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.

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.

Use of a Closed Culture System to Improve the Safety of Lentiviral Vector Production.

We evaluated the possibility of introducing a combination of six oncogenes into primary porcine hepatocytes (PPH) using a lentiviral vector (LV)-mediated gene transfer in order to develop a porcine hepatocellular carcinoma model based on autologous transplantation of ex vivo-transformed hepatocytes. The six oncogenes were introduced into three plasmids, hence enabling the production of LVs encoding a luciferase reporter gene and hTERT+p53(DD), cyclinD1+CDK4(R24C), and c-myc(T58A)+HRas(G21V) genes, respectively. In order to improve the protection of the laboratory personnel manipulating such LVs, we used a compact cell culture cassette (CliniCell(®) device) as a closed cell culture system. We demonstrated that the CliniCell device allows to produce LVs, through plasmid transfection of 293T cells, and, after transfer to a second cassette, to transduce PPH with a similar efficacy as conventional open cell culture systems such as flasks or Petri dishes. Additionally, it is possible to cryopreserve at -80°C the transduced cells, directly in the CliniCell device used for the transduction. In conclusion, the use of a closed culture system for the safe handling of oncogene-encoding LVs lays the foundation for the development of porcine tumor models based on the autologous transplantation of ex vivo-transformed primary cells.

Early immune response against retrovirally transduced herpes simplex virus thymidine kinase-expressing gene-modified T cells coinfused with a T cell-depleted marrow graft: an altered immune response?

Administration of herpes simplex thymidine kinase (HSV-tk)-expressing, gene-modified T cells (GMCs) with T cell-depleted bone marrow transplantation (TCD-BMT) can allow modulation of posttransplantation alloreactivity. Twelve patients received 2 x 10(5) to 2 x 10(6) CD3+ donor GMCs per kilogram with HLA-identical sibling TCD-BMT. Despite extensive T cell depletion of bone marrow, an intensive conditioning regimen, and immunosuppressive graft-versus-host disease (GvHD) prophylaxis, infusion at the time of TCD-BMT of this low number of GMCs sufficed to induce a rapid GMC-specific immune response, as detected by interferon- enzyme- linked immunospot assay in six of eight patients, preferentially targeting HSV-tk. Maximal responses were reached early (median time, 49 [35-68] days post-BMT), with a subsequent rapid and significant decrease in five of six evaluable patients. Immune responses were negatively correlated with the maximal circulating GMC counts. However, such immune response did not result in the elimination of circulating GMCs and was not associated with measurable ex vivo cytotoxic activity against GMCs. Furthermore, alloreactive GMCs still could induce GCV-sensitive GvHD in one patient despite an ongoing immune response. Overall, infusion of HSV-tk-expressing GMCs at the time of BMT results in an early immune response. Such immune response may be altered and may not prevent persistent GCV-sensitive alloreactivity.

Abnormal expression of only the CD34 part of a transgenic CD34/herpes simplex virus-thymidine kinase fusion protein is associated with ganciclovir resistance.

Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.

Transcriptome of retrovirally transduced CD8+ lymphocytes: influence of cell activation, transgene integration, and selection process.

A suicide gene introduced by retroviral means can allow in vivo control of alloreactivity mediated by donor gene-modified T cells (GMTC) after allogeneic hematopoietic stem cell transplantation. The present study establishes the transcriptomic profile of GMTC prepared according to the GMTC production process used in our clinical trial (activation/selection methods, CD3/NeoR), which was previously demonstrated to induce phenotypical and functional alterations. This transcriptomic profile was compared with that of GMTC prepared by a novel process (CD3-CD28/DeltaNGFR-MACS) that limits alterations. Using a human pan-genomic microarray and GeneSpring software, we determined the gene expression profiles of CD8+ T cells from four healthy donors before and after the different steps required for gene modification. This analysis revealed that the gene expression pattern of GMTC is affected mainly by the activation step. Specific analysis of GMTC production processes showed that DeltaNGFR-MACS selection combined with CD3-CD28 activation limits the aberrant expression of genes involved in immunological functions and apoptotic pathways. Furthermore, our results indicate a limited risk of oncogenesis associated with retroviral-mediated gene transfer in CD8+ cells, a lower perturbation of the cell cycle regulation pathway after CD3-CD28 activation than after CD3 activation, and no significant involvement of the DeltaNGFR transduction signaling pathway when DeltaNGFR is used for selection. Moreover, genes that might be targeted to limit T cell functional alterations after ex vivo manipulation and culture were identified. These findings should be relevant to further adoptive T cell immunotherapy trials using ex vivo-expanded, gene-modified or unmodified T cells.

Deletions within the HSV-tk transgene in long-lasting circulating gene-modified T cells infused with a hematopoietic graft.

In our previous phase 1/2 study aimed at controlling graft-versus-host disease, 12 patients received Herpes simplex virus thymidine kinase (HSV-tk(+))/neomycin phosphotransferase (NeoR(+))-expressing donor gene-modified T cells (GMCs) and underwent an HLA-identical sibling T-cell-depleted bone marrow transplantation (BMT). This study's objective was to follow up, to quantify, and to characterize persistently circulating GMCs more than 10 years after BMT. Circulating GMCs remain detectable in all 4 evaluable patients. However, NeoR- and HSV-tk-polymerase chain reaction (PCR) differently quantified in vivo counts, suggesting deletions within the HSV-tk gene. Further experiments, including a novel "transgene walking" PCR method, confirmed the presence of deletions. The deletions were unique, patient-specific, present in most circulating GMCs expressing NeoR, and shown to occur at time of GMC production. Unique patient-specific retroviral insertion sites (ISs) were found in all GMCs capable of in vitro expansion/cloning as well. These findings suggest a rare initial gene deletion event and an in vivo survival advantage of rare GMC clones resulting from an anti-HSV-tk immune response and/or ganciclovir treatment. In conclusion, we show that donor mature T cells infused with a T-cell-depleted graft persist in vivo for more than a decade. These cells, containing transgene deletions and subjected to significant in vivo selection, represent a small fraction of T cells infused at transplantation.

Retrovirus-mediated gene transfer in human primary T lymphocytes induces an activation- and transduction/selection-dependent TCR-B variable chain repertoire skewing of gene-modified cells.

In a clinical trial that we recently reported, a suicide gene transfer in human primary T cells required 12 days of ex vivo culture, including activation of peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody (CD3 mAb), retrovirus-mediated transduction, and selection of gene-modified cells (GMC) by G418. The aim of the present study was to determine the impact of the initial T cell activation and of the transduction/selection on T cell receptor beta variable chain (TCRBV) repertoire of GMC by using the spectratyping method. The TCRBV repertoires of nontransduced, nonselected control (Co) cells and of GMC generated after an initial stimulation with CD3 mAb, CD3/CD28 beads, or allogeneic PBMC or Epstein-Barr virus-transformed B (B-EBV) cells were compared to the ones of their corresponding PBMC. The TCRBV repertoires were skewed in Co cells generated after CD3 mAb or after allogeneic stimulation, and even more so in their corresponding GMC, demonstrating that both culture-dependent and transduction/selection-dependent events led to TCRBV repertoire alterations. However, TCRBV repertoires were not altered, or to a lesser extent, in Co cells or GMC produced after CD3/CD28 bead activation, demonstrating a protective effect on both culture-dependent and transduction/selection-dependent repertoire alterations. Thus, we suggest to replace the initial CD3 mAb stimulation by CD3/CD28 beads for the production of clinical-grade GMC in the setting of future gene therapy trials.