ T cells following 6 hours exposure to K562, K562-HLA-G1 and JEG-3 cells at a E:T ratio of 10:1 (n=6).

Figure 4

Anti-HLA-G CAR-T cells differentiated into long-term memory effector cells after repeated stimulation with HLA-G⁺ tumor cells. (A) Schematic representation of anti-HLA-G CAR-T cells repeated stimulation with K562-HLA-G1 tumor cells. Transduced CAR-LFTT1, CAR-15E7 sets or activated non-transduced T cells were repeatedly stimulated every 12 days with K562-HLA-G1 cells at a 10:1 E:T ratio. Expression of differentiation markers was monitored prior to and 24 hours after the stimulation and cytokines secretion levels were monitored at days 13, 26 and 39. (B) Representative flow cytometry analysis of the expression of CD62L and CD45RA differentiation markers on activated non-transduced, CAR-15E7 and CAR-LFTT1 T cells prior to and 24 hours after stimulations rounds. (C) Day prior stimulation and after each repeated stimulation with K562-HLA-G1 cells, differentiation of T cells was determined on the basis of their CD62L/CD45RA cell-surface expression. CAR-T cells were gated on CD8 and trCD19 reporter co-expression. (D) Co-culture medium was recovered the day after each stimulation with K562-HLA-G1 cells and analyzed for interferon (IFN)γ, interleukin (IL)-2 and tumor necrosis factor (TNF)α concentration (n=3). Columns represent means±SEM. For each panel, after first stimulation round, activated non-transduced T cells were removed from the experiment due to absence of cytotoxicity against K562-HLA-G1 cells.

Figure 5

Anti-HLA-G CAR-T cells are not inhibited by ILT2 expression. Following transduction of T cells, CAR-T cells displaying or not the CAR construct at their surface (respectively CAR^pos and CAR^neg T cells) were analyzed for their ILT2 and programmed cell death protein 1 (PD-1) cell-surface expression after repeated stimulation with K562-HLA-G1 cells at a 10:1 E:T ratio. CH2-CH3 hinge was used to determine the CAR cell-surface expression CAR-LFTT1 or CAR-15E7 T cells. (A) ILT2 expression levels on CAR^neg/pos cells prior and after repeated stimulations with K562-HLA-G1 were compared with activated non-tranduced (NT) T cells. (B) PD-1 expression levels on CAR^neg/pos cells prior and after repeated stimulations with K562-HLA-G1 were compared with activated NT T cells. Significance was determined using a Friedmann test (*p<0,05; **p<0002) (n=6). (C) Differentiation of CAR-15E7 and CAR-LFTT1 T cells expressing or not ILT2 was determined on the basis of their CD62L/CD45RA expression on CAR^pos T cells before and after the repeated stimulations with K562-HLA-G1 cells. (D) 15E7^CH2-CH3 or LFTT1^CH2-CH3 CAR-T cells were generated from ILT2⁺ and ILT2^- sorted human CD8⁺ T cells before being co-cultured with K562-HLA-G1 cells. (E) CD107a degranulation of ILT2^–/+ CAR^neg/pos T cells was monitored by labeling of CD107a at the surface of CAR^neg/pos CD8⁺/trCD19⁺ T cells after 6 hours exposure to K562-HLA-G1 target cells. Columns represent means±SEM. Significance was determined using a Mann-Whitney U test (ns, non significative) (n=3). (F) ILT2 expression impact on anti-HLA-G CAR^neg/pos T cells. T helper 1 cytokines expression profile was evaluated after 18 hours co-incubation with K562-HLA-G1 cells. Cytokines (interferon (IFN)γ, tumor necrosis factor (TNF)α, IL-2) secretion by CAR^neg/pos CD8⁺ cells was determined by FACS and proportion of each combination are represented in cumulative histograms.

Figure 6

Anti-HLA-G CAR-T cells mediated tumor control of K562-HLA-G1-luciferase mouse model. (A) Schema of experimental procedure: NOD/SCID/IL-2Rγc-deficient (NSG) mice were irradiated the day before being intravenously injected with 10⁶ K562-HLA-G1-luciferase cells (K562-HLA-G1-Luc). Mice received activated non-transduced T cells or CAR-T (CAR-15E7^CH2-CH3 or CAR-LFTT1^CH2-CH3) cells on day 3 and were monitored by bioluminescence imaging over time. (B) Representative bioluminescence intensity of mice over time. (C) Total flux of each mouse in the three groups at different points.