CD39 is expressed by a wide range of cutaneous T-cell lymphomas.

CD39, an ectoenzyme in the immunosuppressive CD39/CD73/adenosine pathway, known to promote solid tumour outgrowth and spreading, was investigated in both skin and blood compartments of cutaneous T cell lymphomas. CD39 was overexpressed by peripheral blood T-cells in Sezary syndrome and mycosis fungoides, and in skin-infiltrating lymphocytes of Sezary syndrome, mycosis fungoides, subcutaneous panniculitis-like T-cell lymphoma and primary cutaneous CD30-positive lymphoproliferation. Our study emphasizes the interest in using CD39/CD73/adenosine pathway blocking agents for cutaneous T cell lymphomas treatment.

Identification of CD160-TM as a tumor target on triple negative breast cancers: possible therapeutic applications.

BACKGROUND: Despite major therapeutic advances, triple-negative breast cancer (TNBC) still presents a worth prognosis than hormone receptors-positive breast cancers. One major issue relies in the molecular and mutational heterogeneity of TNBC subtypes that is reinforced by the absence of reliable tumor-antigen that could serve as a specific target to further promote efficient tumor cell recognition and depletion. CD160 is a receptor mainly expressed by NK lymphocytes and presenting two isoforms, namely the GPI-anchored form (CD160-GPI) and the transmembrane isoform (CD160-TM). While CD160-GPI is constitutively expressed on resting cells and involved in the generation of NK cells' cytotoxic activity, CD160-TM is neo-synthesized upon activation and promotes the amplification of NK cells' killing ability. METHODS: CD160 expression was assessed by immunohistochemistry (IHC) and flow cytometry on TNBC patient biopsies or cell lines, respectively. Antibody (Ab)-mediated tumor depletion was tested in vitro by performing antibody-dependent cell cytotoxicity (ADCC) and phagocytosis (ADCP) assays, and in vivo on a TNBC mouse model. RESULTS: Preliminary data obtained by IHC on TNBC patients' tumor biopsies revealed an unconventional expression of CD160 by TNBC tumor cells. By using a specific but conformation-dependent anti-CD160-TM Ab, we established that CD160-TM, but not CD160-GPI, was expressed by TNBC tumor cells. A conformation-independent anti-CD160-TM mAb (22B12; muIgG2a isotype) was generated and selected according to pre-defined specificity and functional criterions. In vitro functional assays demonstrated that ADCC and ADCP could be induced in the presence of 22B12, resulting in TNBC cell line apoptosis. The ability of 22B12 to exert an in vivo anti-tumor activity was also demonstrated on a TNBC murine model. CONCLUSIONS: Our data identify CD160-TM as a tumor marker for TNBC and provide a rational for the use of anti-CD160-TM antibodies as therapeutic tools in this tumor context.

Validation of AAC-11-Derived Peptide Anti-Tumor Activity in a Single Graft Sézary Patient-Derived Xenograft Mouse Model.

Sézary syndrome (SS) is an aggressive cutaneous T cell lymphoma with poor prognosis mainly characterized by the expansion of a tumor CD4+ T cell clone in both skin and blood. So far, the development of new therapeutic strategies has been hindered by a lack of reproducible in vivo models closely reflecting patients' clinical features. We developed an SS murine model consisting of the intravenous injection of Sézary patients' PBMC, together with a mixture of interleukins, in NOD-SCID-gamma mice. Thirty-four to fifty days after injection, mice showed skin disorders similar to that observed in patients, with the detection of epidermis thickening and dermal tumor T cell infiltrates. Although experimental variability was observed, Sézary cells could be tracked in the blood stream, confirming that our model could efficiently exhibit both skin and blood involvement. Using this model, we evaluated the therapeutic potential of RT39, a cell-penetrating peptide derived from the survival protein anti-apoptosis clone 11 (AAC-11), that we previously characterized as specifically inducing apoptosis of Sézary patients' malignant clone ex vivo. Systemic administration of RT39 led to cutaneous tumor T cells depletion, demonstrating efficient malignant cells' targeting and a favorable safety profile. These preclinical data confirmed that RT39 might be an innovative therapeutic tool for Sézary syndrome.

The soluble form of CD160 acts as a tumor mediator of immune escape in melanoma.

Melanoma is responsible for 90% of skin cancer-related deaths. Major therapeutic advances have led to a considerable improvement in the prognosis of patients, with the development of targeted therapies (BRAF or MEK inhibitors) and immunotherapy (anti-CTLA-4 or -PD-1 antibodies). However, the tumor constitutes an immunosuppressive microenvironment that prevents the therapeutic efficacy and/or promotes the development of secondary resistances. CD160 is an activating NK-cell receptor initially described as delineating the NK and CD8+ T-cell cytotoxic populations. Three forms of CD160 have been described: (1) the GPI isoform, constitutively expressed and involved in the initiation of NK-cells' cytotoxic activity, (2) the transmembrane isoform, neo-synthesized upon cell activation, allowing the amplification of NK cells' cytotoxic functions and (3) the soluble form, generated after cleavage of the GPI isoform, which presents an immuno-suppressive activity. By performing immunohistochemistry analyses, we observed a strong expression of CD160 at the primary cutaneous tumor site of melanoma patients. We further demonstrated that melanoma cells express CD160-GPI isoform and constitutively release the soluble form (sCD160) into the tumor environment. sCD160 was shown to inhibit the cytotoxic activity of NK-cells towards their target cells. In addition, it was found in the serum of melanoma patients and associated with increased tumor dissemination. Altogether these results support a role for sCD160 in the mechanisms leading to the inhibition of anti-tumor response and immune surveillance in melanoma.

Macrophage-derived CXCL9 and CXCL11, T-cell skin homing, and disease control in mogamulizumab-treated CTCL patients.

Cutaneous T-cell lymphomas (CTCLs) are rare malignancies involving primarily the skin. Responses to treatment are usually short-lived in advanced CTCL. The determinants of long-term CTCL control are unclear. Mogamulizumab, an anti-human CCR4 antibody that acts by antibody-dependent cell cytotoxicity against CCR4+ CTCL tumor cells and peripheral memory blood regulatory T cells, has been associated with long-lasting remissions and immune adverse events. Here, we reported skin rashes in 32% of 44 patients with CTCL treated with mogamulizumab, associated with significantly higher overall survival (hazard ratio, 0.16; 0.04-0.73; P = .01). Rash occurred in patients with Sézary syndrome and was associated with longer time to progression. These rashes were characterized by a CD163+ granulomatous and/or CD8+ lichenoid skin infiltrate. High-throughput sequencing analysis of T-cell receptor ß genes in skin and blood flow cytometry confirmed the depletion of CTCL tumor cells, as well as the recruitment of new reactive T-cell clones in skin at the time of skin rash. CXCL9 and CXCL11, two macrophage-derived chemokines that recruit CXCR3+ T cells to skin, were overexpressed in skin rashes. A higher frequency of TIGIT+ and PD1+ exhausted reactive blood T cells was observed at baseline in patients with rash, and this frequency decreased with mogamulizumab treatment. These data are consistent with mogamulizumab-induced long-term immune CTCL control by activation of the macrophage and T-cell responses in patients with rash.

PAK1-Dependent Antitumor Effect of AAC-11‒Derived Peptides on Sézary Syndrome Malignant CD4+ T Lymphocytes.

Sézary syndrome is an aggressive form of cutaneous T-cell lymphoma characterized by the presence of a malignant CD4+ T-cell clone in both blood and skin. Its pathophysiology is still poorly understood, and the development of targeted therapies is hampered by the absence of specific target proteins. AAC-11 plays important roles in cancer cell progression and survival and thus has been considered as an anticancer therapeutic target. In this study, we show that a peptide called RT39, comprising a portion of AAC-11‒binding site to its protein partners coupled to the penetratin sequence, induces the specific elimination of the malignant T-cell clone both ex vivo on the circulating cells of patients with Sézary syndrome and in vivo in a subcutaneous xenograft mouse model. RT39 acts by direct binding to PAK1 that is overexpressed, located in the plasma membrane, and constitutively activated in Sézary cells, resulting in their selective depletion by membranolysis. Along with the absence of toxicity, our preclinical efficacy evidence suggests that RT39 might represent a promising alternative therapeutic tool for Sézary syndrome because it spares the nonmalignant immune cells and, contrary to antibody-based immunotherapies, does not require the mobilization of the cellular immunity that shows heavy deficiencies at advanced stages of the disease.

Restoration of T-cell Effector Function, Depletion of Tregs, and Direct Killing of Tumor Cells: The Multiple Mechanisms of Action of a-TIGIT Antagonist Antibodies.

TIGIT is an immune checkpoint inhibitor expressed by effector CD4+ and CD8+ T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown in vitro potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti-PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on γδ T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti-PD(L)-1-like restoration of αß T-cell function. CD155 also mediated inhibition of γδ T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP-enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcγ receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.

Efficient Therapeutic Delivery by a Novel Cell-Penetrating Peptide Derived from Acinus.

In this study, we have identified a novel cell-penetrating sequence, termed hAP10, from the C-terminus of the human protein Acinus. hAP10 was able to efficiently enter various normal and cancerous cells, likely through an endocytosis pathway, and to deliver an EGFP cargo to the cell interior. Cell penetration of a peptide, hAP10DR, derived from hAP10 by mutation of an aspartic acid residue to an arginine was dramatically increased. Interestingly, a peptide containing a portion of the heptad leucine repeat region domain of the survival protein AAC-11 (residues 377-399) fused to either hAP10 or hAP10DR was able to induce tumor cells, but not normal cells, death both ex vivo on Sézary patients' circulating cells and to inhibit tumor growth in vivo in a sub-cutaneous xenograft mouse model for the Sézary syndrome. Combined, our results indicate that hAP10 and hAP10DR may represent promising vehicles for the in vitro or in vivo delivery of bioactive cargos, with potential use in clinical settings.

IPH4102, a first-in-class anti-KIR3DL2 monoclonal antibody, in patients with relapsed or refractory cutaneous T-cell lymphoma: an international, first-in-human, open-label, phase 1 trial.

BACKGROUND: IPH4102 is a first-in-class monoclonal antibody targeting KIR3DL2, a cell surface protein that is expressed in cutaneous T-cell lymphoma, and predominantly in its leukaemic form, Sézary syndrome. We aimed to assess the safety and activity of IPH4102 in cutaneous T-cell lymphoma. METHODS: We did an international, first-in-human, open-label, phase 1 clinical trial with dose-escalation and cohort-expansion parts in five academic hospitals in the USA, France, the UK, and the Netherlands. Eligible patients had histologically confirmed relapsed or refractory primary cutaneous T-cell lymphoma, an Eastern Cooperative Oncology group performance score of 2 or less, were aged 18 years or older, and had received at least two previous systemic therapies. Ten dose levels of IPH4102, administered as an intravenous infusion, ranging from 0·0001 mg/kg to 10 mg/kg, were assessed using an accelerated 3 + 3 design. The primary endpoint was the occurrence of dose-limiting toxicities during the first 2 weeks of treatment, defined as toxicity grade 3 or worse lasting for 8 or more days, except for lymphopenia. Global overall response by cutaneous T-cell lymphoma subtype was a secondary endpoint. Safety and activity analyses were done in the per-protocol population. The study is ongoing and recruitment is complete. This trial is registered with ClinicalTrials.gov, number NCT02593045. FINDINGS: Between Nov 4, 2015, and Nov 20, 2017, 44 patients were enrolled. 35 (80%) patients had Sézary syndrome, eight (18%) had mycosis fungoides, and one (2%) had primary cutaneous T-cell lymphoma, not otherwise specified. In the dose-escalation part, no dose limiting toxicity was reported and the trial's safety committee recommended a flat dose of 750 mg for the cohort-expansion, corresponding to the maximum administered dose. The most common adverse events were peripheral oedema (12 [27%] of 44 patients) and fatigue (nine [20%]), all of which were grade 1-2. Lymphopenia was the most common grade 3 or worse adverse event (three [7%]). One patient developed possibly treatment-related fulminant hepatitis 6 weeks after IPH4102 discontinuation and subsequently died. However, the patient had evidence of human herpes virus-6B infection. Median follow-up was 14·1 months (IQR 11·3-20·5). A confirmed global overall response was achieved in 16 (36·4% [95% CI 23·8-51·1]) of 44 patients, and of those, 15 responses were observed in 35 patients with Sézary syndrome (43% [28·0-59·1]). INTERPRETATION: IPH4102 is safe and shows encouraging clinical activity in patients with relapsed or refractory cutaneous T-cell lymphoma, particularly those with Sézary syndrome. If confirmed in future trials, IPH4102 could become a novel treatment option for these patients. A multi-cohort, phase 2 trial (TELLOMAK) is underway to confirm the activity in patients with Sézary syndrome and explore the role of IPH4102 in other subtypes of T-cell lymphomas that express KIR3DL2. FUNDING: Innate Pharma.

Therapeutic Antibodies to KIR3DL2 and Other Target Antigens on Cutaneous T-Cell Lymphomas.

KIR3DL2 is a member of the killer cell immunoglobulin-like receptor (KIR) family that was initially identified at the surface of natural killer (NK) cells. KIR3DL2, also known as CD158k, is expressed as a disulfide-linked homodimer. Each chain is composed of three immunoglobulin-like domains and a long cytoplasmic tail containing two immunoreceptor tyrosine-based inhibitory motifs. Beside its expression on NK cells, it is also found on rare circulating T lymphocytes, mainly CD8+. Although the KIR gene number varies between haplotype, KIR3DL2 is a framework gene present in all individuals. Together with the presence of genomic regulatory sequences unique to KIR3DL2, this suggests some particular functions for the derived protein in comparison with other KIR family members. Several ligands have been identified for KIR3DL2. As for other KIRs, binding to HLA class I molecules is essential for NK development by promoting phenomena such as licensing and driving NK cell maturation. For KIR3DL2, this includes binding to HLA-A3 and -A11 and to the free heavy chain form of HLA-B27. In addition, KIR3DL2 binds to CpG oligonucleotides (ODN) and ensures their transport to endosomal toll-like receptor 9 that promotes cell activation. These characteristics have implicated KIR3DL2 in several pathologies: ankylosing spondylitis and cutaneous T-cell lymphomas such as Sézary syndrome, CD30+ cutaneous lymphoma, and transformed mycosis fungoides. Consequently, a new generation of humanized monoclonal antibodies (mAbs) directed against KIR3DL2 has been helpful in the diagnosis, follow-up, and treatment of these diseases. In addition, preliminary clinical studies of a novel targeted immunotherapy for cutaneous T-cell lymphomas using the anti-KIR3DL2 mAb IPH4102 are now underway. In this review, we discuss the various aspects of KIR3DL2 on the functions of CD4+ T cells and how targeting this receptor helps to develop innovative therapeutic strategies.

Usefulness of KIR3DL2 to Diagnose, Follow-Up, and Manage the Treatment of Patients with Sézary Syndrome.

Purpose: KIR3DL2 is a recently discovered marker of the malignant clonal cell population in Sézary syndrome. We intended to evaluate the expression of KIR3DL2 on blood T cells as a diagnostic, prognostic, and follow-up marker of Sézary syndrome.Experimental Design: Sixty-four patients diagnosed with Sézary syndrome were included in this monocentric study. We collected the percentage of KIR3DL2+ cells among CD3+ T cells, obtained by flow cytometry, and other classical diagnostic criteria for Sézary syndrome at diagnosis and during the follow-up.Results: Compared with the classical diagnostic factors, KIR3DL2 was the most sensitive diagnostic factor for Sézary syndrome. Univariate and multivariate analyses established that an eosinophil cell count >700/mm3 and a percentage of KIR3DL2+ cells within the CD3+ T cells >85% at diagnosis were associated with a significantly reduced disease-specific survival. Moreover, KIR3DL2 immunostaining allowed the assessment of treatment efficiency and specificity toward tumor cells, the detection of the residual disease following treatment, and the occurrence of relapse, even though patients clinically experienced complete remission and/or undetectable circulating Sézary cells by cytomorphologic analysis.Conclusions: We show that KIR3DL2 expression is the most sensitive diagnostic criterion of Sézary syndrome when compared with all other available biological criteria. It also represents the best independent prognostic factor for Sézary syndrome-specific death and the most relevant feature for the follow-up of Sézary syndrome, showing the invasion of the functional lymphocytes pool by Sézary cells. KIR3DL2 therefore represents a valuable tool for routine use as a clinical parameter at diagnosis, for prognosis and during patient follow-up. Clin Cancer Res; 23(14); 3619-27. ©2017 AACR.

Evaluation of Immunophenotypic and Molecular Biomarkers for Sézary Syndrome Using Standard Operating Procedures: A Multicenter Study of 59 Patients.

Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses.

A novel targeted immunotherapy for CTCL is on its way: Anti-KIR3DL2 mAb IPH4102 is potent and safe in non-clinical studies.

Cutaneous T-cell lymphomas (CTCLs) represent a group of rarely occurring and clinically and pathologically heterogeneous diseases that are considered incurable at advanced stages. Current treatments provide limited clinical benefit and are thus largely amenable to improvement. An antibody-based CTCL-specific immunotherapy targeting the KIR3DL2 receptor expressed by the tumor cells in CTCL is currently under development and has shown encouraging results in pre-clinical studies.