High Ligand Expression Research Articles

A Phase 1 Study of NKX101, a Chimeric Antigen Receptor Natural Killer (CAR-NK) Cell Therapy, with Fludarabine and Cytarabine in Patients with Acute Myeloid Leukemia

Introduction Relapsed or refractory (r/r) acute myeloid leukemia (AML) has a dismal prognosis. Treatment options are limited and include fludarabine (Flu) and cytarabine (Ara-C), +/- anthracyclines (e.g., FLAG-Ida). Recent progress has been limited to targeted therapies, which benefit only a subset of the population; efforts to develop cellular therapies have not been met with success. NKX101 is an NK cell therapy derived from healthy donors and engineered to express an NKG2D ligand-directed chimeric antigen receptor (CAR) to enhance killing of malignant cells, as well as a membrane bound form of interleukin (IL)-15 to promote persistence and activity. Because Ara-C is known to upregulate NKG2D ligand expression, use of Flu/Ara-C as an alternative to standard Flu/cyclophosphamide (Flu/Cy) for lymphodepletion (LD) was tested for NKX101. Methods Six patients with r/r AML were enrolled in this cohort of NKX101-101, a Phase 1 safety study. All patients had received at least one prior line of therapy, as well as approved targeted therapies for those with underlying genetic mutations. Patients received NKX101 at a dose of 1.5 billion (B) viable CAR+ cells/dose on Days 0, 7, and 14 after LD comprising Flu (30 mg/m2) and Ara-C (2 g/m2) each once daily for five days and 2 days of rest. Efficacy assessments were performed on Day 27 and included bone marrow aspiration and biopsy. Additional treatment cycles of LD and NKX101 were given to some patients to deepen or consolidate responses. Pharmacokinetic (PK) and cytokine sampling were performed throughout the treatment cycle(s). Results At baseline, five out of six patients had poor-risk genetic features, including TP53 mutation; the median bone marrow blast count was 35% (range: 20 - 86%). Patients had received a median of two prior lines of therapy, with all patients having previous venetoclax exposure (Table 1). All patients received at least three doses of NKX101 at 1.5 B cells/dose. There were no cases of cytokine release syndrome (CRS), immune cell associated neurotoxicity syndrome (ICANS), or graft-versus-host disease of any grade in any patients (Table 2). Myelosuppression and infection were the most common higher-grade toxicities. Four out of six patients had CR/CRi, with three out of six achieving CR. Three of these patients had no detectable MRD by flow, and one had 0.18% RUNX1-RUNX1T1 translocation via polymerase chain reaction testing on bone marrow. One subject was taken to consolidative hematopoietic cell transplant (HCT) and remains in CR. Another subject received a consolidative cycle of therapy and remains in CR. A third subject had three cycles of treatment with successive decrease in disease burden and remains in CRi. The fourth subject had CR after one cycle of treatment. PK profiling showed that NKX101 was consistently detected and correlated with infusion days (Figure 1). Cytokine data showed minimal post-infusion elevations above baseline; no association was observed between clinical response and elevation of serum cytokines (including IL-15, IL-6, IFNγ, IL-10, and IL-8). Data on the expression of NKG2D ligands (MICA, MICB, ULBP1, and ULBP3) in subject bone marrow samples were obtained via multiplex immunohistochemistry. This was evaluated as a composite H-score. Responders were split between those with relatively low ligand expression and those with higher ligand expression, suggesting that further factors may contribute to NK cell resistance. Conclusions Alternative LD with Flu/Ara-C followed by NKX101 shows promising early responses including MRD negativity in r/r AML. The toxicity profile of this regimen is manageable and was consistent with underlying AML and exposure to LD, without any events of CRS or ICANS. Expansion of enrollment and longer follow-up are needed. Despite being an allogeneic, non-HLA-matched cell product, NKX101 persisted for up to three weeks in PK testing. Directly comparing the data from patients treated with Flu/Cy with NKX101 versus Flu/Ara-C with NKX101 suggests that Flu/Ara-C can replace the Flu/Cy regimen common to CAR T cell therapy as LD without compromising overall NKX101 exposure.

Multiparametric Flow Cytometry Evaluation of CD200L/CD200R- LSC/NK Synapse Including Leukemia Stem Cell (LSC) Fraction As a Potential Therapeutic Target and Marker of NK Cell Exhaustion in Pediatric AML-Conect-AML French Collaborative Network

BACKGROUNDNK cells play a crucial role in the immune surveillance of malignant hemopathies. They undergo fine regulation by the microenvironment and by integrating activating and inhibiting signals trough several receptor/ligand couple interactions, hereafter referred to as “NK synapse”. The ligands are expressed by a variety of cell types in the hematopoietic niche, including most immature leukemic stem cells CD34+CD38-. High expression of inhibiting ligands on AML (acute myeloid leukemia) blasts was associated with adverse clinical outcome . This observation highlights the relevance of identifying new ligand/receptor (L/R) pairs that could be targeted to prevent inhibiting interactions at the NK synapse. Relevant interactions to be blocked would display both ligand and receptor expressions on the leukemic cells and NK cells respectively. PATIENTS AND METHODS23 pediatric AML patients from the pediatric MyeChild01 protocol including in CONECT-AML French national collaborative network project diagnosed between 2018 and 2019 were included in this study. Reference bone marrows used were regenerative (4) or healthy bone marrows (5) . Multicolour flowcytometry protocole used fresh EDTA bone marrow at AML diagnosis and immunostaining with fluorochrome-coupled antibodies using 14 colour panel of L/R couples (Figure 1). Data was acquired on the FORTESSA Becton Dickinson with the Diva software and analysis using script R-PCA and FlowJo . RESULTSWe studied 5 inhibiting NK synapses (iinhibitory ligand/receptor pairs) . Four out of five inhibiting synapses (TIGIT/CD155; PD1-1/PD-L1; CD94/HLA-E and KIR2DL/HLA-A-B-C), showed not significant expression of ligand associated with the corresponding receptor expression. The CD200/CD200R synapse was the only one in which high ligand expression in blasts was significantly associated with high receptor expression on NK cells (Figure 2). This synapse could thus be of interest to develop targeting therapies for CD200-positive pediatric AML, with the strong advantage that patient eligibility could be easily identified at diagnosis. We then realized a principal component analysis, using the R software (PCA), integrating the MFIs of the 5 inhibiting NK synapses and 6 activating NK synapses (Figure 1) for the pediatric AML cohort (ID #1 to #23 ) together with reference bone marrows (healthy donors (n=5; ID #24 to #28) and regenerative bone marrows (n=4; ID #29 to #32)) . The CD200/CD200R synapse was identified as the main variable, explaining the distribution of patients and healthy donors as both CD200 and CD200R expressions happened to be among the most contributive to PCA axes. Interestingly, healthy donors clustered together, close to regenerative bone marrows. Pediatric AML patients distributed heterogeneously (Figure 3). In parallel, we evaluated whether CD200 expression on bulk leukemia blasts including most immature CD34+CD38- LSC was associated with exhaustion markers on NK cells. We found that patients with high and intermediate expression of CD200 on blasts (MFI > 3 rd quartile and comprised between 2 nd and 3 rd quartile, respectively) displayed strong PD-1 and TIGIT expressions on NK cells. Reciprocally, patients with low CD200 expression (MFI< 2 nd quartile) displayed a moderate PD-1 expression on NK cells, and TIGIT expression was more heterogeneous among individuals (Figure 4). CONCLUSIONSHere, we identified CD200 expression in AML blasts including LSC as a marker that could be associated with NK cell exhaustion. at diagnosis. A PCA strategy allowed to observe that this marker differentiated pediatric AML patients NK synapse profiles from healthy donors and regenerative bone marrows sugesting a potential deregulation of bone marrow niche including NK-LSC escape. This suggests that CD200 expression assessment on blasts at diagnosis could be a tool to evaluate NK cell antitumor potential. Indeed, direct NK cell assessment by flow cytometry can be challenging because of blast invasion in the bone marrow. Nevertheless, it remains to be elucidated whether this clustering and exhaustion markers on NK cells correlated with patient clinical outcomes and MRD kinetics including CD34+CD38- LSC flow frequency evaluation that should be useful in most clinical trials to overcome chemoresistance of LSC. These results should be confirmed in a prospectively larger cohort of patients in future clinical trials. [Display omitted] DisclosuresRenard: Jazz Pharmaceuticals: Research Funding.

Artificial Intelligence–Assisted Amphiregulin and Epiregulin IHC Predicts Panitumumab Benefit in RAS Wild-Type Metastatic Colorectal Cancer

Purpose High tumor mRNA levels of the EGFR ligands, amphiregulin (AREG) and epiregulin (EREG) are associated with anti-EGFR agent response in metastatic colorectal cancer (mCRC). However, ligand RNA assays have not been adopted into routine practice due to issues with analytical precision and practicality. We investigated whether AREG/EREG immunohistochemistry (IHC) could predict benefit from the anti-EGFR agent, panitumumab. Experimental Design Artificial intelligence algorithms were developed to assess AREG/EREG IHC in 274 patients from the PICCOLO trial of irinotecan+/-panitumumab (Ir vs IrPan) in RAS wild-type (-wt) mCRC. The primary endpoint was progression-free survival (PFS). Secondary endpoints were Response Evaluation Criteria in Solid Tumors (RECIST) response rate (RR) and overall survival (OS). Models were repeated adjusting separately for BRAF mutation status and primary tumor location (PTL). Results High ligand expression was associated with significant PFS benefit from IrPan compared with Ir (8·0 vs 3·2 months; HR 0·54, 95% CI 0·37-0·79; p=0·001); whereas low ligand expression was not (3·4 vs 4·4 months; HR 1·05, 95% CI 0·74-1·49; p=0·78). The ligand-treatment interaction was significant (pinteraction=0·02) and remained significant after adjustment for BRAF-mutation status and PTL. Likewise, RECIST RR was significantly improved in patients with high ligand expression (IrPan vs Ir: 48% vs 6%; p<0·0001) but not those with low ligand expression (25% vs 14%; p=0·10) (pinteraction=0·01). The effect on OS was similar but not statistically significant. Conclusion AREG/EREG IHC identified patients who benefitted from the addition of panitumumab to irinotecan chemotherapy. IHC is a practicable assay that may be of use in routine practice.

Artificial intelligence-assisted immunohistochemical (IHC) evaluation of tumor amphiregulin (AREG) and epiregulin (EREG) expression as a combined predictive biomarker for panitumumab (Pan) therapy benefit in RAS wild-type (wt) metastatic colorectal cancer (mCRC): Analysis within the phase III PICCOLO trial.

111 Background: High tumor mRNA levels of the EGFR ligands, AREG and EREG are associated with anti-EGFR agent response in patients (pts) with RAS-wt mCRC, regardless of tumor location. However, ligand RNA assays have not been adopted into routine clinical practice due to issues with analytical precision and practicality. Here we test whether AREG and EREG expression assessed by IHC can predict benefit from Pan. Methods: A retrospective biomarker study within the PICCOLO trial (NCT00389870; irinotecan [Ir] ± Pan in fluoropyrimidine-resistant RAS-wt mCRC). AREG and EREG positive tumor cells were assessed by IHC in all RAS-wt patients with available tumor tissue. Pathologists annotated tumor areas on digital images of glass slides. Artificial intelligence (AI) algorithms calculated the percentage of tumor cells staining positive for AREG and EREG within the tumor areas. More than 50% AREG and/or EREG tumor cell positivity was regarded as high ligand expression. The primary endpoint was progression-free survival (PFS) and secondary endpoints were RECIST response rate (RR) and overall survival (OS). Results: 274 RAS-wt pts had available tumor tissue. High ligand expression (n = 132) was associated with significant PFS benefit from IrPan compared with Ir (8.0 vs 3.2 months; HR 0.54 [0.37-0.79]; p = 0.001); whereas low ligand expression (n = 142) was not (3.4 vs 4.4 months; HR 1.05 [95% CI, 0.74-1.49]; p = 0.78). The ligand-treatment interaction was significant (p = 0.02) and independent of BRAF-mutation status and primary tumor location. Likewise RR was significantly improved in pts with high ligand expression (IrPan vs Ir: 48% vs 6%; risk ratio, 7.8 [2.90-20.69]; p &lt; 0.0001) but not those with low ligand expression (IrPan vs Ir: 25% vs 14%; risk ratio, 1.8 [95% CI, 0.89-3.65]; p = 0.10) (interaction p = 0.01). Lesser effect was seen on OS. Conclusions: IHC assessment of AREG and EREG identified pts who did or did not benefit from Pan, as has been previously demonstrated through mRNA quantification. IHC represents a more practicable technique as it can be provided at the point of care and is associated with shorter turn-around times. AREG and EREG IHC may be of use in routine practice to identify patients who would benefit from anti-EGFR therapy and those for whom alternative treatment strategies should be explored.

Detection of Cell Surface Ligands for Human Synovial γδ T Cells.

Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1β. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum-Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.

Combined Epiregulin and Amphiregulin Expression Levels as a Predictive Biomarker for Panitumumab Therapy Benefit or Lack of Benefit in Patients With RAS Wild-Type Advanced Colorectal Cancer.

RAS wild-type (wt) status is necessary but not sufficient for response to anti-epidermal growth factor receptor (EGFR) agents in advanced colorectal cancer (aCRC). RNA expression of EGFR ligands epiregulin (EREG) and amphiregulin (AREG) may correlate with EGFR-targeted therapy efficacy in aCRC, so may represent a much-needed additional predictive marker for these drugs. To examine a novel ligand model in a randomized clinical trial of panitumumab, irinotecan, and ciclosporin in colorectal cancer (PICCOLO) with with the a priori hypothesis that high tumor expression of either AREG or EREG would predict panitumumab therapy benefit in RAS-wt patients; and low expression, lack of efficacy. Prospectively planned retrospective biomarker study from the PICCOLO trial, which tested the addition of panitumumab to irinotecan therapy in patients with KRAS wt aCRC who experienced failure with prior fluoropyrimidine treatment. The analysis was conducted between 2012 and 2014. A predefined dichotomous model classified tumors as "high expressor" (either EREG or AREG in top tertile for messenger RNA level) or "low expressor" (neither EREG nor AREG in top tertile). Ligand expression was assessed as a prognostic and predictive biomarker. Expression of AREG/EREG and RAS and BRAF mutations were assessed in archival tumor tissue. Primary end point was progression-free survival (PFS); secondary end points were response rate and overall survival (OS). Of the 696 PICCOLO trial patients in the irinotecan-vs-irinotecan with panitumumab randomization, 331 had sufficient tumor tissue available and measurement of ligand expression was successful in 323. High ligand expression was not prognostic for OS (hazard ratio [HR], 0.79 [95% CI, 0.58-1.09]; P = .15) or PFS (HR, 0.93 [95% CI, 0.68-1.27]; P = .64). The primary population had RAS wt aCRC (n = 220); for RAS wt patients with high ligand expression, median (interquartile range [IQR]) PFS was 8.3 [4.0-11.0] months (irinotecan with panitumumab) vs 4.4 [2.8-6.7] months (irinotecan alone); HR, 0.38 [95% CI, 0.24-0.61]; P < .001). In RAS wt patients with low ligand expression, median (IQR) PFS was 3.2 [2.7-8.1] months (irinotecan with panitumumab) vs 4.0 [2.7-7.5] months (irinotecan); HR, 0.93 [95% CI, 0.64-1.37]; P = .73; interaction test results were significant [P = .01]). Less marked effects were seen for response rate (interaction P = .17) and OS (interaction P = .11). High ligand expression is a predictive marker for panitumumab therapy benefit on PFS in RAS wt patients; conversely, patients with low ligand expression gained no benefit. The current "opt-in" strategy for anti-EGFR therapy in all patients with RAS wt aCRC should be questioned. Expression of EREG/AREG is a useful biomarker for anti-EGFR therapy; optimization for clinical use is indicated. isrctn Identifier: ISRCTN93248876.

Competition in Notch Signaling with Cis Enriches Cell Fate Decisions

Notch signaling is involved in cell fate choices during the embryonic development of Metazoa. Commonly, Notch signaling arises from the binding of the Notch receptor to its ligands in adjacent cells driving cell-to-cell communication. Yet, cell-autonomous control of Notch signaling through both ligand-dependent and ligand-independent mechanisms is known to occur as well. Examples include Notch signaling arising in the absence of ligand binding, and cis-inhibition of Notch signaling by titration of the Notch receptor upon binding to its ligands within a single cell. Increasing experimental evidences support that the binding of the Notch receptor with its ligands within a cell (cis-interactions) can also trigger a cell-autonomous Notch signal (cis-signaling), whose potential effects on cell fate decisions and patterning remain poorly understood. To address this question, herein we mathematically and computationally investigate the cell states arising from the combination of cis-signaling with additional Notch signaling sources, which are either cell-autonomous or involve cell-to-cell communication. Our study shows that cis-signaling can switch from driving cis-activation to effectively perform cis-inhibition and identifies under which conditions this switch occurs. This switch relies on the competition between Notch signaling sources, which share the same receptor but differ in their signaling efficiency. We propose that the role of cis-interactions and their signaling on fine-grained patterning and cell fate decisions is dependent on whether they drive cis-inhibition or cis-activation, which could be controlled during development. Specifically, cis-inhibition and not cis-activation facilitates patterning and enriches it by modulating the ratio of cells in the high-ligand expression state, by enabling additional periodic patterns like stripes and by allowing localized patterning highly sensitive to the precursor state and cell-autonomous bistability. Our study exemplifies the complexity of regulations when multiple signaling sources share the same receptor and provides the tools for their characterization.

Regulation of 2B4 (CD244)‐mediated NK cell activation by ligand‐induced receptor modulation

Natural killer (NK) cell activity can be stimulated by different surface receptors. 2B4 is a member of the signaling lymphocyte activation molecule (SLAM)-related receptor family and is important for stimulating human NK cell cytotoxicity and cytokine production. Here we show that stimulation of human NK cells by antibody-mediated 2B4 cross-linking or incubation with target cells expressing the 2B4 ligand CD48 results in a strong down-modulation of 2B4 surface expression. This down-modulation is observed in NK cell lines, purified human NK cells and NK cell clones, and is accompanied by an internalization of 2B4. The modulation of 2B4 is dependent on the activity of Src-family kinases, but independent of PI3 K activity or actin polymerization. Inhibitory receptors can interfere with 2B4-mediated signals and NK cell activation. However, co-engagement of inhibitory killer cell Ig-like receptors has no influence on the down-modulation of 2B4. This suggests that the modulation of 2B4 expression is independent of inhibitory receptors. The lower surface expression of 2B4 after ligand-induced down-modulation results in reduced 2B4-mediated NK cell activation and cytotoxicity. The modulation of activating surface receptors may therefore be another mechanism for the fine-tuning of NK cell activity and may lead to the adaptation of NK cell cytotoxicity in tissues with high ligand expression.

The roles of cis-inactivation by Notch ligands and of neuralized during eye and bristle patterning in Drosophila.

BackgroundThe receptor protein Notch and its ligand Delta are expressed throughout proneural regions yet non-neural precursor cells are defined by Notch activity and neural precursor cells by Notch inactivity. Not even Delta overexpression activates Notch in neural precursor cells. It is possible that future neural cells are protected by cis-inactivation, in which ligands block activation of Notch within the same cell. The Delta-ubiquitin ligase Neuralized has been proposed to antagonize cis-inactivation, favoring Notch activation. Cis-inactivation and role of Neuralized have not yet been studied in tissues where neural precursor cells are resistant to nearby Delta, however, such as the R8 cells of the eye or the bristle precursor cells of the epidermis.ResultsOverexpressed ligands could block Notch signal transduction cell-autonomously in non-neural cells of the epidermis and retina, but did not activate Notch nonautonomously in neural cells. High ligand expression levels were required for cis-inactivation, and Serrate was more effective than Delta, although Delta is the ligand normally regulating neural specification. Differences between Serrate and Delta depended on the extracellular domains of the respective proteins. Neuralized was found to act cell nonautonomously in signal-sending cells during eye development, inconsistent with the view that Neuralized antagonizes cis-inactivation in non-neural cells.ConclusionsDelta and Neuralized contribute cell nonautonomously to Notch signaling in neurogenesis, and the model that Neuralized antagonizes cis-inactivation to permit Notch activity and specification of non-neural cells is refuted. The molecular mechanism rendering Notch insensitive to paracrine activation in neural precursor cells remains uncertain.