Immunoglobulin-like Transcript Research Articles

Tumor-derived ILT3 inhibition to reshape the immunosuppressive microenvironment and potentiate the PD-L1 blockade immunotherapy in lung adenocarcinoma.

e14647 Background: The low response of immune checkpoint inhibitors against PD-1/PD-L1 in cancer patients calls for further development of new immune targets. Immunoglobulin-like transcript (ILT)3 and its ligand ApoE have been demonstrated to be highly expressed in a variety of malignant tumor cells and involved in tumor biological behaviors, including invasion and angiogenesis. ILT3 targeting drugs are undergoing increasing preclinical and clinical trials. However, the role of tumor-derived ILT3 in immune regulation is unclear. The aim of this study was to investigate the immunomodulatory effect of tumor-derived ILT3 and its immunotherapy therapeutic efficacy that combination with PD-L1 blockade in NSCLC. Methods: The correlations between ILT3/ApoE expression and prognosis of NSCLC patients were analyzed basing on public databases. Multiplex immunofluorescence was performed to analyze the correlations between tumor-derived ILT3/ApoE expression and intratumoral immune cells infiltration in human adenocarcinoma tissues. The impact of ILT3 and ApoE on tumor-associated macrophage (TAM) recruitment and polarization were evaluated by transwell migration assay, flow cytometry, and RT-PCR, while their direct impact on T cell survival and cytotoxicity was detected by flow cytometry. Using lung cancer xenograft murine models, we further confirmed the pro-tumoural activity of tumor-derived gp49B and analyzed its correlations with immune cells in vivo. Tumor immunotherapy models targeting at gp49B and/or PD-L1 were established in C57BL/6 mice inoculated with LLC cells. Gp49b inhibition was implemented by infection of specific knockdown lentivirus and PD-L1 was blocked using mouse neutralizing antibodies. Flow cytometry was performed to detect the frequency and phenotype of macrophages and T cells in the tumors, spleen, and blood of the mice. Results: Patients with high expression of ILT3 had significantly worse prognosis in LUAD, but not in LUSC. In vitro assays demonstrated that tumor-derived ILT3/ApoE promoted recruitment and M2-like polarization of TAMs and directly inhibited the proliferation and cytotoxicity of T cells, which had been further confirmed in vivo models. Moreover, ILT3 (gp49B) inhibition enhanced anti-tumor immunity and suppressed tumor progression by counteracting TAM- and dysfunctional T cell- induced immunosuppressive TME; the combined inhibition of gp49B and PD-L1 showed the most dramatic tumor retraction in LLC xenograft models. Conclusions: Tumor-derived ILT3 overexpression suppressed anti-tumor immunity by recruiting M2-like TAMs and directly impairing T cell activities, while ILT3 inhibition counteracted above immunosuppression and potentiated the efficacy of PD-L1 blockade in LUAD. Our study verified that ILT3 may be a promising novel immunotherapeutic target for LUAD patients.

Immunoglobulin-like transcript 2 as an impaired anti-tumor cytotoxicity marker of natural killer cells in patients with hepatocellular carcinoma.

Natural killer (NK) cells play a pivotal role in immune surveillance in the liver. We aimed to identify potential targets for NK cell-mediated immune intervention by revealing the functional molecules on NK cells in HCC patients. To evaluate the impact of aging on NK cell phenotypes, we examined NK cells from healthy volunteers (HVs) of various ages. Because ILT2 expression on CD56dim NK cells increased with increasing age, we enrolled age-matched HCC patients and HVs. We determined the NK cell phenotypes in blood mononuclear cells (PBMCs) and intrahepatic lymphocytes (IHLs) from cancerous and non-cancerous tissues. We evaluated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells in vitro. ILT2-positive CD56dim NK cells in PBMCs were increased in HCC patients compared with HVs. In HCC patients, ILT2-positive CD56dim NK cells were increased in cancerous IHLs compared with non-cancerous IHLs and PBMCs. We examined the impact of macrophage migration inhibitory factor (MIF) on ILT2 expression in co-cultures of HCC cells and NK cells. The enhanced expression of ILT2 on CD56dim NK cells from HCC patients was inhibited by masking antibodies against MIF and CXCR4. ILT2-positive CD56dim NK cells exhibited lower capacities for cytotoxicity and ADCC than ILT2-negative cells, which were partially restored by ILT2 blockade. In HCC patients, ILT2 is a signature molecule for cancerous CD56dim NK cells with impaired cytolytic capacity. The MIF-CXCR4 interaction is associated with ILT2 induction on CD56dim NK cells and ILT2 serves as a target for functional NK cell restoration.

Abstract 1364: Characterization of CHS-1000, an Fc-modified anti-ILT4 monoclonal antibody for reprogramming suppressive myeloid cells in solid tumors

Abstract Tumor resident immunosuppressive myeloid cells play a significant role in inducing resistance to cancer immunotherapy and are associated with poor prognosis in several solid tumors. The leukocyte immunoglobulin-like receptor B2 (LILRB2), also known as ILT4, is primarily expressed on myeloid cells and is a negative regulator of myeloid cell activation. ILT4 and its primary ligands (HLA-G, HLA-A) are widely expressed in various solid tumors and are associated with disease progression, tumor metastasis and poor clinical outcome. Abrogating inhibitory signals mediated through ILT4 and its ligands is a promising strategy to reprogram suppressive myeloid cells and enhance efficacy of immune checkpoint therapies. Here we report IND-enabling preclinical characterization of CHS-1000, a novel recombinant humanized Fc-modified IgG1 antibody with attenuated Fc-gamma receptor (FcγR) binding, that specifically targets immunoglobulin-like transcript 4 (ILT4). CHS-1000 selectively binds to human ILT4 with high affinity and potently blocks ILT4 interactions with its primary ligands (HLA-A, HLA-G). Functionally, CHS-1000 promotes re-polarization of suppressive myeloid cells to a pro-inflammatory phenotype and enhances activation of dendritic cells and T cells. Combinations of CHS-1000 with PD-1 inhibitors or other immunotherapy agents hold particular promise for enhanced anti-tumor activity. These preclinical characterization data support clinical development of CHS-1000 in combination with toripalimab (a commercial stage anti-PD-1 antibody) as an immunotherapy treatment for patients with solid tumors. Citation Format: Narendiran Rajasekaran, Xiaoguang Wang, Scott Klakamp, Sruthi Ravindranathan, Suzanna Tatarewicz, Julia Du, Brian Nguyen, Kate Widmann, Nessa Hawkins, Daniel J. Chin, Theresa LaVallee, Varun Kapoor. Characterization of CHS-1000, an Fc-modified anti-ILT4 monoclonal antibody for reprogramming suppressive myeloid cells in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1364.

ILT4 facilitates angiogenesis in non-small cell lung cancer.

Antiangiogenic therapy targeting VEGF-A has become the standard of first-line therapy for non-small cell lung cancer (NSCLC). However, its clinical response rate is still less than 50%, and most patients eventually develop resistance, even when using combination therapy with chemotherapy. The major cause of resistance is the activation of complex bypass signals that induce angiogenesis and tumor progression. Therefore, exploring novel proangiogenic mechanisms and developing promising targets for combination therapy are crucial for improving the efficacy of antiangiogenic therapy. Immunoglobulin-like transcript (ILT) 4 is a classic immunosuppressive molecule that inhibits myeloid cell activation. Recent studies have shown that tumor cell-derived ILT4 drives tumor progression via the induction of malignant biologies and creation of an immunosuppressive microenvironment. However, whether and how ILT4 participates in NSCLC angiogenesis remain elusive. Herein, we found that enriched ILT4 in NSCLC is positively correlated with high microvessel density, advanced disease, and poor overall survival. Tumor cell-derived ILT4 induced angiogenesis both invitro and invivo and tumor progression and metastasis invivo. Mechanistically, ILT4 was upregulated by its ligand angiopoietin-like protein 2 (ANGPTL2). Their interaction subsequently activated the ERK1/2 signaling pathway to increase the secretion of the proangiogenic factors VEGF-A and MMP-9, which are responsible for NSCLC angiogenesis. Our study explored a novel mechanism for ILT4-induced tumor progression and provided a potential target for antiangiogenic therapy in NSCLC.

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

Exogenous α-ketoglutarate Modulates Redox Metabolism and Functions of Human Dendritic Cells, Altering Their Capacity to Polarise T Cell Response.

Alpha-ketoglutarate (αKG) emerged as a key regulator of energetic and redox metabolism in cells, affecting the immune response in various conditions. However, it remained unclear how the exogenous αKG modulates the functions of dendritic cells (DCs), key cells regulating T-cell response. Here we found that non-toxic doses of αKG display anti-inflammatory properties in human APC-T cell interaction models. In a model of monocyte-derived (mo)DCs, αKG impaired the differentiation, and the maturation of moDCs induced with lipopolysaccharide (LPS)/interferon (IFN)-γ, and decreased their capacity to induce Th1 cells. However, αKG also promoted IL-1β secretion by mature moDCs, despite inflammasome downregulation, potentiating their Th17 polarizing capacity. αKG induced the expression of anti-oxidative enzymes and hypoxia-induced factor (HIF)-1α in moDCs, activated Akt/FoxO1 pathway and increased autophagy flux, oxidative phosphorylation (OXPHOS) and glycolysis. This correlated with a higher capacity of immature αKG-moDCs to induce Th2 cells, and conventional regulatory T cells in an indolamine-dioxygenase (IDO)-1-dependent manner. Additionally, αKG increased moDCs' capacity to induce non-conventional T regulatory (Tr)-1 and IL-10-producing CD8+T cells via up-regulated immunoglobulin-like transcript (ILT3) expression in OXPHOS-dependent manner. These results suggested that exogenous αKG-altered redox metabolism in moDCs contributed to their tolerogenic properties, which could be relevant for designing more efficient therapeutic approaches in DCs-mediated immunotherapies.

Soluble CD83 modulates human-monocyte-derived macrophages toward alternative phenotype, function, and metabolism.

Alterations in macrophage (Mφ) polarization, function, and metabolic signature can foster development of chronic diseases, such as autoimmunity or fibrotic tissue remodeling. Thus, identification of novel therapeutic agents that modulate human Mφ biology is crucial for treatment of such conditions. Herein, we demonstrate that the soluble CD83 (sCD83) protein induces pro-resolving features in human monocyte-derived Mφ biology. We show that sCD83 strikingly increases the expression of inhibitory molecules including ILT-2 (immunoglobulin-like transcript 2), ILT-4, ILT-5, and CD163, whereas activation markers, such as MHC-II and MSR-1, were significantly downregulated. This goes along with a decreased capacity to stimulate alloreactive T cells in mixed lymphocyte reaction (MLR) assays. Bulk RNA sequencing and pathway analyses revealed that sCD83 downregulates pathways associated with pro-inflammatory, classically activated Mφ (CAM) differentiation including HIF-1A, IL-6, and cytokine storm, whereas pathways related to alternative Mφ activation and liver X receptor were significantly induced. By using the LXR pathway antagonist GSK2033, we show that transcription of specific genes (e.g., PPARG, ABCA1, ABCG1, CD36) induced by sCD83 is dependent on LXR activation. In summary, we herein reveal for the first time mechanistic insights into the modulation of human Mφ biology by sCD83, which is a further crucial preclinical study for the establishment of sCD83 as a new therapeutical agent to treat inflammatory conditions.

Overcoming Radio-Immunotherapy Treatment Resistance through ILT4 Blockade and Reversal of HFRT Induced CXCL1-CXCR2 Axis Activation and Tumor-Associated Macrophage Immunosuppression

Immunotherapy combined with radiotherapy (iRT) has unlimited potential, but up to 60% of cancer patients do not benefit from it. Enhancing the anti-tumor immune stimulatory effect triggered by radiotherapy is the key to overcome iRT resistance. Immunoglobulin-like transcript (ILT) 4 is a potential immune checkpoint molecule, highly expressed in various tumor cells, but its role in radiotherapy is still unknown. This study confirmed the role and molecular mechanism of ILT4 in suppressing radiotherapy immunosuppressive microenvironment formation and promoting tumor radiotherapy resistance. We propose a new therapeutic strategy that block ILT4 to enhance the efficacy of radiotherapy, and cooperate with radiotherapy to reverse immunotherapy resistance. Using multiplex immunohistochemistry, we analyzed ILT4 expression, tumor-associated macrophage (TAM) /T cell phenotype and quantity in tumor patient treated with SBRT. Using mice subcutaneous tumor models, Single-cell RNA sequencing and multiplex flowcytometry, we assessed the role of ILT4 inhibition and hyper-fractionated radiotherapy (HFRT) on preventing tumor growth and immune escape. The molecular signaling and cytokines regulated by ILT4 under HFRT were analyzed by transcriptome sequencing and further verified by molecular experiments. By establishing cancer cell/TAM co-culture system in vitro, using CXCL1 protein or CXCR2 inhibitor and macrophage/CD8+ T cell deletion antibody in vivo, we identified the downstream pathway and cytokine of ILT4 to enhancing HFRT -induced TAM immune response. In the tumor specimens of NSCLC patients treated with SBRT, we found that high ILT4 expression predicted poor progression-free survival and more M2-TAM recruitment. Among the C57BL/6 mice model, ILT4 inhibition in cancer cells reduced HFRT mediated M2-TAMs accumulation, and to sustain activation and proliferation of CD8+ T cells, and eventually suppressed tumor progression. Mechanistically, RT promoted ILT4 expression, which subsequently induced NF-κB pathway activation and CXCL1 secretion to enhance M2-TAMs migration in vitro. Using CXCL1 protein or CXCR2 inhibitor administration, inferring that ILT4 promotes TAMs migration via NF-κB-CXCL1-CXCR2 axis. Consistently, depletion of TAMs blocked the T cell function impairment and radiotherapy resistance induced by ILT4 in vivo. Importantly, targeting ILT4 potentiated the effect of radiotherapy, overcomes radio-immunotherapy treatment resistance. ILT4 mediates HFRT-induced M2-like TAMs recruitment and subsequently T cell response impairment by regulating NF-κB-CXCL1-CXCR2 axis. ILT4 is an attractive drug target for enhancing radiotherapy and overcomes radio-immunotherapy treatment resistance.

Increased ILT2+ natural killer T cells correlate with disease activity in systemic lupus erythematosus.

Numerous immune cell types, such as B and T lymphocytes, natural killer cells (NK), and NKT cells, are related to the pathogenesis of diseases in systemic lupus erythematosus (SLE). Our goal in this investigation is to examine the phenotype of NK cells and NKT cells alterations in individuals with SLE. Typically, 50 SLE patients and 24 age-matched healthy people had their PBMCs obtained. Employing flow cytometry, the phenotype of NK and NKT cells and immunoglobulin-like transcript 2 (ILT2) expressions were identified. ELISA was utilized to evaluate the amounts of interleukin-15 (IL-15) and sHLA-G in the serum. The frequencies of the circulating NK and NKT cells in individuals with SLE were decreased compared to healthy controls. Furthermore, ILT2 expression was significantly increased in NKT cells, but showed no obvious change in NK cells. Clinical severity and active nephritis were substantially associated with ILT2+ NKT cell frequencies. The correlation study showed that the upregulation of ILT2 expression was related to sHLA-G in plasma but not to IL-15. ILT2+ NKT cells have a vital function in the immune abnormalities of SLE, which can also supply a viable goal for therapeutic intervention. Key Points •ILT2 expression was significantly increased in NKT cells in SLE patients. •ILT2+ NKT cell frequencies were associated with clinical severity which may be used as an indicator for evaluating disease activity in patients with SLE.

ILT4 reprograms glucose metabolism to promote tumor progression in triple negative breast cancer.

Triple negative breast cancer (TNBC) is the most aggressive and poorly-treated subtype of breast cancer. Identifying novel drivers and mechanisms for tumor progression is quite essential for precise targeted therapy of TNBC. Immunoglobulin-like transcript (ILT) 4 is a classic myeloid suppressor for their activation and immune response. Our recent results found that ILT4 is also high expressed in lung cancer cells where has a role in promoting immune evasion and thus tumor formation. However, the expression and function of ILT4 in breast cancer remains elusive. Here, using our patient cohort and public database analysis, we found that TNBC displayed the most abundant ILT4 expression among all breast cancer subtypes. Functionally, enriched ILT4 promoted TNBC cell proliferation, migration and invasion in vitro, as well as tumor growth and metastasis in vivo. Further mechanistic analysis revealed that ILT4 reprogrammed aerobic glycolysis of tumor cells via AKT/mTOR signaling-mediated glucose transporter 3 (GLUT3) and Pyruvate kinase muscle 2 (PKM2) overexpression. ILT4 inhibition in TNBC reduced tumor progression and GLUT3/PKM2 expression in vivo. Our study identified a novel driver for TNBC progression and proposed a promising strategy to combat TNBC by targeting ILT4.

Detailed Analysis of Simultaneous Renal and Liver Allografts in the Presence of DSA.

We reevaluated allograft biopsies from simultaneous liver-kidney transplant recipients who had both allografts biopsied within 60 d of one another and within 30 d of DSA being positive in serum (positive: mean florescence intensity ≥5000). Routine histology, C4d staining, and specialized immunohistochemistry for Kupffer cells (KCs; CD163) and a C4d receptor immunoglobulin-like transcript-4 were carried out in 4 patients with 6 paired biopsies. Overt antibody-mediated rejection was found in 3 of 4 renal and liver allografts. One patient had biopsy-confirmed renal and liver allograft antibody-mediated rejection despite serum clearance of DSA. All biopsies showed KC hypertrophy (minimal: 1; mild: 2; moderate: 1; severe: 2) and cytoplasmic C4d KC staining was easily detected in 2 biopsies from 2 patients; minimal and negative in 2 biopsies each. Implications of which are discussed. Control 1-y protocol liver allograft biopsies from DSA- recipients showed neither KC hypertrophy nor KC C4d staining (n = 6). Partial renal allograft protection by a liver allograft from the same donor may be partially mediated by phagocytosis/elimination of antibody and complement split products by KCs, as shown decades ago in controlled sensitized experimental animal experiments.

CDX-585, a Bispecific Antibody with Dual Targeting of ILT4 and PD-1 Checkpoint Pathways

Immunoglobulin-like transcript 4 (ILT4) is an immunosuppressive molecule predominantly expressed on myeloid cells. Recent studies combining ILT4 suppression with programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) blockade have shown promising signs of activity in immune checkpoint inhibitor refractory patients. We theorized that coupling ILT4 and PD-1/PD-L1 blockade in a bispecific antibody (bsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced bridging of APCs to T cells. To test this approach, we developed CDX-585, a tetravalent ILT4xPD-1 IgG1-scFv bsAb from novel PD-1 and ILT-4 mAbs. CDX-585 is a potent antagonist of both PD-1 and ILT4. CDX-585 promotes M1 macrophage polarization and enhances pro-inflammatory cytokine secretion in response to lipopolysaccharide or CD40 agonist mAb treatment. In mixed lymphocyte reaction (MLR) assays, CDX-585 is more potent than the combination of parental antibodies. In a humanized NCG mouse SK-MEL-5 tumor model, CDX-585 exhibits greater antitumor activity than the combination of parental mAbs. A pilot study of CDX-585 in cynomolgus macaques confirmed a mAb-like pharmacokinetic profile without noted toxicities. These studies demonstrate that CDX-585 effectively combines ILT4 and the PD-1 blockade into one molecule that is more potent than the combination of the parental antibodies, providing the rationale to advance this bsAb into clinical studies.

Elevated sHLA-G plasma levels post chemotherapy combined with ILT-2 rs10416697C allele status of the sHLA-G-related receptor predict poorest disease outcome in early triple-negative breast cancer patients.

Triple negative breast cancer (TNBC) shows an aggressive growing and spreading behavior and has limited treatment options, often leading to inferior disease outcome. Therefore, surrogate markers are urgently needed to identify patients at high risk of recurrence and more importantly, to identify additional therapeutic targets enabling further treatment options. Based on the key role of the non-classical human leukocyte antigen G (HLA-G) and its related receptor immunoglobulin-like transcript receptor-2 (ILT-2) in immune evasion mechanisms of tumors, members of this ligand-receptor axis appear to be promising tool for both, defining risk groups and potential therapeutic targets. To follow this, sHLA-G levels before and after chemotherapy (CT), HLA-G 3' UTR haplotypes, and allele variations rs10416697 at the distal gene promoter region of ILT-2 were defined in healthy female controls and early TNBC patients. The results obtained were associated with clinical status, presence of circulating tumor cell (CTC) subtypes, and disease outcome of patients in terms of progression-free or overall survival. sHLA-G plasma levels were increased in TNBC patients post-CT compared to levels of patients pre-CT or controls. High post-CT sHLA-G levels were associated with the development of distant metastases, the presence of ERCC1 or PIK3CA-CTC subtypes post-CT, and poorer disease outcome in uni- or multivariate analysis. HLA-G 3' UTR genotypes did not influence disease outcome but ILT-2 rs10416697C allele was associated with AURKA-positive CTC and with adverse disease outcome by uni- and multivariate analysis. The prognostic value of the combined risk factors (high sHLA-G levels post-CT and ILT-2 rs10416697C allele carrier status) was an even better independent indicator for disease outcome in TNBC than the lymph nodal status pre-CT. This combination allowed the identification of patients with high risk of early progression/death with positive nodal status pre-CT or with non-pathological complete therapy response. The results of this study highlight for the first time that the combination of high levels of sHLA-G post-CT with ILT-2 rs10416697C allele receptor status is a promising tool for the risk assessment of TNBC patients and support the concept to use HLA-G/ILT-2 ligand-receptor axis as therapeutic targets.

Fibronectin on target cells attenuates natural cytotoxicity of NK cells via myeloid immune checkpoint ILT3/LILRB4/gp49B.

Natural killer (NK) cells play pivotal roles in innate immunity as well as in anti-tumor responses via natural killing, while their activity is tightly regulated by cell-surface inhibitory receptors. Immunoglobulin-like transcript 3/leukocyte immunoglobulin-like receptor B4 (ILT3/LILRB4, also known as gp49B in mice) is an inhibitory receptor expressed on activated NK cells as well as myeloid-lineage cells. The common physiologic ligand of human LILRB4 and gp49B was identified very recently as fibronectin, particularly the N-terminal 30 kDa domain (FN30). We hypothesized that LILRB4 could bind fibronectin on target cells in trans together with integrins, classical fibronectin receptors, in cis and deliver an inhibitory signal in NK cells, leading to attenuated natural killing. Flow cytometric and confocal microscopic analyses of NK cell-surface gp49B and integrins suggested that these novel and classical fibronectin receptors, respectively, co-engage fibronectin immobilized on a culture plate. Biochemical analyses indicated that tyrosine phosphorylation of spleen tyrosine kinase was augmented in gp49B-deficient NK cells upon binding to the immobilized fibronectin. While surface fibronectin-poor YAC-1 cells were evenly sensitive as to natural killing of both gp49B-positive and -negative NK cells, the killing of fibronectin-rich Lewis lung carcinoma cells, but not the FN30-knockout cells, was augmented among gp49B-deficient NK cells. These results suggest that the natural cytotoxicity of NK cells is negatively regulated through LILRB4/gp49B sensing fibronectin on target cells, which sheds light on the unexpected role of LILRB4 and fibronectin as a potential attenuator of NK cell cytotoxicity in the tumor microenvironment.

Abstract CT046: A randomized, phase 2 study of pembrolizumab plus chemotherapy with or without anti—immunoglobulin-like transcript 4 monoclonal antibody MK-4830 as neoadjuvant treatment for high-grade serous ovarian cancer

Abstract Background: Combination therapy with the anti-PD-1 monoclonal antibody (mAb) pembrolizumab and chemotherapy in advanced solid tumors has shown efficacy in the (neo)adjuvant and first-line setting, including high-grade serous ovarian cancer (HGSOC). The immunoglobulin-like transcript 4 (ILT4) receptor expressed on myeloid cells is involved in immune tolerance and suppression within the tumor microenvironment, and its blockade may improve treatment outcomes with combination therapies. Pembrolizumab plus chemotherapy with or without MK-4830 (a novel humanized anti-ILT4 mAb) will be investigated as neoadjuvant therapy for HGSOC in a randomized, open-label, phase 2 study (NCT05446870). Specifically, this trial is designed to evaluate the utility of circulating tumor DNA (ctDNA) as a biomarker of response and minimal residual disease in patients with treatment-naive HGSOC. Methods: Key eligibility criteria include adult patients with treatment-naive International Federation of Gynecology and Obstetrics Stage IIIA-C/IV HGSOC, primary peritoneal cancer, or fallopian tube cancer who are candidates for neoadjuvant and adjuvant chemotherapy and interval debulking surgery (IDS), and have an ECOG performance status of 0 or 1. Approximately 160 patients will be randomly assigned (1:1) to receive 3 cycles each of neoadjuvant and adjuvant therapy with patients in arm 1 receiving chemotherapy (carboplatin AUC 5 or 6 plus paclitaxel 175 mg/m2 or docetaxel 75 mg/m2) + pembrolizumab 200 mg and patients in arm 2 receiving chemotherapy + pembrolizumab + MK-4830 800 mg. Bevacizumab (or biosimilar) may be added to either arm in the adjuvant setting. All treatments will be given on day 1 of each 3-week cycle. For patients who undergo IDS, the last dose of neoadjuvant therapy will be administered 3-6 weeks before IDS, and adjuvant therapy will commence <7 weeks after IDS. Adjuvant therapy will be administered for up to 3 cycles or until disease progression, unacceptable toxicity, or withdrawal. Upon completion of adjuvant therapy, patients may undertake maintenance therapy per investigator discretion and local guidelines. Tumor imaging will be performed at baseline, ≥21 days after neoadjuvant therapy or prior to IDS, after IDS or prior to the start of adjuvant therapy, and at the end of treatment. The primary objective is to evaluate the change from baseline in ctDNA, defined as the continuous mean mutant/tumor molecules/mL as measured in a blood sample, at cycle 3. Secondary objectives are to evaluate the association between change from baseline in ctDNA at cycle 3 and surgical outcomes (including pathologic complete response and chemotherapy response score), and safety and tolerability. Enrollment is ongoing. Citation Format: Jung-Yun Lee, Maria Bell, Mariusz Bidzinski, Lubomir Bodnar, Jack Junjie Chan, Marta Gill-Martin, Carolina Ibanez, Domenica Lorusso, Chien-Hsing Lu, Ora Rosengarten, Steven M. Townson, Julie Kobie, Yiwei Zhang, Scott Pruitt, Ronnie Shapira-Frommer. A randomized, phase 2 study of pembrolizumab plus chemotherapy with or without anti—immunoglobulin-like transcript 4 monoclonal antibody MK-4830 as neoadjuvant treatment for high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT046.

Phase 1b Trial of Anti-Immunoglobulin-like Transcript 3 Monoclonal Antibody MK-0482 Monotherapy in Relapsed/Refractory Acute Myeloid Leukemia and Chronic Myelomonocytic Leukemia

Background: Immunoglobulin-like transcript 3 (ILT3) is a cell surface inhibitory receptor expressed on various immune cells including dendritic cells, monocytic myeloid cells, and macrophages. The expression of ILT3 on dendritic cells and myeloid cells is thought to be involved in immune suppression and antigen-specific immune tolerance, contributing to an immunosuppressive tumor microenvironment. Acute myeloid leukemia (AML) is a type of cancer characterized by bone marrow that makes a large number of abnormal blood cells. In monocytic AML, ILT3 is highly expressed, and its signaling can mediate T-cell suppression and promote tumor infiltration (Deng M et al. Nature. 2018;562:605-609). Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm characterized by dysplasia, abnormal production and accumulation of monocytic cells, and with an elevated risk of transforming into AML. Based on the M2GEN database, ILT3 is also highly expressed in CMML. Thus, targeting ILT3 may exhibit antitumor activity in patients with AML and CMML. Currently, limited effective treatments are available for AML and CMML, especially in the relapse and refractory setting. MK-0482, a humanized IgG4 anti-ILT3 monoclonal antibody, has been evaluated as monotherapy and in combination with pembrolizumab at doses ≤2250 mg every 3 weeks (Q3W) in patients with advanced solid tumors. Patients treated with MK-0482 had a manageable safety profile, and modest antitumor activity was observed with MK-0482 plus pembrolizumab (Gutierrez M et al. J Clin Oncol. 2022;40. Abstract 2505). In this phase 1b study (NCT05038800), we are evaluating the safety and the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of MK-0482 in relapsed or refractory (r/r) AML and CMML. Study Design and Methods: Adult patients with AML with myelomonocytic or monoblastic/monocytic differentiation or CMML and with confirmed refractory or relapsed disease after treatment with available therapies will be eligible for enrollment. All patients must have an ECOG performance status of 0-2, adequate organ function, and a white blood cell count ≤20 × 109/L. Patients with active central nervous system leukemia, isolated extramedullary disease (ie no leukemic involvement), acute promyelocytic leukemia, or who have previously received allogeneic stem cell or organ transplant <60 days of treatment will be excluded. In part 1 (dose escalation), MK-0482 will be evaluated at dose levels ranging from 7.5 mg to 750 mg Q3W for ≤35 cycles with an accelerated titration design followed by a modified toxicity probability interval design in ~20 patients. After the recommended phase 2 dose (RP2D) is determined, 10 to 15 additional patients with AML with myelomonocytic or monoblastic/monocytic differentiation will be enrolled in part 2 (dose expansion). The primary objective of the study is to determine safety and tolerability and RP2D. Secondary objectives include characterizing the PK profile of MK-0482 monotherapy and antileukemia activities in AML as assessed by complete remission (CR), composite CR, and objective response per the 2017 European Leukemia Net AML response criteria. Exploratory objectives include assessment of receptor occupancy in peripheral blood and tumor biopsies, molecular biomarkers, and antileukemic activities in patients with CMML. Dose-limiting toxicities will be observed for 21 days after the first dose in cycle 1 and adverse events (AEs) will be assessed through 30 days after treatment discontinuation (90 days for serious AEs; 30 days if new anticancer therapy is initiated) per National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0. Disease assessment will be performed by investigator at baseline and prior to day 1 of cycles 3, 5, and 7, followed by every 3 cycles until disease progression or confirmed CR. Samples for PK analyses will be collected before and after treatment on day 1 of cycles 1-4, 6, 8, and every 4 cycles thereafter. The study is currently ongoing.

Preclinical Characterization of NGM936, a Novel Bispecific T Cell Engager Targeting ILT3 for the Treatment of Acute Myeloid Leukemia with Monocytic Differentiation

Acute myeloid leukemia (AML) with monocytic differentiation [M4 and M5 AML per the French-American-British (FAB) classification] accounts for 25-30% of all AML cases and has a particularly poor prognosis, with a <15% five-year overall survival rate and an increased risk of bone marrow and extramedullary relapse after stem cell transplant compared with other AML subtypes. The use of T cell engagers in AML has been limited by the lack of suitable target antigens with high specificity for AML cells and minimal expression on healthy bone marrow progenitors. Immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) is a highly specific marker of AML with monocytic differentiation and is not expressed on non-monocytic blood cells or hematopoietic progenitor cells. This highly restricted expression profile makes ILT3 a promising target for monocytic AML, with the potential to enable effective elimination of AML cells with less toxicity than current therapies. Here, we report the pre-clinical characterization of NGM936, an ILT3-based T cell engager for the treatment of monocytic AML and other ILT3+ hematologic malignancies. NGM936 is a monovalent scFv-Fab bispecific on an effectorless human IgG1 backbone (LALA) with a knob-in-hole Fc design for heterodimeric assembly. NGM936 demonstrates sub-nM affinity towards ILT3 of human origin (KD = 0.2 nM at 25°C by surface plasmon resonance) and is not cross-reactive towards other LILRA/B family members. A panel of more than 30 ILT3 x CD3 engagers in various formats was generated and screened in assays to measure both T cell-dependent cytotoxicity and cytokine release. NGM936 was identified for its ability to potently induce T cell-dependent cytotoxicity (TDCC) against ILT3+ AML cells while inducing minimal cytokine release. In both whole blood cytokine release assays and in TDCC assays in which cytokine secretion was measured after target engagement, induction of TNF-α, IL-6, IFN-γ, and IL-2 by NGM936 was considerably lower than that induced by a vibecotamab biosimilar (CD123 x CD3). NGM936 induced potent cytotoxicity when both expanded and naïve T cells were used as effectors. Importantly, NGM936 efficiently ablated tumor cells with a range of ILT3 expression, from ~1500 copies/cell to ~40,000 copies/cell. In addition, NGM936 failed to induce T cell-dependent cytotoxicity against CD34+ hematopoietic stem cells or non-monocytic immune cells, consistent with the lack of ILT3 expression on these cell types. In ex vivo cultures of primary M5 AML bone marrow, NGM936 induced a dose-dependent depletion of AML cells and a concordant increase in T cell proliferation and activation. Finally, NGM936 induced a dose-dependent depletion of circulating tumor cells in a xenograft model in which irradiated, immunodeficient NSG mice were engrafted with human PBMCs and human ILT3+ AML cells. NGM936 thus represents a promising new treatment strategy for monocytic AML, with the potential to eliminate monocytic leukemia cells while minimizing the myelotoxicity associated with ablation of healthy bone marrow.

BND-22, a first-in-class humanized ILT2-blocking antibody, promotes antitumor immunity and tumor regression

BackgroundCancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments...

Increased ILT2 expression contributes to dysfunction of CD56dimCD16+NK cells in chronic hepatitis B virus infection

Natural killer (NK) cells play a crucial role in the control of human viral infections but their activity is significantly impaired in patients infected with chronic hepatitis B (CHB). The mechanism that contributes to NK cell dysfunction in CHB needs further elucidation. In this study, we analyzed the expression and function of the novel inhibitory receptor immunoglobulin-like transcript-2 (ILT2) on NK cells from 131 CHB patients and 36 healthy controls. We observed that ILT2 expression on circulating CD56dimCD16+NK cells was increased in immune-tolerant, immune-active and HBeAg-negative hepatitis patients compared with inactive carriers and controls. The frequency of ILT2+CD56dimNK cells was positively correlated with serum viral load in immune-tolerant patients. The percentage of ILT2+CD56dimNK cells decreased along with HBV load in CHB patients who received antiviral therapy. Functional analysis showed that ILT2+CD56dimNK cells in CHB patients had significantly reduced degranulation and IFN-γ production. Upregulation of ILT2 was associated with high levels of apoptosis in CD56dimCD16+NK cells from CHB patients. ILT2 blockade was shown to increase the cytotoxicity and IFN-γ production of CD56dimNK cells in some CHB patients. Finally, ILT2 was found to be moderately upregulated by TGF-β1, which was increased in immune-tolerant, immune-active and HBeAg-negative hepatitis patients. Our results show that chronic HBV infection increases the levels of the inhibitory receptor ILT2 on CD56dimNK cells and inhibits their functions, providing a new mechanism of NK-cell disability in CHB patients.

A highly diverse set of novel immunoglobulin-like transcript (NILT) genes in zebrafish indicates a wide range of functions with complex relationships to mammalian receptors.

Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout, and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby limiting our predictions for their evolutionary origin and function. In order to better understand the diversity of NILTs and their possible roles in immune function, we identified five NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3-Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternative haplotype for this same genomic region. We then identified transcripts encoded by 43 different NILT genes which reflect an unprecedented diversity of Ig domain sequences and combinations for a family of non-recombining receptors within a single species. Zebrafish NILTs include a sole putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. These results reveal a higher level of genetic complexity, interindividual variation, and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity.