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

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.

HLA-G Neo-Expression on Tumors.

HLA-G is known to modulate the immune system activity in tissues where physiological immune-tolerance is necessary (i.e., maternal-fetal interface, thymus, and cornea). However, the frequent neo-expression of HLA-G in many cancer types has been previously and extensively described and is correlated with a bad prognosis. Despite being an MHC class I molecule, HLA-G is highly present in tumor context and shows unique characteristics of tissue restriction of a Tumor Associated Antigen (TAA), and potent immunosuppressive activity of an Immune CheckPoint (ICP). Consequently, HLA-G appears to be an excellent molecular target for immunotherapy. Although the relevance of HLA-G in cancer incidence and development has been proven in numerous tumors, its neo-expression pattern is still difficult to determine. Indeed, the estimation of HLA-G's actual expression in tumor tissue is limited, particularly concerning the presence and percentage of the new non-canonical isoforms, for which detection antibodies are scarce or inexistent. Here, we summarize the current knowledge about HLA-G neo-expression and implication in various tumor types, pointing out the need for the development of new tools to analyze in-depth the HLA-G neo-expression patterns, opening the way for the generation of new monoclonal antibodies and cell-based immunotherapies.

CD28/4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is associated with a remarkably poor prognosis and with no treatment consensus. The identification of relevant therapeutic targets is challenging. Here, we investigated the immune functions, antileukemia efficacy and safety of CD28/4-1BB CAR T cells targeting CD123 the interleukin (IL)-3 receptor alpha chain which is overexpressed on BPDCN. We demonstrated that both retroviral and lentiviral engineering CD28/4-1BB CD123 CAR T cells exhibit effector functions against BPDCN cells through CD123 antigen recognition and that they efficiently kill BPDCN cell lines and BPDCN-derived PDX cells. In vivo, CD28/4-1BB CD123 CAR T-cell therapy displayed strong efficacy by promoting a decrease of BPDCN blast burden. Furthermore we showed that T cells from BPDCN patient transduced with CD28/4-1BB CD123 CAR successfully eliminate autologous BPDCN blasts in vitro. Finally, we demonstrated in humanized mouse models that these effector CAR T cells exert low or no cytotoxicity against various subsets of normal cells with low CD123 expression, indicating a potentially low on-target/off-tumor toxicity effect. Collectively, our data support the further evaluation for clinical assessment of CD28/4-1BB CD123 CAR T cells in BPDCN neoplasm.

Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC.

Invariant Natural Killer T (iNKT) cells are a small and distinct population of T cells crucial in immunomodulation. After activation by alpha-GalactosylCeramide (αGC), an exogenic glycolipid antigen, iNKT cells can rapidly release cytokines to enhance specific anti-tumor activity. Several human clinical trials on iNKT cell-based anti-cancer are ongoing, however results are not as striking as in murine models. Given that iNKT-based immunotherapies are dependent mainly on antigen-presenting cells (APC), a human tolerogenic molecule with no murine homolog, such as Human Leucocyte Antigen G (HLA-G), could contribute to this discrepancy. HLA-G is a well-known immune checkpoint molecule involved in fetal-maternal tolerance and in tumor immune escape. HLA-G exerts its immunomodulatory functions through the interaction with immune inhibitory receptors such as ILT2, differentially expressed on immune cell subsets. We hypothesized that HLA-G might inhibit iNKT function directly or by inducing tolerogenic APC leading to iNKT cell anergy, which could impact the results of current clinical trials. Using an ILT2-transduced murine iNKT cell line and human iNKT cells, we demonstrate that iNKT cells are sensitive to HLA-G, which inhibits their cytokine secretion. Furthermore, human HLA-G+ dendritic cells, called DC-10, failed at inducing iNKT cell activation compared to their autologous HLA-G‒ DCs counterparts. Our data show for the first time that the HLA-G/ILT2 ICP is involved in iNKT cell function modulation.

Human Hepatocytes and Differentiated Adult-Derived Human Liver Stem/Progenitor Cells Display In Vitro Immunosuppressive Properties Mediated, at Least in Part, through the Nonclassical HLA Class I Molecule HLA-G.

One of the main challenges in liver cell therapy (LCT) is the induction of a tolerogenic microenvironment to promote graft acceptance in the recipient. Little is known about the immunomodulatory potential of the hepatic cells used in liver cell therapy. In this work, we wanted to evaluate the immunosuppressive properties of human hepatocytes and adult-derived human liver stem/progenitor cells (ADHLSCs), as well as the potential involvement of the immunomodulatory molecule HLA-G. We demonstrated that both cell types were capable of inhibiting the proliferative response of PBMCs to an allogenic stimulus and that the immune inhibitory potential of ADHLSCs, although lower than that of hepatocytes, increased after hepatogenic differentiation. We demonstrated that liver cells express HLA-G and that the immune inhibition pattern was clearly associated to its expression. Interestingly, HLA-G expression increased after the third step of differentiation, wherein oncostatin M (OSM) was added. A 48 hr treatment with OSM was sufficient to induce HLA-G expression in ADHLSCs and result in immune inhibition. Surprisingly, blocking HLA-G partially reversed the immune inhibition mediated by hepatocytes and differentiated ADHLSCs, but not that of undifferentiated ADHLSCs, suggesting that additional immune inhibitory mechanisms may be used by these cells. In conclusion, we demonstrated that both hepatocytes and ADHLSCs present immunomodulatory properties mediated, at least in part, through HLA-G, which can be upregulated following hepatogenic differentiation or liver cell pretreatment with OSM. These observations open up new perspectives for the induction of tolerance following LCT and for potential therapeutic applications of these liver cells.

Anticancer DNA vaccine based on human telomerase reverse transcriptase generates a strong and specific T cell immune response.

Human telomerase reverse transcriptase (hTERT) is overexpressed in more than 85% of human cancers regardless of their cellular origin. As immunological tolerance to hTERT can be overcome not only spontaneously but also by vaccination, it represents a relevant universal tumor associated antigen (TAA). Indeed, hTERT specific cytotoxic T lymphocyte (CTL) precursors are present within the peripheral T-cell repertoire. Consequently, hTERT vaccine represents an attractive candidate for antitumor immunotherapy. Here, an optimized DNA plasmid encoding an inactivated form of hTERT, named INVAC-1, was designed in order to trigger cellular immunity against tumors. Intradermal injection of INVAC-1 followed by electrogene transfer (EGT) in a variety of mouse models elicited broad hTERT specific cellular immune responses including high CD4+ Th1 effector and memory CD8+ T­cells. Furthermore, therapeutic INVAC­1 immunization in a HLA-A2 spontaneous and aggressive mouse sarcoma model slows tumor growth and increases survival rate of 50% of tumor-bearing mice. These results emphasize that INVAC-1 based immunotherapy represents a relevant cancer vaccine candidate.

Synthetic HLA-G proteins for therapeutic use in transplantation.

The human leukocyte antigen (HLA)-G is a tolerogenic molecule, whose expression by allografts is associated with better acceptance. An increasing interest in producing HLA-G as a clinical-grade molecule for therapy use is impaired by its complexity and limited stability. Our purpose was to engineer simpler and more stable HLA-G-derived molecules than the full-length HLA-G trimolecular complex that are also tolerogenic, functional as soluble molecules, and compatible with good manufacturing practice (GMP) production conditions. We present two synthetic molecules: (α3-L)x2 and (α1-α3)x2 polypeptides. We show their capability to bind the HLA-G receptor LILRB2 and their functions in vitro and in vivo. The (α1-α3)x2 polypeptide proved to be a potent tolerogenic molecule in vivo: One treatment of skin allograft recipient mice with (α1-α3)x2 was sufficient to significantly prolong graft survival, and four weekly treatments induced complete tolerance. Furthermore, (α1-α3)x2 was active as a soluble molecule and capable of inhibiting the proliferation of tumor cell lines, as does the full length HLA-G trimolecular complex. Thus, the synthetic (α1-α3)x2 polypeptide is a stable and simpler alternative to the full-length HLA-G molecule. It can be produced under GMP conditions, it functions as a soluble molecule, and it is at least as tolerogenic as HLA-G in vivo.

Multimeric structures of HLA-G isoforms function through differential binding to LILRB receptors.

The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α1-α2-α3 non-covalently bound to ß-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α1-α3 structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the α1-α3-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.