Receptor-expressing Cells Research Articles

P-436 Uterine NK cells underexpress KIR2DL1/S1 and LILRB1 receptors recognizing HLA-C2 and HLA-G on extravillous trophoblasts in reproductive failure

Abstract Study question Is there a difference in frequency, receptor expression and cytokine production of uterine NK cells (uNK) subpopulations in women with reproductive failure compared to controls? Summary answer Reproductive failure is associated with global underexpression of uNK receptors interacting with extravillous trophoblasts (EVT) that may result in defective implantation and placentation. What is known already A significant proportion of infertility, recurrent miscarriage and recurrent implantation failure are unexplained. This may be caused by immunological dysfunction at the maternal-fetal interface resulting in defective implantation and placentation. uNK is thought to play an important role in early pregnancy by cross-talking with EVT via KIR/HLA-C, LILRB1/HLA-G and CD94/HLA-E interactions to regulate EVT invasion and spiral arteriole remodeling in the decidua. Dysregulation of uNK function may cause reproductive failure; however it is unclear whether NK cell over- or under-activation is the pathology. Previous studies have been conducted on uNK obtained after miscarriage, which may be confounded by inflammatory processes. Study design, size, duration Here, we conducted a cross-sectional study to assess frequency, phenotype and function of matched pNK and uNK during the window of implantation in 16 women with reproductive failure compared to 11 controls. Women with reproductive failure cohort included unexplained subfertility (9), recurrent miscarriage (4) and recurrent implantation failure (3). The women were subsequently followed up after IVF and comparison made between women who did and did not fall pregnant. Participants/materials, setting, methods Matched peripheral blood and endometrial tissue were obtained from participants during the mid-secretory phase. Samples were accurately timed for 7-9 days after ovulation by urine LH test or histological dating. Flow cytometry was used to assess the frequency, phenotype and function. Phenotype was measured by KIR, LILRB1 and CD94 and receptor expression. Function was measured by degranulation (anti-CD107a internalization) and cytokine production (TNF-α, GM-CSF, IFN-γ and IL-8), with and without stimulation by PMA and ionomycin. Main results and the role of chance The expression of KIR2DL1/S1 and LILRB1 were significantly lower in reproductive failure group for both uNK (total uNK, uNK2 and 3) and pNK. The common finding in both uNK and pNK suggests that this is not tissue specific and may have an immunogenetic aetiology. We have previously found that degranulation peaks during secretory phase and in first trimester pregnancy. TNF-α production was also found to peak in secretory phase. Here, we found that degranulation activity of total uNK, as well as TNF-α production all uNK subsets was significantly lower in reproductive failure compared to control group. However, no significant difference was found in women in the reproductive failure cohort who fell pregnant compared to women who did not fall pregnant or miscarried after IVF. Contrary to findings from previous studies, we have not found any difference in uNK or pNK frequencies in any of the groups that were examined. Taken together, our findings suggest that reproductive failure is associated with global underexpression of receptors important for interaction with HLA-C and HLA-G on EVT, resulting in reduced uNK activation. This in turn may lead to dysregulation of implantation and placenta formation in early stage of pregnancy. Limitations, reasons for caution KIR2DL1/S1 antibody used does not distinguish between KIR2DL1 and KIR2DS1 which exerts inhibitory and activating effects respectively. Larger cohort studies on both pNK and uNK that consider KIR2DL1 and KIR2DS1 separately is needed to confirm which of these receptors are specifically reduced in reproductive failure. Wider implications of the findings Overall, our findings suggest that reproductive failure is associated with global underexpression of NK cell receptors leading to impaired implantation and placentation. This is the first study to examine uNK subsets during window of implantation and will serve as a platform to focus on particular uNK subsets in future studies. Trial registration number Not applicable

Multi-modal generative modeling for joint analysis of single-cell T cell receptor and gene expression data

Recent advances in single-cell immune profiling have enabled the simultaneous measurement of transcriptome and T cell receptor (TCR) sequences, offering great potential for studying immune responses at the cellular level. However, integrating these diverse modalities across datasets is challenging due to their unique data characteristics and technical variations. Here, to address this, we develop the multimodal generative model mvTCR to fuse modality-specific information across transcriptome and TCR into a shared representation. Our analysis demonstrates the added value of multimodal over unimodal approaches to capture antigen specificity. Notably, we use mvTCR to distinguish T cell subpopulations binding to SARS-CoV-2 antigens from bystander cells. Furthermore, when combined with reference mapping approaches, mvTCR can map newly generated datasets to extensive T cell references, facilitating knowledge transfer. In summary, we envision mvTCR to enable a scalable analysis of multimodal immune profiling data and advance our understanding of immune responses.

CTLA-4-expressing ILC3s restrain interleukin-23-mediated inflammation.

Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1-4. However, the mechanisms thatshape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5-7, contributes to chronic inflammation.

In Vitro Assessment of 177Lu-Labeled Trastuzumab-Targeted Mesoporous Carbon@Silica Nanostructure for the Treatment of HER2-Positive Breast Cancer.

This study assessed the effectiveness of a trastuzumab-targeted 177Lu-labeled mesoporous Carbon@Silica nanostructure (DOTA@TRA/MC@Si) for HER2-positive breast cancer treatment, focusing on its uptake, internalization, and efflux in breast cancer cells. The synthesized PEI-MC@Si nanocomposite was reacted with DOTA-NHS-ester, confirmed by the Arsenazo(III) assay. Following this, TRA was conjugated to the DOTA@PEI-MC@Si for targeting. DOTA@PEI-MC@Si and DOTA@TRA/MC@Si nanocomposites were labeled with 177Lu, and their efficacy was evaluated through in vitro radiolabeling experiments. According to the results, the DOTA@TRA/MC@Si nanocomposite was successfully labeled with 177Lu, yielding a radiochemical yield of 93.0 ± 2.4%. In vitro studies revealed a higher uptake of the [177Lu]Lu-DOTA@TRA/MC@Si nanocomposite in HER2-positive SK-BR-3 cells (44.0 ± 4.6% after 24 h) compared to MDA-MB-231 cells (21.0 ± 2.3%). The IC50 values for TRA-dependent uptake in the SK-BR-3 and BT-474 cells were 0.9 µM and 1.3 µM, respectively, indicating affinity toward HER-2 receptor-expressing cells. The lipophilic distribution coefficients of the radiolabeled nanocomposites were determined to be 1.7 ± 0.3 for [177Lu]Lu-DOTA@TRA/MC@Si and 1.5 ± 0.2 for [177Lu]Lu-DOTA@PEI-MC@Si, suggesting sufficient passive transport through the cell membrane and increased accumulation in target tissues. The [177Lu]Lu-DOTA@TRA/MC@Si nanocomposite showed an uptake into HER2-positive cell lines, marking a valuable step toward the development of a nanoparticle-based therapeutic agent for an improved treatment strategy for HER2-positive breast cancer.

Dynamic network biomarker of treg cells differentiation critical state with neoadjuvant treatment in LUSC.

e20063 Background: Regulatory T cells (Tregs) suppress immune responses and can promote tumor growth and metastasis during cancer progression. Studies have shown that tumor-infiltrating Tregs upregulate inhibitory gene programs after immune checkpoint blockade therapy. Targeting Tregs is believed to be a valuable approach to enhance the effectiveness of immunotherapy. However, the precise role of Tregs in the tumor microenvironment, their connection to treatment resistance mechanisms, and how changes in their immune activity impact tumor treatment are still not well understood. Methods: Single-cell data and clinical information were obtained from the Gene Expression Omnibus database with the accession number GSE176021 for 4 patients diagnosed with lung squamous cell carcinoma (LUSC) who received neoadjuvant anti-PD-1 therapy, including 2 patients with major pathologic response (MPR) and 2 patients with non-MPR. BioTIP was employed to identify critical transition signals of the Treg cell differentiation trajectory constructed by Monocle2. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set variation analysis were performed to understand biological processes and pathways. Additionally, potential transcription factors were identified using Single-Cell rEgulatory Network Inference and Clustering, and CellChat analysis was conducted to evaluate possible interactions among cell populations. Results: The study constructed a pseudo-temporal differentiation trajectory of Tregs in LUSC patients, and Treg subpopulations during the critical transition period were identified based on the dynamic network biomarker method. Notably, subpopulations of Treg cells clustering MPR or non-MPR samples were located at different ends of the differentiation trajectory, showing variations in Treg-related pathways and transcription factor regulation. The critical transition subpopulation exhibited high expression of glycolysis, TCA cycle, and natural killer cell receptors, along with upregulation of the CEBPB transcription factor activity and downregulation of ARNTL activity. SELPLG-SELL, CD99-CD99, and MHC-II-CD4 were identified as potential key signals that may affect therapeutic effect, primarily influencing cell adhesion, the immune suppressive activity of Treg cells, and T cell response. Conclusions: In conclusion, this study utilized single-cell data analysis to uncover the dynamic regulation of Treg cells in LUSC, identifying critical transition Treg subpopulations. Notably, our findings suggest the involvement of the transcription factors CEBPB and ARNTL in regulating Treg activity. These novel insights shed light on potential strategies for targeted therapies and provide valuable information regarding resistance mechanisms associated with Treg cells.

T cell receptor and immune gene expression pharmacodynamics for durvalumab monotherapy and in combination with tremelimumab or bevacizumab in unresectable hepatocellular carcinoma (uHCC).

T cell receptor and immune gene expression pharmacodynamics for durvalumab monotherapy and in combination with tremelimumab or bevacizumab in unresectable hepatocellular carcinoma (uHCC).

A first-in-human phase 1, multicenter, open-label study of CB-012, a next-generation CRISPR-edited allogeneic anti-CLL-1 CAR-T cell therapy for adults with relapsed/refractory acute myeloid leukemia (AMpLify).

TPS6586 Background: CAR-T cell therapies have shown significant clinical benefit in treating adults with many hematologic malignancies. In acute myeloid leukemia (AML), a challenge in the development of CAR-T cell therapies has been the limitation of suitable target antigens since many are also expressed on hematopoietic stem cells (HSCs). The target antigen C-type lectin-like molecule-1 (CLL-1) has emerged as an attractive therapeutic target due to its expression on AML mature blasts and leukemic stem cells and its absence on HSCs. CB-012 is an allogeneic CAR-T cell therapy that targets CLL-1. CB-012 is derived from healthy donor T cells by using Cas12a CRISPR hybrid RNA-DNA (chRDNA) technology to introduce 5 genome edits: (1) TRAC gene knockout (KO), which eliminates T cell receptor expression to reduce the risk of graft-versus-host disease, (2) site-directed insertion of a fully human anti-CLL-1 CAR into the TRAC locus, (3) KO of PD-1 to enhance antitumor activity by reducing T cell exhaustion, (4) β2-microgobulin ( B 2M) gene KO, which eliminates HLA class I presentation to mitigate host T cell-mediated rejection, and (5) site-directed insertion of a B2M–HLA-E-peptide fusion into the B 2M locus to reduce NK cell-mediated rejection. In murine xenograft models of AML, CB-012 significantly reduced the tumor burden and increased the survival of mice bearing CLL-1+PD-L1+ tumors. Methods: CB-012 is being evaluated in a multicenter, Phase 1 clinical trial in patients with relapsed/refractory (r/r) AML. A 3+3 dose escalation design is being utilized with the primary objectives to determine the safety and tolerability of CB-012 and the recommended Phase 2 dose (RP2D). An expansion phase will follow dose escalation at the recommended dose(s) for expansion. Additional objectives include preliminary antitumor activity and pharmacokinetics. Key inclusion criteria include nonproliferative disease defined as ≤25% blasts in the bone marrow and peripheral blood, ECOG performance status ≤1, and adequate organ function. After receiving lymphodepletion therapy with cyclophosphamide (750 mg/m2/d) and fludarabine (30 mg/m2/d) administered concurrently for 3 days followed by a 2-day break, patients receive a single-dose infusion of CB-012 and are followed for safety and efficacy. The dose escalation portion of the study is actively enrolling patients. The AMpLify trial is registered at clinicaltrials.gov (NCT06128044). Clinical trial information: NCT06128044 .

Abstract PO2-08-04: Unraveling alterations in the pre-neoplastic breast microenvironment of BRCA1/2 mutation carriers using spatial transcriptomics

Abstract Breast cancer is the most common cancer in females, affecting one in every eight women and accounting for the majority of cancer-related deaths in women worldwide. Germline mutations in the BRCA1 and BRCA2 genes are significant risk factors for specific subtypes of breast cancer. BRCA1 mutations are associated with basal-like breast cancers, whereas BRCA2 mutations are associated with luminal-like disease. There are currently few chemoprevention strategies available for BRCA1/2 mutation carriers, and irreversible prophylactic mastectomy is the primary option. Designing chemo-preventive strategies requires an in-depth understanding of the physiological processes underlying tumor initiation. Here, we employ spatial transcriptomics to investigate defects in mammary epithelial cell differentiation accompanied by distinct microenvironmental alterations in preneoplastic breast tissues from BRCA1/2 mutation carriers and normal breast tissues from non-carrier controls. We uncovered spatially defined receptor-ligand interactions in these tissues for the investigation of autocrine and paracrine signaling. We discovered that β1-integrin-mediated autocrine signaling in BRCA2-deficient mammary epithelial cells differs from BRCA1-deficient mammary epithelial cells. In addition, we found that the epithelial-to-stromal paracrine signaling in the breast tissues of BRCA1/2 mutation carriers is greater than in control tissues. More integrin-ligand pairs were differentially correlated in BRCA1/2-mutant breast tissues than non-carrier breast tissues with more integrin receptor-expressing stromal cells. These results reveal alterations in the communication between mammary epithelial cells and the microenvironment in BRCA1 and BRCA2 mutation carriers, laying the foundation for designing innovative breast cancer chemo-prevention strategies for high-risk patients. Citation Format: Mihriban Karaayvaz, Anthony Caputo, Kavya Vipparthi, Peter Bazeley, Erinn Downs-Kelly, Patrick McIntire, Ying Ni, Bo Hu, Ruth Keri. Unraveling alterations in the pre-neoplastic breast microenvironment of BRCA1/2 mutation carriers using spatial transcriptomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-04.

Abstract Wrk2-02: Modelling the effects of cell-cell interactions in the tumor microenvironment from single cell variability

Abstract Multicellular programs underly emergent phenotypes in health and disease. Yet, deciphering the functional effects of interactions amongst cell types remains a major challenge. Cells typically send messages to their microenvironment by emitting ligands, which bind to complimentary receptors on the surfaced of target cells; then triggering a change in the behavior of the target cell. Complimentary ligand-receptor-pairs are well annotated and methods have been developed to infer cell-cell interactions from single cell expression data based on their mutual expression. However, these methods do not assess the effect of ligand-receptor-mediated interactions on the microenvironment. Thus, we developed ContactTracing – a fundamentally new, systems level approach that exploits inter- and intra-sample variability to infer ligand effects on gene expression in target (receptor-expressing) cells without prior knowledge of downstream signaling (Nature, 2023). Foundational work towards benchmarking and validation of this method was performed in isogenic breast cancer models distinguished by tumor cell-intrinsic rates of chromosome missegregation (a cellular process called chromosomal instability, or CIN). Through this, we identified tumor ligands emanating from an endoplasmic reticulum (ER)-stress response as potential mediators of immune suppression in chromosomally unstable tumors. Indeed, CIN-induced chronic STING activation led to rapid interferon-selective desensitization and a switch to ER-stress-dependent transcription. Moreover, inhibition of chronic STING - or key mediators of its unfolded protein response to ER-stress - suppressed metastasis in syngeneic models of melanoma, breast and colorectal cancer; validating this innovative methodology and identifying a targetable mediator of cancer metastasis. Citation Format: A. Laughney. Modelling the effects of cell-cell interactions in the tumor microenvironment from single cell variability [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr Wrk2-02.

Effect of estrogens on apoptosis in the pituitary of viscachas (Lagostomus maximus maximus)

Estrogens, acting through their receptors (ERα and ERβ), regulate cell turnover in the pituitary gland, influencing cell proliferation and apoptosis across various species. However, their role in pituitary processes in seasonally reproducing animals remains poorly understood. This study aims to investigate the influence of estrogens, through the expression of their specific receptors, on the apoptosis of PD cells in relation to sexual maturity, the reproductive cycle, and pregnancy in a seasonal reproductive rodent (Lagostomus maximus maximus). ERα and caspase-3-cleaved (CASP3c) immunoreactive (-ir) cells were identified through immunohistochemistry. Apoptotic cells were detected using the TUNEL technique, with quantitative analysis facilitated by image analysis software, alongside measurement of serum estradiol levels using radioimmunoassay The immunostaining pattern for ERα included nuclear (ERαn) and cytoplasmic (ERαc) staining. In male viscachas, ERα expression significantly increases from immature to adult animals, correlating with the rise in serum estradiol levels and a decrease in the percentage of apoptotic cells. During the gonadal regression period in adult males, a decrease in the number of ER-ir cells and serum levels of estradiol corresponds with an increase in the number of apoptotic cells. In females, serum levels of estradiol peaked during mid-pregnancy, coinciding with a significant decrease in the number of apoptotic cells in the PD. Simultaneously, the percentage of ERαn-ir cells reaches its maximum value during late pregnancy, indicating the need to maintain the protective action of this gonadal hormone throughout the extensive pregnancy in these rodents. Regional ERα receptor expression and apoptotic cells appear to be associated with distinct PD cell populations and their hormonal responses. Finally, elevated estradiol levels coincide with diminished apoptotic cells in the male reproductive cycle and during pregnancy, suggesting an antiapoptotic role of estradiol in this species.

Thymocyte migration and emigration

Hematopoietic precursors (HPCs) entering into the thymus undergo a sequential process leading to the generation of a variety of T cell subsets. This developmental odyssey unfolds in distinct stages within the thymic cortex and medulla, shaping the landscape of T cell receptor (TCR) expression and guiding thymocytes through positive and negative selection. Initially, early thymic progenitors (ETPs) take residence in the thymic cortex, where thymocytes begin to express their TCR and undergo positive selection. Subsequently, thymocytes transition to the thymic medulla, where they undergo negative selection. Both murine and human thymocyte development can be broadly classified into distinct stages based on the expression of CD4 and CD8 coreceptors, resulting in categorizations as double negative (DN), double positive (DP) or single positive (SP) cells. Thymocyte migration to the appropriate thymic microenvironment at the right differentiation stage is pivotal for the development and the proper functioning of T cells, which is critical for adaptive immune responses. The journey of lymphoid progenitor cells into the T cell developmental pathway hinges on an ongoing dialogue between the differentiating cell and the signals emanating from the thymus niche. Herein, we review the contribution of the key factors mentioned above for the localization, migration and emigration of thymocytes.

Development of KoRV-pseudotyped lentiviral vectors for efficient gene transfer into freshly isolated immune cells

Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need.

Abstract LB313: Immunosuppressive effect of tumor-infiltrating IL-22 receptor-expressing myeloid cells in early-stage pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a poor survival rate, largely due to the lack of diagnostic biomarker for early detection. Here, we found enriched IL-10R2+/IL-22R1+ myeloid cells in peripheral blood and specifically PDAC tissues respectively using xenograft/orthotropic PDAC mouse models. The subcutaneous injection of PDAC cell line in mouse showed a positive correlation of pancreatic tumor growth with increased IL-10R2+/IL-22R1+ myeloid cells by flow cytometry. We also verified that IL-10R2+CD45+ and IL-22R1+CD45+ myeloid cells were infiltrated in tumor tissues after 2 weeks and 3 weeks of injection in orthotropic murine PDAC model. Notably, the IL-10R2+IL-22R1+ cells were highly detected in tumor tissues after 7 days of injection, an earlier time point of PDAC progression. To define whether these cells have immunosuppressive functions to contribute further PDAC progression, IL-10R2+/IL-22R1+ myeloid cells were sorted from splenocytes after 3 days of tumor implantation and co-cultured with T cells in the presence of T cell receptor triggering. The proliferation and cytotoxicity of CD8+ T cells were alleviated by co-culture with IL-10R2+, IL-22R1+, or IL-10R2+IL-22R1+ myeloid cells, compared to that with IL-10R2-IL-22R1- cells. In addition, the population of regulatory T cells was increased when they were co-cultured with IL-22R1+ myeloid cells. Furthermore, the proliferation of human T cells in vitro was also inhibited by co-culture with IL-10R2+/IL-22R1+ myeloid cells from human peripheral blood, indicating the immunosuppressive function of IL-10R2+/IL-22R1+ cell population. Therefore, IL-10R2+/IL-22R1+ myeloid cell population could be a possible promising early detection marker for PDAC, contributing to the non-immunogenic features of tumor microenvironment during PDAC progression. Citation Format: Yoolim Sung, Ji-Eun Kim, Hyo-Jin Min, Soo-Hyun Chung, Seong Who Kim, Eun-Ju Chang. Immunosuppressive effect of tumor-infiltrating IL-22 receptor-expressing myeloid cells in early-stage pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB313.

A novel dihydroacridine derivative targets epidermal growth factor receptor-expressing cancer cells in vitro and in vivo.

Small molecules are considered a source of novel medicines targeting carcinogenic intracellular pathways including epidermal growth factor receptor (EGFR) signaling. The main goal of the study is to assess whether LHT-17-19 could be considered an effective target molecule against EGFR-expressing tumor cells in silico, in vitro, and in vivo. This was an in vivo, ex vivo, and in vivo experimental study. LHT-17-19 affinity to EGFR's kinase domain was assessed by the ligand's molecular docking. EGFR-expressing Hs746T human gastric cancer cell culture and patient-derived organoid (PDO) model of EGFR-positive breast cancer (BC) were used for in vitro assessment of the molecule anticancer property. IC50 and GI50 indexes were estimated using MTT- and MTS-based tests, respectively. Anticancer activity of LHT-17-19 against EGFR-expressing mutant lung carcinoma was studied on patient-derived xenograft (PDX) model established in 10 humanized BALB/c male mice. Continuous variables were presented as a mean ± standard deviation. Intergroup differences were assessed by two-way t-test. Kaplan-Meier's curves were used for survival analysis. High affinity of LHT-17-19 for the EGFR kinase domain with dG score -7.9 kcal/mol, EDoc-5.45 kcal/mol, and Ki 101.24 uM was due to intermolecular π-σ bonds formation and the ligand intramolecular transformation. LHT-17-19 induced anti-EGFR-expressing gastric cancer cells cytotoxicity with IC50 0.32 µM (95% confidence interval [CI] 0.11-0.54 µM). The derivative inhibited growth of EGFR-expressing BC PDO with GI50 16.25 µM (95% CI 4.44-28.04 µM). 2 mg/kg LHT-17-19 intravenously daily during 7 days inhibited PDX tumor growth and metastatic activity, prolonged animals' survival, and eliminated EGFR-mutant lung cancer cells from residual tumor's node. LHT-17-19 may be considered a molecular platform for further search of promising molecules, EGFR-expressing cancer cell inhibitors.

Abstract 5229: Enhancing solid tumor treatment with engineered blood-derived Vδ1 platform: Safe and effective targeting of mesothelin-expressing tumors with Vδ1 γδT cells

Abstract Mesothelin (MSLN) is a solid tumor-associated antigen that has been thoroughly studied and is expressed in several tumor indications that have significant unmet therapeutic needs. Vδ1-enriched γδT cells differentiate themselves from other MSLN-targeted modalities by mitigating the challenges of targeting tumors with heterogeneous MSLN expression through their inherent capability to detect malignant cells while sparing normal cells. The ability to effectively combat cancer cells that express mesothelin is significantly increased by the expression of a chimeric antigen receptor (CAR). Novel armoring is being evaluated to improve engineered Vδ1 persistence in the solid tumor microenvironment. Cytokine armoring, through novel engineering, enhances sensitivity to cytokines and recovers cell fitness that is impaired by 'traditional' cytokine armoring options. Extensive in vitro and in vivo safety and efficacy studies were carried out to validate the product's potency. These studies aimed to determine cytotoxicity, expansion, and persistence. MSLN CAR cytokine-armored Vδ1 cells exhibited high post-thaw viability and survival. Furthermore, cytokine-armored Vδ1 cells displayed elevated innate receptor expression and gene-engineered cells maintained their baseline innate phenotype, indicating that they can maintain their ‘innate’ effectiveness over time. In vitro studies demonstrated broad anti-tumor activity against multiple tumor cell lines expressing various levels of mesothelin. Additionally, in long-term cytotoxicity assays, cytokine-armored cells demonstrated improved tumor-clearance capabilities. The effector cells also exhibited an enhanced innate cytotoxic phenotype that equips the cells with the capability to persist and proliferate in response to soluble cytokines and upon cytokine trans-presentation by adjacent cells. In vitro studies further revealed that there is a distinction in the killing activity between tumor and normal cells, which is important for ensuring that the product is safe for use in patients. An orthotopic lung tumor-bearing mouse model was developed to confirm their anti-tumor effectiveness in vivo. The model's outcomes demonstrated that MSLN CAR cytokine-armored Vδ1 cells exhibit higher in vivo efficacy in comparison to their unarmored counterparts at low effector-to-target ratios. With no significant healthy tissue damage found, the histopathology report confirmed an excellent safety profile. Terminal biodistribution studies demonstrated that armored drug candidates preferentially home to and persist in tumor-bearing tissues. Overall, these findings imply that the product might be highly effective in treating tumors that express mesothelin. This strategy could greatly advance our knowledge of how Vδ1-enriched γδT cells function as a platform against solid tumors. Citation Format: Nandhu Mohan Sobhana, Andre Simoes, Sarah Edwards, Sweta Mishra, Gary Gu, George Papadopoulos, Stefan Kiesgen, Rebecca Alade, njing Li, Andrea Venuso, Jyothi Kumaran, Kristy Waddington, Tabassum Huseni, Rajeev Karattil, Lilly Reynolds, Maria Sharif, David Wotherspoon, Corina Tudor, Istvan Kovacs, Mei Rosa Ng, Yana Wang, Lan Cao. Enhancing solid tumor treatment with engineered blood-derived Vδ1 platform: Safe and effective targeting of mesothelin-expressing tumors with Vδ1 γδT cells [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 5229.

Abstract 2722: CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms

Abstract Many therapeutic monoclonal antibodies mediate the immune effector functions through the engagement of their fragment crystallizable (Fc) domain and Fcγ receptors on the immune cells. However, these functions are limited due to the low affinity of their interactions and the requirement of abundancy of antigens for the avidity effects. Recently, mutation-based Fc engineered antibodies showed their enhanced immune effector functions mostly through the NK cell-mediated ADCC. To overcome the limited improvement of current Fc engineering, we developed an innovated platform, Twin Fc-Immune Cell Engager (Twin Fc-ICE) composed of a single Fab domain and two Fc domain with drug-like properties. Here we demonstrated the superior efficacy of CTN001, a first Twin Fc-ICE, in in vitro and in vivo Her2+ tumor models. CTN001 bound to various types of Fcγ receptor-expressing immune cells with stronger affinity, supported by its better in vitro affinity to all types of Fcγ receptors than wild type and Fc-engineered antibodies. To evaluate the pharmacological efficacies, CTN001 was tested for its functions of in vitro antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) and in vivo efficacy with Her2+ xenograft models. In Her2+ models, both CTN001 and trastuzumab induced ADCC-mediated effects, but CTN001 showed better anti-tumor cytotoxicity through ADCP and CDC than trastuzumab. Importantly, CTN001 also showed cytotoxic effects on Her2-low tumor cells. Consistently, CTN001 showed better anti-tumor efficacy than trastuzumab or its combination with pertuzumab in Her2+ tumor xenograft model and, surprisingly, even in Her2-low or resistant models. These results suggest that CTN001 can provide new therapeutic options for treating Her2-low patients differentiated from antibody-drug conjugates (ADCs), and that the Twin Fc-ICE can be an innovative platform to overcome limitations of existing therapeutics. Citation Format: Deokjae Lee, Sujin Yoon, Jiseon Bae, Minju Kim, Ki-June Lee, Eunmi Kim, Hyunkyu Park, Eun S. Choi, Gi-Hyeok Yang. CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms [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 2722.

Abstract 1340: Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™

Abstract Natural Killer (NK) cells are highly responsive cytotoxic immune cells of the innate immune system with well characterized anti-tumor properties. Their ability to directly kill malignant cells and elicit an adaptive immune response makes them a promising candidate for a precision guided immunotherapy in oncology. Bicycle® peptides are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle® platform technology to discover and evaluate a new class of fully synthetic molecules termed NK tumor immune cell agonists (NK-TICA™). The NK-TICA™ consists of chemically coupled Bicycle® peptides that bind specifically to the key activating receptor, NKp46, and to tumor antigens, that results in highly potent, antigen-dependent receptor activation and NK cell function. We demonstrate potent, selective binding of our Bicycle® peptides to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner. In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle® NK-TICA™ as a multifunctional immune cell engager will promote the modulation and anti-tumor activity of peripheral and intra-tumoral NK cells to solid tumors. Citation Format: Fay Dufort, Tucker Ezell, Alexandra Rezvaya, Kathleen Ho, Christopher Leitheiser, Philip Brandish, Kevin McDonnell, Nicholas Keen. Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™ [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 1340.

The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αβ T cell counts at birth persisted over time, with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells, autoimmune conditions were more frequent in these patients compared with the general population.

Evaluation of New Folate Receptor-mediated Mitoxantrone Targeting Liposomes In Vitro.

<p>Background: Ligand-mediated liposomes targeting folate receptors (FRs) that are overexpressed on the surface of tumor cells may improve drug delivery. However, the properties of liposomes also affect cellular uptake and drug release. <p> Objective: Mitoxantrone folate targeted liposomes were prepared to increase the enrichment of drugs in tumor cells and improve the therapeutic index of drugs by changing the route of drug administration. <p> Methods: Liposomes were prepared with optimized formulation, including mitoxantrone folatetargeted small unilamellar liposome (MIT-FSL), mitoxantrone folate-free small unilamellar liposome (MIT-SL), mitoxantrone folate-targeted large unilamellar liposome (MIT-FLL), mitoxantrone folate-free large unilamellar liposomes (MIT-LL). Cells with different levels of folate alpha receptor (FRα) expression were used to study the differences in the enrichment of liposomes, the killing effect on tumor cells, and their ability to overcome multidrug resistance. <p> The results of the drug release experiment showed that the particle size of liposomes affected their release behavior. Large single-compartment liposomes could hardly be effectively released, while small single-compartment liposomes could be effectively released, MIT-FSL vs MIT-FLL and MIT-SL vs MIT-LL had significant differences in the drug release rate (P<0.0005). Cell uptake experiments results indicated that the ability of liposomes to enter folic acid receptor-expressing tumor cells could be improved after modification of folic acid ligands on the surface of liposomes and it was related to the expression of folate receptors on the cell surface. There were significant differences in cell uptake rates (p<0.0005) for cells with high FRα expression (SPC-A-1 cells), when MIT-FSL vs MIT-SL and MIT-FLL vs MIT-LL. For cells with low FRα expression (MCF-7 cells), their cell uptake rates were still different (p<0.05), but less pronounced than in SPC-A-1 cells. The results of the cell inhibition experiment suggest that MIT-FLL and MIT-LL had no inhibitory effect on cells, MIT-FSL had a significant inhibitory effect on cells and its IC50 value was calculated to be 4502.4 ng/mL, MIT-SL also had an inhibitory effect, and its IC50 value was 25092.1 ng/mL, there was a statistical difference (p<0.05), MIT-FSL had a higher inhibitory rate than MIT-SL at the same drug concentration. Afterward, we did an inhibitory experiment of different MIT-loaded nanoparticles on MCF-7 cells compared to the drug-resistant cells (ADR), Observing the cell growth inhibition curve, both MIT-FSL and MIT-SL can inhibit the growth of MCF-7 and MCF-7/ADR cells. For MCF- 7 cells, at the same concentration, there is little difference between the inhibition rate of MITFSL and MIT-SL, but for MCF-7/ADR, the inhibition rate of MIT-FSL was significantly higher than that of MIT-SL at the same concentration (P<0.05). <p> Conclusion: By modifying folic acid on the surface of liposomes, tumor cells with high expression of folic acid receptors can be effectively targeted, thereby increasing the enrichment of intracellular drugs and improving efficacy. It can also change the delivery pathway, increase the amount of drug entering resistant tumor cells, and overcome resistance.</p>.

Dopamine Receptor-Expressing Neurons Are Differently Distributed throughout Layers of the Motor Cortex to Control Dexterity.

The motor cortex comprises the primary descending circuits for flexible control of voluntary movements and is critically involved in motor skill learning. Motor skill learning is impaired in patients with Parkinson's disease, but the precise mechanisms of motor control and skill learning are still not well understood. Here we have used transgenic mice, electrophysiology, in situ hybridization, and neural tract-tracing methods to target genetically defined cell types expressing D1 and D2 dopamine receptors in the motor cortex. We observed that putative D1 and D2 dopamine receptor-expressing neurons (D1+ and D2+, respectively) are organized in highly segregated, nonoverlapping populations. Moreover, based on ex vivo patch-clamp recordings, we showed that D1+ and D2+ cells have distinct morphological and electrophysiological properties. Finally, we observed that chemogenetic inhibition of D2+, but not D1+, neurons disrupts skilled forelimb reaching in adult mice. Overall, these results demonstrate that dopamine receptor-expressing cells in the motor cortex are highly segregated and play a specialized role in manual dexterity.