Receptor Chain Research Articles

Dual ON/OFF-switch chimeric antigen receptor controlled by two clinically approved drugs

The ability to remotely control the activity of chimeric antigen receptors (CARs) with small molecules can improve the safety and efficacy of gene-modified T cells. Split ON- or OFF-switch CARs involve the dissociation of tumor-antigen binding from T cell activation (i.e., CD3ζ) on the receptor (R-) and signaling (S-) chains, respectively, that either associate or are disrupted in the presence of a small molecule. Here, we have developed an inducible (i)ON-CAR comprising the anti-apoptotic B cell lymphoma protein 2 protein in the ectodomain of both chains which associate in the presence of venetoclax. We showed that inducible ON (iON)-CAR T cells respond to target tumors cells in the presence of venetoclax or the BH3 mimetic navitoclax in a dose-dependent manner, while there is no impact of the drugs on equivalent second generation-CAR T cells. Within 48 h of venetoclax withdrawal, iON-CAR T cells lose the ability to respond to target tumor cells in vitro as evaluated by Interferon-gamma (IFNγ) production, and they are reliant upon the presence of venetoclax for in vivo activity. Finally, by fusing a degron sequence to the endodomain of the iON-CAR S-chain we generated an all-in-one ON/OFF-switch CAR, the iONØ-CAR, down-regulated by lenalidomide within 4 to 6 for functionally inactive T cells (no IFNγ production) within 24 h. We propose that our remote-control CAR designs can reduce toxicity in the clinic. Moreover, the periodic rest of iON and iONØ-CAR T cells may alleviate exhaustion and hence augment persistence and long-term tumor control in patients.

Calcitonin gene-related peptide promotes epithelial reparative and anti-colitic functions of IL-4 educated human macrophages.

Interleukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect in a murine model. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn's disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, raising the issue of neural modulation of the M(IL4)s reparative function. Thus, human (MIL4)s were treated with CGRP and the cells phagocytotic, epithelial wound repair and anti-colitic functions were assessed. Initial studies confirmed up-regulation of expression of the CGRP receptor, which was localized to the cell surface and was functional as determined by CGRP-evoked increases in cAMP. M(IL4,CGRP)s had increased mannose receptor (CD206) and FcgRIIa (CD32a) mRNA expression, a subtle, but significant increase in phagocytosis, and decreased chemokine production following exposure to E. coli. When delivered systemically (106 cells, ip.) to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same blood donor. Conditioned medium (CM) from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM in the human CaCo2 epithelial cell line in vitro wounding assay. Moreover, M(IL4,CGRP)s displayed increased cyclooxygenase (COX)-1 and prostaglandin D2, and CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers. These data confirm the human M(IL4)s' anti-colitic effect that was enhanced by CGRP, and may be partially dependent on macrophage COX1/PDG2 activity. Thus, input from neurone-derived molecules is a local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer.

Limits on inferring T cell specificity from partial information

A key challenge in molecular biology is to decipher the mapping of protein sequence to function. To perform this mapping requires the identification of sequence features most informative about function. Here, we quantify the amount of information (in bits) that T cell receptor (TCR) sequence features provide about antigen specificity. We identify informative features by their degree of conservation among antigen-specific receptors relative to null expectations. We find that TCR specificity synergistically depends on the hypervariable regions of both receptor chains, with a degree of synergy that strongly depends on the ligand. Using a coincidence-based approach to measuring information enables us to directly bound the accuracy with which TCR specificity can be predicted from partial matches to reference sequences. We anticipate that our statistical framework will be of use for developing machine learning models for TCR specificity prediction and for optimizing TCRs for cell therapies. The proposed coincidence-based information measures might find further applications in bounding the performance of pairwise classifiers in other fields.

An super sensitive Multi-Modal controlled release biosensor with label- and enzyme- free assisted single step simultaneously detect multiple breast cancer biomarkers

Despite the fact that homogeneous biosensing strategies have been proven to be advantageous, it remains a difficult task to identify multiple tumor biomarkers. Our proposition entails the creation of a groundbreaking biosensor that combines electrochemical and fluorescent techniques, enabling simultaneous detect multiple biomarkers with label- and enzyme-free assistance in a single step. Porous UIO-66-NH2 nanovessels loaded with 3,3′,5,5′ −tetramethylbenzidine (TMB) and methylene blue (MB) double signal dyes, along with double-stranded DNA (dsDNA) formed by the aptamer chain of human epidermal growth factor receptor-2 (HER2) and estrogen receptor (ER) and its complementary chain as a gated switch to the envelope MOFs, were utilized to create functional MOFs (dsDNA@MB@UIO and dsDNA@TMB@UIO). When HER2 and ER targets are present, the chain replacement reaction led to the formation of a biomarker-aptamer complex, resulting in the destruction of the dsDNA structure on the surface of MOF and the liberation of MB and TMB. The concentration of the two target biomarkers determines the strength of the electrochemical and fluorescent signals produced. By combining targeted biomarkers, signal enhancement, and dual-signal detection into one unified process, the amount of test error caused by the multi-step detection needed in prior studies is significantly decreased. We executed the detect HER2 and ER, achieving detection limits of 4.7 and 5.9 fg/mL in 4.7–1000 pg/mL linear range with good selectivity, stability, reproducibility, and acceptable applicability. The proposed biosensor for developing dsDNA@MB@UIO and dsDNA@TMB@UIO is promising for the early and sensitive detection of cancer biomarkers.

Generation of antitumor chimeric antigen receptors incorporating T cell signaling motifs.

Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor.

Computational Study of Lactucine and its Derivatives to Investigate its Anti-cancerous Properties Targeting Apoptosis-inducing Protein

Background: Lactucine is related to the sesquiterpene lactone group of naturally occurring compounds and has a variety of pharmacological effects including anticancer properties found in Chicory, Wormwood, Laurus nobilis, Pyrethrum, Chamomile, etc. Lactucine has an anticancer effect which may induce apoptosis in cancerous cells and protect other cells from getting infected. Objective: In this study, Lactucine and its derivatives were screened, and performed their in silico docking study with the proteins involved in the apoptosis-inducing effect in human leukemia cancer. Methods: The three-dimensional structure of lactucine and its derivatives were retrieved in the SDF format. Active sites of protein structures were determined by Sitemap. LigPrep module was used for geometrical refining of chemical structures of lactucine and its derivatives. The protein preparation wizard of Maestro (Schrodinger) was used for protein preparation. From the receptor-complex structure, the cocrystallized ligands were removed from the active site position of the receptor chain. All ligands were docked using default Glide settings for a grid centered on the ligand and structure. Flexible docking was performed using the extra precision (XP) feature of Glide module. The best docking poses for the lactucine and their derivatives were selected based on their docking score. The ADMET properties of lactucine 15- oxalate have been predicted by admetSAR software. Results: Proteins and ligands three-dimensional structures were retrieved from PDB and Pubchem databases, respectively. All lactucine derivatives suitably docked on the apoptosis-inducing proteins with ample Glide scores Lactucin 15-oxalate interacts with proteins which are responsible for apoptosis with a maximum of six H-bonds. Other types of interactions are also involved, like Pi-cation, Pi-Pi stacking, salt bridges, and halogen bonds. Protein CDK-4 has shown the highest number of H-bond (LYS142 salt bridges), ALA16, VAL14, ASP99, LYS35, TYR17, and ASN145) with the Lactucin 15-oxalate. ADMET properties of lactucin 15-oxalate met with the criteria of being eligible to be a novel drug for the treatment of human leukemia cancer. The Dock score of both the Dasatinib drug and the lactucine-15-oxalate with the apoptosis-inducing protein stipulates that the selected ligand has equitable interaction with the target proteins. Conclusion: In this study, lactucine derivatives were docked with apoptosis-inducing proteins for the prediction of its anticancer effect. Lactucin15-oxalate has shown the highest binding affinity for the CDK-4 target and can be used as a lead compound for cancer treatment. Glide and Dock score for docking of lactucin 15-oxalate with CDK-4, well as the number of hydrogen bonding, is in agreement to use this ligand for study. These in silico results are valuable to proceed with the in vitro and in vivo studies related to the anti-cancer role of lactucin 15-oxalate.

Camelid-derived Tcell engagers harnessing human γδ Tcells as promising antitumor immunotherapeutic agents.

In the last decade, there has been a surge in developing immunotherapies to enhance the immune system's ability to eliminate tumor cells. Bispecific antibodies known as Tcell engagers (TCEs) present an attractive strategy in this pursuit. TCEs aim to guide cytotoxic Tcells toward tumor cells, thereby inducing a strong activation and subsequent tumor cell lysis. In this study, we investigated the activity of different TCEs on both conventional alpha-beta (αβ) Tcells and unconventional gamma delta (γδ) Tcells. TCEs were built using camelid single-domain antibodies (VHHs) targeting the tumor-associated antigen CEACAM5 (CEA), together with Tcell receptor chains or a CD3 domain. We show that Vγ9Vδ2 Tcells display stronger in vitro antitumor activity than αβ Tcells when stimulated with a CD3xCEA TCE. Furthermore, restricting the activation of fresh human peripheral Tcells to Vγ9Vδ2 Tcells limited the production of protumor factors and proinflammatory cytokines, commonly associated with toxicity in patients. Taken together, our findings provide further insights that γδ Tcell-specific TCEs hold promise as specific, effective, and potentially safe molecules to improve antitumor immunotherapies.

High common-γ cytokine receptor levels promote expression of Interleukin-2/Interleukin-7 receptor α-chains with implications on T-cell differentiation and function.

Cytokines of the common-γ receptor chain (γc) family are crucial for T-cell differentiation and dysregulation of γc cytokine pathways is involved in the pathogenesis of autoimmune diseases. There is increasing evidence that the availability of the γc receptor (CD132) for the associated receptor chains has implications for T-cell functions. Here we studied the influence of differential γc expression on the expression of the IL-2Rα (CD25), the IL-7Rα (CD127) and the differentiation of activated naïve T cells. We fine-tuned the regulation of γc expression in human primary naïve T cells by lentiviral transduction using small hairpin (sh)RNAs and γc cDNA. Differential γc levels were then analysed for effects on T-cell phenotype and function after activation. Differential γc expression markedly affected IL-2Rα and IL-7Rα expression on activated naïve T cells. High γc expression (γc-high) induced significantly higher expression of IL-2Rα and re-expression of IL-7Rα after activation. Inhibition of γc caused lower IL-2Rα/IL-7Rα expression and impaired proliferation of activated naïve T cells. In contrast, γc-high T cells secreted significantly higher concentrations of effector cytokines (i.e., IFN-γ, IL-6) and showed higher cytokine-receptor induced STAT5 phosphorylation during initial stages as well as persistently higher pSTAT1 and pSTAT3 levels after activation. Finally, accelerated transition towards a CD45RO expressing effector/memory phenotype was seen especially for CD4+ γc-high naïve T cells. These results suggested that high expression of γc promotes expression of IL-2Rα and IL-7Rα on activated naïve T cells with significant effects on differentiation and effector cytokine expression.

Local chicken breeds exhibit abundant TCR-V segments but similar repertoire diversity

The thymus-derived lymphocytes of jawed vertebrates have four T-cell receptor (TCR) chains that play a significant role in immunity. As chickens have commercial value, their immune systems require a great deal of attention. Local chicken breeds are an essential part of poultry genetic resources in China. Here, we used high-throughput sequencing to analyze the TCRα and TCRβ repertoires and their relative expression levels in the native chicken breeds Baier Buff, Longyou Partridge, Xiaoshan, and Xianju. We found that TCR Vα and TCR Vβ were expressed and included 17, 19, 17, and six segments of the Vα2, Vα3, Vβ1, and Vβ2 subgroups, respectively. V-J pairing was biased; Jα11 was utilized by nearly all Vα segments and was the most commonly used. Breed-specific V segments and V-J pairings were detected as well. The results of the principal coordinate analysis (PCoA) as well as the V-J pairing and CDR3 diversity analyses suggested that the four local chicken breeds did not significantly differ in terms of TCR diversity. Hence, they expressed not significant differentiation, and they are rich genetic resources for the development and utilization of immune-related poultry breeding.

Unpaired cysteine insertions favor transmembrane dimerization and induce ligand-independent constitutive cytokine receptor signaling.

Naturally occurring gain-of-function (GOF) mutants have been identified in patients for a variety of cytokine receptors. Although this constitutive activation ofcytokine receptors is strongly associated with malignant disorders, ligand-independent receptor activation is also a useful tool in synthetic biology e.g. to improve adoptive cellular therapies with genetically modified T-cells. Balanced Interleukin (IL-)7 signaling via a heterodimer of IL-7 receptor (IL-7Rα) and the common γ-chain (γc) controls T- and B-cell development and expansion, whereas uncontrolled IL-7 signaling can drive acute lymphoid leukemia (ALL) development. The ALL-driver mutation PPCL in the transmembrane domain of IL-7Rα is a mutational insertion of the four amino acids proline-proline-cysteine-leucine and leads to ligand-independent receptor dimerization and constitutive activation. We showed here in the cytokine-dependent pre-B-cell line Ba/F3 that the PPCL-insertion in a synthetic version of the IL-7Rα induced γc-independent STAT5 and ERKphosphorylation and also proliferation of the cells and that booster-stimulation by arteficial ligands additionally generated non-canonical STAT3 phosphorylation via the synthetic IL-7Rα-PPCL-receptors. Transfer of theIL-7Rα transmembrane domain with the PPCL insertion intonatural and synthetic cytokine receptor chains of the IL-6, IL-12 and Interferon families also resulted in constitutive receptor signaling. In conclusion, our data suggested that the insertion of the mutated PPCL IL-7Rα transmembrane domain is an universal approach to generate ligand-independent, constitutively active cytokine receptors.

Loganin exerts neuroprotective effect by inhibiting neuronal pyroptosis in rat with cerebral haemorrhage.

Intracerebral haemorrhage (ICH) presents significant challenges in clinical management because of the high morbidity and mortality, necessitating novel therapeutic approaches. This study aimed to assess the neuroprotective effects of loganin in a rat ICH model. Sprague-Dawley rats were used, subjected to a collagenase-induced ICH model, followed by loganin treatment at doses of 2.5, 5 and 10 mg/kg. Neurological functions were evaluated using the modified neurological severity score (mNSS) and a rotarod test. Results indicated a significant improvement in neurological functions in loganin-treated groups, evident from the mNSS and rotarod tests, suggesting dose-dependent neuroprotection. Loganin also effectively reduced the blood-brain barrier (BBB) permeability and cerebral oedema. Additionally, it mitigated cellular pyroptosis, as shown by terminal deoxynucleotidyl transferase dUTP nick-end labelling staining and western blot analysis, which indicated reduced levels of pyroptosis markers in treated rats. Furthermore, loganin's regulatory effects on the adenosine A2A receptor and myosin light chain kinase pathways were observed, potentially underpinning its protective mechanism against ICH. The study concludes that loganin exhibits significant neuroprotective properties in a rat ICH model, highlighting its potential as a novel therapeutic strategy. Despite promising results, the study needs further research to determine loganin's therapeutic potential in human ICH patients. This research paves the way for further exploration into loganin's clinical applications, potentially revolutionizing treatment strategies for patients suffering from intracerebral haemorrhage.

FEATURES OF THE T-IMMUNE SYSTEM IN PATIENTS WITH GLOMERULONEPHRITISES WITH NEPHROTIC SYNDROME

The study of issues related to glomerulonephritises with nephrotic syndrome is one of the urgent problems of medicine due to their prevalence worldwide, mainly in the young age group. Medical workers distinguish primary (idiopathic) nephrotic syndrome, which occurs in 80–90% of cases, and secondary nephrotic syndrome, mainly associated with systemic autoimmune diseases, diabetes mellitus and neoplasms. Glomerulonephritises, manifested by nephrotic syndrome (membranous nephropathy, focal segmental glomerulosclerosis, nephropathy with minimal changes), are known to be autoimmune diseases. To date, the immunological mechanisms of the pathogenesis of glomerulonephritises with nephrotic syndrome associated with the T-system of adaptive immunity remain unexplored. The aim of the study was to study the role of the T–immune system in the pathogenesis of primary nephrotic syndrome based on the study of immunoregulatory, activated T-cell subsets in patients with this pathology. Material and methods. 136 patients with chronic glomerulonephritis with nephrotic syndrome were selected for the study. The assessment of the T-immune system included determination of the lymphocyte phenotype of immunoregulatory T-cell subsets (T-helper/inducers, cytotoxic T-lymphocytes), various subpopulations of activated T-cells (activated T-lymphocytes; activated T-lymphocytes expressing CD25–alpha chain of IL-2 receptor; activated cytotoxic T-lymphocytes expressing HLA-DR and CD38) and regulatory T-cells (Treg cells). Study results. In the patients of the examined cohort, an increase in the number of T-lymphocytes and T-helper cells, as well as activated T-lymphocytes expressing HLA-DR antigens, was found. At this, the content of cytotoxic T-cells and the number of activated T-cells expressing the IL-2 – CD25 receptor did not differ from similar indicators in healthy individuals. The levels of Treg cells and activated cytotoxic T-lymphocytes with the CD3+CD8brightCD38+ phenotype were reduced. The immunoregulatory index (T-helpers/cytotoxic T-lymphocytes) was increased, due to an increase in the number of T-helper cells against the background of an unchanged number of cytotoxic T-lymphocytes. Conclusions. The results of the study indicate that the main features of the T-system of the immune response in primary nephrotic syndrome are imbalance in the ratio of the content of immunoregulatory cells due to predominance of T-helper cells and a decrease in the number of Treg cells.

Abstract 3129: BYON4413, an in vivo active CD123-targeting antibody-drug conjugate, combines effectively with azacitidine and venetoclax in acute myeloid leukemia cell lines

Abstract Background: CD123, the alpha chain of the interleukin-3 receptor (IL-3Rα), is overexpressed in multiple hematologic malignancies and found particularly highly expressed on the surface of acute myeloid leukemia (AML) cells. Furthermore, increased CD123 expression on leukemic stem cells relative to non-neoplastic hematopoietic stem cells makes CD123 an attractive tumor-associated target for an antibody-drug conjugate (ADC). BYON4413 is an ADC that consists of a novel humanized IgG1 anti-CD123 antibody site-specifically conjugated with Byondis' proprietary duocarmazine linker-drug technology, ByonZine® and ByonShieLD®. The efficacy of BYON4413 is studied as monotherapy and in combination with azacitidine and venetoclax (AZA/VEN), since the AZA/VEN regimen is the standard of care (SOC) for AML patients who are ineligible for intensive chemotherapy. Method: A panel of CD123+ and CD123- hematological cell lines and AML patient-derived bone marrow mononuclear cells (BMMCs)/peripheral blood mononuclear cells (PBMCs) were utilized in in vitro studies to determine the cytotoxic effects of BYON4413. The induction of apoptosis and cell killing by BYON4413 in a triple combination with AZA/VEN was studied in vitro using the CD123+ AML cell lines MOLM-13 and MV4-11. AML patient and cell line-derived xenograft models were used to investigate the in vivo efficacy of BYON4413. Results: In vitro studies with human AML cell lines show that BYON4413 is highly effective in eradicating CD123+ cells while having little impact on CD123- cells. These results were confirmed ex vivo with AML patient-derived BMMCs/PBMCs. BYON4413 effectively killed AML patient-derived blasts while largely sparing healthy hematopoietic cells. The triple combination of BYON4413 with AZA/VEN enhanced both apoptosis and cell killing of AML cell lines compared to the singular BYON4413 treatment. In vivo studies demonstrate that BYON4413 is remarkably efficient at reducing the tumor burden in AML patient- and cell line-derived xenograft models. Conclusions: Preclinical studies show that BYON4413 has potential to be an effective targeted therapy against CD123+ hematological malignancies such as AML. The enhanced efficacy of BYON4413 together with AZA/VEN suggests that a clinical benefit from the triple combination may be achievable and is worth further exploration. A first-in-human dose-escalation and expansion trial with BYON4413, designed to enroll R/R AML and high-risk MDS patients, is scheduled to start in Q2 2024. Citation Format: Miranda M.C. van der Lee, Tanja van Achterberg, Kaylee Hersmus, Annet Brouwers-Vos, Monique van der Vleuten, Maurien Pruis, Wendy Kappers, Gijs Verheijden, Gerwin Huls, Glenn van Wigcheren, Ruud Ubink, Aloys Sesink, Alyson MacInnes, Jan Jacob Schuringa, Wim Dokter. BYON4413, an in vivo active CD123-targeting antibody-drug conjugate, combines effectively with azacitidine and venetoclax in acute myeloid leukemia cell lines [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 3129.

Abstract 2661: Identification of a novel morphology of tertiary lymphoid structure in patients with hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade

Abstract Background: Tertiary lymphoid structures (TLS) are ectopic lymphoid follicles that arise in non-lymphoid tissue. TLS may contribute to response to immune checkpoint blockade (ICB) in solid tumors, but understanding of the life cycle of these structures, particularly the circumstances of their resolution and the functional contribution of this stage to the adaptive immune response, remains incomplete. Methods: We employed a multi-omics approach to evaluate TLS in the tumors of patients with hepatocellular carcinoma (HCC) treated with neoadjuvant ICB (n = 19) and untreated controls (n = 14). TLS density was assessed by immunohistochemistry and correlated with pathologic response, relapse free survival, and overall survival. Imaging mass cytometry was used to characterize distinct TLS morphologies in areas of tumor regression bed and viable tumor. Individual TLS from treated tumors were microdissected and characterized by T cell receptor (TCR) and immunoglobulin heavy chain (IGH) sequencing, and TCR clonotypes identified in TLS were further characterized by comparison against matched single cell RNA and TCR sequencing of peripheral blood and TIL. Results: Intratumoral TLS density was significantly increased in HCC tumors treated with neoadjuvant ICB compared to untreated controls (p = 0.05). Tumors with major or complete pathologic response to treatment had significantly higher intratumoral TLS density compared to tumors with partial pathologic response (p = 0.000246) and non-response (p = 0.0129). In the treatment group, patients with tumors in the top tertile of intratumoral TLS density had significantly longer relapse free survival (p = 0.021 by log-rank test). In the regression bed of treated tumors, an involuted TLS morphology was identified which was notable for dispersion of the B cell germinal center, retention of a T cell zone enriched for T-DC interactions, and increased expression of markers of T cell memory. In a subset of patients, immune repertoire sequencing demonstrated increased TCRβ clonality and BCR somatic hypermutation at involuted TLS. Highly expanded T cell clonotypes identified in TLS were found in the GZMK+ and GZMB+ CD8 T effector memory clusters in matched single cell data. Conclusions: These data suggest that in HCC tumors treated with neoadjuvant ICB, treatment may induce formation of intratumoral TLS, which are associated with superior pathologic response and relapse free survival. An involuted TLS morphology is identified in tumor regression bed with features consistent with dissolution of the B cell germinal center, persistent antigen presentation to T cells, and increased T cell memory formation. Taken together, these findings suggest a potential role for late-stage TLS in supporting consolidation of the intratumoral T cell repertoire after elimination of viable tumor. Citation Format: Daniel H. Shu, Won Jin Ho, Luciane T. Kagohara, Alexander Girgis, Sarah M. Shin, Ludmila Danilova, Jae W. Lee, Dimitrios N. N. Sidiropoulos, Sarah Mitchell, Kabeer Munjal, Kathryn Howe, Kayla J. Bendinelli, Hanfei Qi, Guanglan Mo, Janelle Montagne, Tamara Y. Lopez-Vidal, Qingfeng Zhu, Amanda L. Huff, Xuan Yuan, Alexei Hernandez, Erin M. Coyne, Neeha Zaidi, Daniel J. Zabransky, Logan L. Engle, Aleksandra Ogurtsova, Marina Baretti, Daniel Laheru, Jennifer N. Durham, Hao Wang, Robert Anders, Elizabeth M. Jaffee, Elana J. Fertig, Mark Yarchoan. Identification of a novel morphology of tertiary lymphoid structure in patients with hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade [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 2661.

Roles of IL-4, IL-13, and Their Receptors in Lung Cancer.

Interleukin (IL)-4 and IL-13 are the main effectors of innate lymphoid cells (ILC2) of the type 2 innate immune response, which can carry out specific signal transmission between multiple cells in the tumor immune microenvironment. IL-4 and IL-13 mediate signal transduction and regulate cellular functions in a variety of solid tumors through their shared receptor chain, the transmembrane heterodimer interleukin-4 receptor alpha/interleukin-13 receptor alpha-1 (type II IL-4 receptor). IL-4, IL-13, and their receptors can induce the formation of a variety of malignant tumors and play an important role in their progression, growth, and tumor immunity. In order to explore possible targets for lung cancer prediction and treatment, this review summarizes the characteristics and signal transduction pathways of IL-4 and IL-13, and their respective receptors, and discusses in depth their possible role in the occurrence and development of lung cancer.

Specific Targeting of Zinc Transporter LIV-1 with Immunocytokine Containing Anti-LIV-1 VHH and Human IL-2 and Evaluation of its In vitro Antitumor Activity.

Interleukin 2 (IL-2) is a vital cytokine in the induction of T and NK cell responses, the proliferation of CD8+ T cells, and the effective treatment of human cancers such as melanoma and renal cell carcinoma. However, widespread use of this cytokine is limited due to its short half-life, severe toxicity, lack of specific tumor targeting, and activation of Treg cells mediated by high-affinity interleukin-2 receptors. In this study, a tumor-targeting LIV-1 VHH-mutIL2 immunocytokine with reduced CD25 (α chain of the high-affinity IL-2 receptor) binding activity was developed to improve IL-2 half-life by decreasing its renal infiltration in comparison with wild and mutant IL-2 molecules. The recombinant immunocytokine was designed and expressed. The biological activity of the purified fusion protein was investigated in in vitro and in vivo experiments. The fusion protein represented specific binding to MCF7 (the breast cancer cell line) and more efficient cytotoxicity than wild-type IL-2 and mutant IL-2. The PK parameters of the recombinant immunocytokine were also improved in comparison to the IL-2 molecules. The observed results showed that LIV1-mIL2 immunocytokine could be considered as an effective agent in the LIV-1-targeted treatment of cancers due to its longer half-life and stronger cytotoxicity.

A191 THE NEURAL SIGNAL, CALCITONIN GENE-RELATED PEPTIDE (CGRG) ENHANCES A REGULATORY PHENOTYPE IN THE HUMAN IL-4-TREATED MACROPHAGE

Abstract Background Interlukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect in a murine model. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn’s disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, raising the issue of neural modulation of the M(IL4)s reparative function. Aims To determine if CGRP-RAMP1 signalling in human IL-4 treated macrophages enhances the anti-colitic phenotype. Methods Peripheral blood mononuclear cells from healthy volunteers (HD) and individuals with Crohn’s disease were cultured on plastic (2h, 37°C) and non-adherent cells removed. The adherent cells were cultured with recombinant hM-CSF (10 ng/ml) for 7 days. The resultant macrophages (2.5×105) were differentiated with IL-4 (48h 10 ng/mL). In other cells, CGRP (10 nM) was added 24h after IL-4 and phenotype assessed by qPCR. Rag1-/- mice were treated with M(IL4)±CGRP (1x106 i.p.): 48h later, colitis was induced by oxazolone. FITC dextran was used to evaluate epithelial barrier integrity. A wound healing assay was conducted using CaCo2 epithelial cells and conditioned medium (CM,1:2 dilution) from the different treatments. Prostaglandin D2 production was evaluated by ELISA and cyclooxygenase (COX)-1 and -2 activity inhibited using SC560 and indomethacin. Results Compared to non-treated macrophages (M(0)), M(IL4)s from HD had increased RAMP1 and CLR mRNA. M(IL4)s treated with CGRP showed enhanced expression of CD206, CCL26, RAMP1 and CCL18. When delivered systemically to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same healthy blood donor. Use of the the human colon CaCo2 cell in vitro wounding model revealed that CM from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM. Moreover, M(IL4,CGRP)s displayed increased COX-1 and PGD2. CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers. Conclusions Neuronal input is revealed as an important local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer to treat enteric inflammation. Funding Agencies CCCHelmsley Charity

Cryo-EM structure of the extracellular domain of murine Thrombopoietin Receptor in complex with Thrombopoietin

Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric Class I cytokine receptor) and initiating cell proliferation or differentiation. Here we characterise the murine Tpo:TpoR signalling complex biochemically and structurally, using cryo-electron microscopy. Tpo uses opposing surfaces to recruit two copies of receptor, forming a 1:2 complex. Although it binds to the same, membrane-distal site on both receptor chains, it does so with significantly different affinities and its highly glycosylated C-terminal domain is not required. In one receptor chain, a large insertion, unique to TpoR, forms a partially structured loop that contacts cytokine. Tpo binding induces the juxtaposition of the two receptor chains adjacent to the cell membrane. The therapeutic agent romiplostim also targets the cytokine-binding site and the characterisation presented here supports the future development of improved TpoR agonists.

Targeted siRNA delivery to lung epithelia reduces airway inflammation in a mouse model of allergic asthma

Asthma is a chronic inflammatory disease triggered by allergic reactions in the bronchia. These reactions lead to swelling of mucous membranes, hypersecretion of mucus, and bronchoconstriction, resulting in a restricted opening of the lung airway. Allergic pulmonary inflammation and airway hyperresponsiveness are induced when Th2 cytokines, such as interleukin (IL)-4 and IL-13, bind to their cognate receptors on lung epithelial cells. Specifically, IL-13 stimulates inflammation through a multi-subunit receptor, mainly the alpha chain of the IL-4 receptor (IL-4Rα), which also plays a role in IL-4 signaling. In this study, we employed a lung epithelial cell-targeting siRNA carrier composed of a rabies virus glycoprotein-derived small peptide coupled with cationic nona-arginine and trileucine before cysteine peptide (RVG9R3LC). This carrier was complexed with siRNA, enabling targeted delivery of therapeutic siRNA to IL-4Rα (siIL4Rα) expressed in lung epithelial cells within an asthma model in vivo. Our approach demonstrated efficient gene knockdown in cultured lung epithelial cells and in vivo. Furthermore, two administrations of therapeutic siIL4Rα protected the ovalbumin-sensitized and challenged asthma mouse model from airway inflammation and excessive mucus secretion. Our findings suggest that the peptide-siRNA carrier system presents a promising therapeutic approach for respiratory inflammation.Graphical abstract

Spatial transcriptomics of B cell and T cell receptors reveals lymphocyte clonal dynamics.

The spatial distribution of lymphocyte clones within tissues is critical to their development, selection, and expansion. We have developed spatial transcriptomics of variable, diversity, and joining (VDJ) sequences (Spatial VDJ), a method that maps B cell and T cell receptor sequences in human tissue sections. Spatial VDJ captures lymphocyte clones that match canonical B and T cell distributions and amplifies clonal sequences confirmed by orthogonal methods. We found spatial congruency between paired receptor chains, developed a computational framework to predict receptor pairs, and linked the expansion of distinct B cell clones to different tumor-associated gene expression programs. Spatial VDJ delineates B cell clonal diversity and lineage trajectories within their anatomical niche. Thus, Spatial VDJ captures lymphocyte spatial clonal architecture across tissues, providing a platform to harness clonal sequences for therapy.