High CD38 Research Articles

P0431 Multiparametric Assessment of IBD, SpA and IBD-SpA patients to explore disease biomarkers and response predictors

Abstract Background Immune-mediated inflammatory diseases (IMID) can coexist within the same patient and their pathogenesis could overlap. Inflammatory bowel disease (IBD) [Crohn’s disease (CD) and ulcerative colitis (UC)], and spondylarthritis (SpA), often associate, but disease biomarkers are not fully characterized. This project aims to investigate a possible molecular signature of disease and predictor markers of response Methods We performed an observational prospective study, enrolling consecutive IMID patients (pts) starting a target therapy. We principally focused on 3 cohorts: IBD, SpA and IBD-SpA. At baseline, we performed a test for serum cytokines and also collected intestinal biopsies, to stain for immune cell markers. Clinical intestinal and articular response (CR) was assessed at 4, 12 and 24 months. CR was defined using clinical scores: Patient Reported Ouctome-2 (PRO-2) for UC and CD, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Ankylosing Spondylitis Disease Activity Score-C-Reactive Protein (ASDAS-CRP) for SpA Results We enrolled 134 pts (59 IBD, 75 SpA). All the SpA pts were seronegative with prevalent peripheric articular involvement. In the IBD cohort, 48% of the pts were naïve to advanced therapies, while the remaining part had been exposed to biologics. 34% pts were on concomitant steroid therapy at baseline. At baseline, we found lower serum levels of IL-6, IL-12/23, IL-7, IL-10, IL-33, IL-35 and IL-37 in IBD pts compared to SpA pts and that levels presented a direct or inverse correlation with the clinical scores (figure). In the IBD-SpA group, compared to the IBD group, we found higher levels of IL-6(p=0.001), sTREM1(p=0.027), IL10(p=0.002) and IL-37(p=0.016). According to PRO-2, 70% of pts were in steroid-free clinical remission at 12 months, and 81.8% at 24 months. Considering CR at 12 months, statistically significant differences were found in the groups of responders versus no responders. In the IBD cohort we found that 100% of responders, had low levels of IL-8(<8.6 pg/mL) and low CD117 cell count in intestinal tissue at baseline; besides, 80% of responders had high CD20 count in intestinal tissue at baseline. Kaplan-Meier curves for achieving CR at 24 months, in the entire cohort of pts, showed no statistically significant differences stratifying pts on demographic characteristics and type of disease Conclusion In IBD pts, we found results which could suggest a different systemic inflammation signature between IBD and SpA pts and the presence of possible biomarkers which, if confirmed, could help the physician in the early and non-invasive identification of the disease phenotype at diagnosis and response to therapies

The CD123 antibody-drug conjugate pivekimab sunirine exerts profound activity in preclinical models of pediatric acute lymphoblastic leukemia.

Antibody-drug conjugates (ADCs) combining monoclonal antibodies with cytotoxic payloads are a rapidly emerging class of immune-based therapeutics with the potential to improve the treatment of cancer, including children with relapse/refractory acute lymphoblastic leukemia (ALL). CD123, the α subunit of the interleukin-3 receptor, is overexpressed in ALL and is a potential therapeutic target. Here, we show that pivekimab sunirine (PVEK), a recently developed ADC comprising the CD123-targeting antibody, G4723A, and the cytotoxic payload, DGN549, was highly effective in vivo against a large panel of pediatric ALL patient-derived xenograft (PDX) models (n = 39). PVEK administered once weekly for 3 weeks resulted in a median event-free survival (EFS) of 57.2 days across all PDXs. CD123 mRNA and protein expression was significantly higher in B-lineage (n = 65) compared with T-lineage (n = 25) ALL PDXs (p < 0.0001), and mice engrafted with B-lineage PDXs achieved significantly longer EFS than those engrafted with T-lineage PDXs (p < 0.0001). PVEK treatment also resulted in significant clearance of human leukemia cells in hematolymphoid organs in mice engrafted with B-ALL PDXs. Notably, our results showed no direct correlation between CD123 expression and mouse EFS, indicating that CD123 is necessary but not sufficient for in vivo PVEK activity. Importantly, a PDX with very high CD123 cell surface expression but resistant to in vivo PVEK treatment, failed to internalize the G4723A antibody while remaining sensitive to the PVEK payload, DGN549, suggesting a novel mechanism of resistance. In conclusion, PVEK was highly effective against a large panel of B-ALL PDXs supporting its clinical translation for B-lineage pediatric ALL.

Unraveling the pathophysiology of type 2 diabetes with a new selectively bred animal model, the Oikawa-Nagao mouse.

Type 2 diabetes (T2D) is a polygenic disease, and the development of animal models by selective breeding is crucial for understanding its etiology, pathophysiology, complications, and treatments. We recently developed a new T2D model, the Oikawa-Nagao (ON) mouse, by selectively breeding mice with inferior glucose tolerance [diabetes-prone (ON mouse DP®; ON-DP) strain] and superior glucose tolerance [diabetes-resistant (ON mouse DR®; ON-DR) strain] on a high-fat diet. ON-DP mice are predisposed to develop diabetes and obesity after being fed a high-fat diet, compared to ON-DR mice. These phenotypes provide valuable insights into the genetic and environmental interactions for the etiology of T2D. Our studies revealed that the emergence of these phenotypes is associated with novel pathophysiological mechanisms, such as low insulin secretion capacity associated with high CD36 expression in pancreatic β-cells and hypoleptinemia preceding obesity due to low leptin secretion capacity in adipocytes. In addition, ON-DP mice fed an atherogenic diet exhibit accelerated atherosclerosis, likely related to blood glucose fluctuations. These findings provide new perspectives on the pathogenesis of T2D and suggest potential prevention and treatment strategies. This review will present the development strategy of the ON mouse strain, representative metabolic phenotypes, and discuss the mechanisms driving these traits, and explore their relevance to human T2D and obesity.

CD38-specific immunoPET imaging for multiple myeloma diagnosis and therapeutic monitoring: preclinical and first-in-human studies.

CD38 is a glycoprotein highly specific to multiple myeloma (MM). Therapeutics using antibodies targeting CD38 have shown promising efficacy. However, the efficient stratification of patients who may benefit from daratumumab (Dara) therapy and timely monitoring of therapeutic responses remain significant clinical challenges. To address these issues, we developed a novel nanobody-based PET tracer, [68Ga]Ga-TOHP-CD3813, which exhibits rapid clearance from the blood and rapid accumulation in targeted tumor lesions, facilitating the detection of CD38 expression in murine models of MM and lymphoma. Furthermore, we conducted the world's first-in-human trials using CD38-targeted nanobodies to validate and assess the clinical imaging effectiveness of [68Ga]Ga-TOHP-CD3813 in guiding cancer immunotherapy. We prepared a new PET imaging probe based on a CD38-targeted nanobody CD3813, [68Ga]Ga-TOHP-CD3813, via the site-specific radiolabeling for noninvasive PET imaging of CD38 expression. [68Ga]Ga-TOHP-CD3813 was assessed for its affinity and specificity to CD38 and its ability to image CD38 expression in MM and lymphoma xenograft models. Biodistribution and the relationship between tumor uptake and CD38 expression were evaluated. Subsequently, we conducted a translational PET imaging of 2 MM patients using [68Ga]Ga-TOHP-CD3813, while compared with [18F]FDG PET/CT head-to-head. Dosimetry was also calculated based on the animal data. TOHP-CD3813 retained a high affinity for CD38 with a KD of 0.0826 nmol/L. [68Ga]Ga-TOHP-CD3813 was successfully synthesized at room temperature within 10min, exhibiting optimal radiochemical properties. Preclinical assessments revealed rapid blood clearance, high CD38 affinity, and significant uptake in CD38-positive xenograft mouse models (6.50 ± 2.69%ID/g). [68Ga]Ga-TOHP-CD3813 showed pronounced accumulation in the kidneys and bladder, with moderate liver uptake, indicating its potential as a viable clinical PET radiotracer for diagnosing MM. Additionally, in first-in-human trials, [68Ga]Ga-TOHP-CD3813 PET/CT provides a substantial improvement over [18F]FDG PET/CT for the visualization of MM. [68Ga]Ga-TOHP-CD3813, with its high affinity, specificity, and robust imaging capabilities, rapidly and specifically accumulates in tumors with high CD38 expression, offering a significant advantage over [18F]FDG PET/CT for visualizing MM and enabling same-day PET imaging. Initial human trial results are promising, suggesting its potential as a companion diagnostic tool for optimizing CD38-targeted treatments in tumors. Ongoing larger trials aim to further confirm these findings.

Proteomic profiling identifies classic Hodgkin lymphoma patients at risk of bleomycin pulmonary toxicity

Advances in treating classic Hodgkin lymphoma (cHL) have improved cure rates, with overall survival exceeding 80%, resulting in a growing population of survivors at risk of long-term complications, particularly cardiac and pulmonary toxicity. Bleomycin, a key component of combination chemotherapy, is associated with bleomycin-induced pulmonary toxicity (BPT). Using label-free quantification nano liquid chromatography-tandem mass spectrometry, protein expression in diagnostic lymphoma samples from patients with and without BPT was compared. Results showed differential protein expression and disrupted cellular pathways, suggesting biological differences in BPT risk. Immunohistochemical analysis revealed higher expression of JAK3, BID, and MMP9, and lower expression of CD20, TPD52, and PIK3R4 in patients with BPT. High BID and low CD20 expression were associated with inferior overall survival, while high BID and low JAK3 and CD20 expression were linked to poorer progression-free survival. These findings highlight altered protein profiles in pretreatment cHL biopsies associated with BPT development.

Expanded CD8+CD38+ T Cell Population in Patients With Systemic Lupus Erythematosus Is Linked to Increased Infection Rates: A Prospective Study.

One of the leading causes of morbidity and mortality among patients with systemic lupus erythematosus (SLE) is infections. The expression of the ectonucleotidase CD38 on the surface of CD8+ T cells has been linked to compromised cytotoxic function. The aim of this prospective study was to assess whether the presence of CD8+CD38+ in the peripheral blood of patients with SLE can serve as a biomarker for infectious complications. A cohort of 80 patients with SLE were recruited over 18 months. The rate of clinically significant infections and presence of CD8+CD38+ T cells in the peripheral blood were monitored at each clinic visit. The patients were classified into high CD38+ and low CD38+ CD8+ T cells using flow cytometry and a previously established cutoff rate of 28.4%. A total of 20 infections were registered over the study period. We observed that the patients with an expanded CD8+CD38+ T cell population in the peripheral blood had a higher rate of recurrent infections and a higher likelihood of infection compared with patients with a low CD8+CD38+ T cell population. The levels of CD38 in CD8+ T cells remained stable over time in the studied subjects. High levels of CD8+CD38+ T cells in the peripheral blood of patients with SLE identify a subgroup prone to infections for whom proper clinical measures should be applied.

Genetic and therapeutic heterogeneity shape the baseline and longitudinal immune ecosystem of ovarian clear cell carcinoma

BackgroundOvarian clear cell carcinoma (OCCC) is a rare and chemo-resistant subtype of ovarian cancer. While immunotherapy has demonstrated effectiveness in some OCCC cases, the mechanisms for heterogeneous immunoreactivity and potential...

Single-cell landscape of innate and acquired drug resistance in acute myeloid leukemia

Deep single-cell multi-omic profiling offers a promising approach to understand and overcome drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML). Here, we combine single-cell ex vivo drug profiling (pharmacoscopy) with single-cell and bulk DNA, RNA, and protein analyses, alongside clinical data from 21 rrAML patients. Unsupervised data integration reveals reduced ex vivo response to the Bcl-2 inhibitor venetoclax (VEN) in patients treated with both a hypomethylating agent (HMA) and VEN, compared to those pre-exposed to chemotherapy or HMA alone. Integrative analysis identifies both known and unreported mechanisms of innate and treatment-related VEN resistance and suggests alternative treatments, like targeting increased proliferation with the PLK inhibitor volasertib. Additionally, high CD36 expression in VEN-resistant blasts associates with sensitivity to CD36-targeted antibody treatment ex vivo. This study demonstrates how single-cell multi-omic profiling can uncover drug resistance mechanisms and treatment vulnerabilities, providing a valuable resource for future AML research.

Multidimensional profiling of human T cells reveals high CD38 expression, marking recent thymic emigrants and age-related naive T cell remodeling

Thymic involution is a key factor in human immune aging, leading to reduced thymic output and a decline in recent thymic emigrant (RTE) naive Tcells in circulation. Currently, the precise definition of human RTEs and their corresponding cell surface markers lacks clarity. Analysis of single-cell RNA-seq/ATAC-seq data distinguished RTEs by the expression of SOX4, IKZF2, and TOX and CD38 protein, whereby surface CD38hi expression universally identified CD8+ and CD4+ RTEs. We further determined the dynamics of RTEs and mature cells in a cohort of 158 individuals, including age-associated transcriptional reprogramming and shifts in cytokine production. Spectral cytometry profiling revealed two axes of aging common to naive CD8+ and CD4+ Tcells: (1) a decrease in CD38++ cells (RTEs) and (2) an increase in CXCR3hi cells. Identification of RTEs enables direct assessment of thymic health. Furthermore, resolving the dynamics of naive Tcell remodeling yields insight into vaccination and infection responsiveness throughout aging.

Post-transplantation monitoring and quantitation of microparticles in allogeneic hematopoietic cell transplantation

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HCT) represents a curative treatment for various blood-related disorders, including hematological malignancies and genetic disorders. The success of this procedure hinges on the efficacy of the conditioning regimen and the graft's ability to engraft and function properly. Microparticles (MPs), small vesicles produced from stimulated, apoptotic, or activated cells, are involved in both physiological and pathological processes. However, the impact of MPs on allo-HCT remains poorly understood. ObjectivesThis study aimed to investigate the presence of MPs from different cell types in grafts and patient plasma after allo-HCT, as well as their association with various parameters. We measured MPs from CD34+, CD56+, CD3+, CD19+, and CD33+ cells in grafts and patient plasma from day 0 to day 60 after transplantation. Methods224 blood samples were collected from 19 consecutive allo –HCT recipients at 0, +4, +14,+30 and + 60 day as well as from their grafts. MPs isolated from the plasma and quantified by flow cytometry analysis. ResultsMP levels varied over time. Notably, CD34+ MP levels were linked to both early and late engraftment of neutrophils and platelets. Furthermore, grafts with high CD34+ and CD56+ MP levels in patient plasma on days 0 and + 4 were associated with late engraftment, whereas high CD33+ MP levels in both graft and patient plasma on day +4 were associated with early engraftment. Conditioning regimen affected CD19+ MP levels at day +14, and the number of CD34+, CD56+, and CD19+ MPs 30 days after transplantation was correlated with acute graft-versus-host disease. ConclusionThese findings suggest that MPs derived from hematopoietic cells may play a significant role in the clinical course of patients following allo-HCT.

Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer

BackgroundMajor histocompatibility complex class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and...

CD19+CD73+ B cells infiltration indicates poor prognosis and unfavorable responses to immunotherapy in gastric cancer

ObjectivesCluster of Differentiation 73 (CD73) is expressed on immune cells and plays a significant role in tumor inhibition by suppressing antitumor immunity. The objectives of this study were to explore the expression and functional mechanisms of CD73 on B cells in patients with gastric cancer (GC). MethodsThe prognostic significance of CD19+CD73+ B cells was evaluated in 390 GC patients through dual immunohistochemistry staining. Flow cytometry was employed to analyze the phenotype of the CD19 subpopulation using fresh tumor and non-tumor tissue samples from 8 GC patients. A bioinformatics analysis of CD19+CD73+ B cells was also performed within the scRNA-seq cohort, and the CD19+ B cell subtype was assessed using multiple immunofluorescence staining. ResultsThe infiltration of CD19+CD73+ B cells was observed to be elevated in gastric cancer (GC) tissue compared to normal tissues. A strong correlation was observed between high CD19+CD73+ B cell infiltration, poor overall survival, and diminished responsiveness to neoadjuvant immunotherapy in GC. These cells emerged as a novel subset of regulatory B cells (Bregs) linked to adenosine metabolism and the exhaustion of CD8+ T cells. The CD19+CD73+ B cells also correlated with the production of immunosuppressive cytokines IL-10 and TGFB1. Further analysis indicated an association between CD19+CD73+ B cells and advanced-stage GC. ConclusionsThe presence of CD19+CD73+ B cells in GC may serve as a prognostic indicator for clinical outcomes and a predictive marker for poor responsiveness to neoadjuvant immunotherapy. The correlation between the presence of CD19+CD73+ B cells and CD8+ T cell exhaustion, along with immunosuppression, highlights the tumor-promoting function of these cells.

Neu5Gc-mediated high-affinity interaction is dispensable for CD22 cis-ligands to regulate B cell signaling

CD22 (also known as Siglec-2) is an inhibitory receptor expressed in B cells. CD22 specifically recognizes α2,6 sialic acid and interacts with α2,6 sialylated membrane proteins expressed on the same cell (cis-ligands) and those derived from outside of the cell (trans-ligands). Previously, CD22 cis-ligands were shown to regulate the activity of CD22, thereby regulating both BCR ligation-induced signaling and low-level "tonic" signaling in the absence of BCR ligation that regulates the survival and differentiation of B cells. Mouse CD22 prefers Neu5Gc to Neu5Ac thereby binding to α2,6-linked Neu5Gc with high affinity. Although human CD22 binds to a distinct α2,6 sialylated glycan with high affinity, expression of high-affinity ligands is regulated in a conserved and stringent manner. However, how high- versus low-affinity CD22 ligands regulate B cells is poorly understood. Here we demonstrate that the interaction of CD22 with the endogenous ligands enhances BCR ligation-induced signaling but reduces tonic signaling in Cmah-/- mouse B cells deficient in Neu5Gc as well as wild-type B cells. Moreover, Cmah-/- B cells do not show alterations in the phenotypes correlated to tonic signaling. These results indicate that low-affinity interaction of the CD22 cis-ligands with CD22 is sufficient for the regulation of B cell signaling, and suggest that expression of high-affinity CD22 ligands might be involved in the regulation of B cells by competing for the binding of CD22 with exogenous trans-ligands of CD22.

Spatial tertiary lymphoid structures imply response to anti-PD-1 plus anlotinib in advanced non-small cell lung cancer.

Despite breakthroughs of immunotherapy synergistically combined with blockade of vascular endothelial growth factor receptor, several patients with advanced non-small cell lung cancer (NSCLC) experience non-response or followed relapse. Organized lymphoid aggregates, termed tertiary lymphoid structures (TLSs), are found to be associated with improved response to immunotherapy. Here, we explore the landscapes of TLSs in tumour tissues from a real-world retrospective study. Our investigation showed that with a median follow-up of 11.2 months, the ORR was 28.6% (18/63, 95% CI 17.9-41.3) and the median PFS was 6.1 (95% CI 5.5-6.6) months in NSCLC patients treated with PD-1 blockade combined with anlotinib. By multiplex immunofluorescence (mIF) analysis, spatially, more TLSs and high CD20+ B-cell ratio in TLSs were associated with higher ORR. High density of intratumoral CD8+ T cells showed better ORR and PFS. The numbers of CD8+ T cells with a distance within 20 μm and 20-50 μm between tumour cells were higher in responders than non-responders. But responders had significantly higher TLSs within 20 μm rather than within 20-50 μm of tumour cells than non-responders. The inflamed immunophenotyping occupied higher proportions in responders and was associated with better PFS. Besides, tumour cells in non-responders were found more temporal cell-in-cell structures than responders, which could protect inner cells from T-cell attacks. Taken together, landscape of TLSs and proximity architecture may imply superior responses to PD-1 blockade combined with anlotinib for patients with advanced non-small cell lung cancer.

Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche.

CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche.

Development and validation of a prognostic immunoinflammatory index for patients with gastric cancer

BACKGROUND Studies have demonstrated the influence of immunity and inflammation on the development of tumors. Although single biomarkers of immunity and inflammation have been shown to be clinically predictive, the use of biomarkers integrating both to predict prognosis in patients with gastric cancer remains to be investigated. AIM To investigate the prognostic and clinical significance of inflammatory biomarkers and lymphocytes in patients undergoing surgical treatment for gastric cancer. METHODS Univariate COX regression analysis was performed to identify potential prognostic factors for patients with gastric cancer undergoing surgical treatment. Least absolute shrinkage and selection operator-COX (LASSO-COX) regression analysis was performed to integrate these factors and formulate a new prognostic immunoinflammatory index (PII). The correlation between PII and clinical characteristics was statistically analyzed. Nomograms incorporating the PII score were devised and validated based on the time-dependent area under the curve and decision curve analysis. RESULTS Patients exhibiting elevated neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and systemic immune inflammatory index displayed inferior progression-free survival (PFS) and overall survival (OS). Conversely, low levels of CD3(+), CD3(+) CD8(+), CD4(+)CD8(+), and CD3(+)CD16(+)CD56(+) T lymphocytes were associated with improved PFS and OS, while high CD19(+) T lymphocyte levels were linked to worse PFS and OS. The PII score demonstrated associations with tumor characteristics (primary tumor site and tumor size), establishing itself as an independent prognostic factor for both PFS and OS. Time-dependent area under the curve and decision curve analysis affirmed the effectiveness of the PII-based nomogram as a robust prognostic predictive model. CONCLUSION PII may be a reliable predictor of prognosis in patients with gastric cancer undergoing surgical treatment, and it offers insights into cancer-related immune-inflammatory responses, with potential significance in clinical practice.

Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer.

MHC class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. We used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFNγ-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis. Spatial quantitation of tumor cell MHC-I expression revealed intra- and inter-tumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (DFS) (HR=0.58, p=0.064) and overall survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1×10-3). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3+CD8+ T cells and CD3-CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single cell neighborhood profiles to delineate the cellular environments of IFNγ+/- NK cells and T cells. We discovered that both IFNγ+ NK and CD8 T cells were more frequently associated with other IFNγ+ lymphocytes in comparison to IFNγ- NK cells and CD8 T cells (p<1×10-30). Moreover, IFNγ+ lymphocytes were most often found clustered near MHC-I+ tumor cells. Tumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Co-association of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent co-localization of IFNγ+ NK cells with other IFNγ+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated.

Multiplex analysis of the immune environment before and after neoadjuvant durvalumab as a prognostic factor in resectable non-small cell lung cancer (NSCLC) in the IFCT-1601 IONESCO phase 2 trial.

8022 Background: Immune checkpoint inhibitors (ICI) are currently included in the peri-operative standard of care for NSCLC with the objective of a curative strategy. In early stages of NSCLC, biomarkers predicting ICI efficacy should be more stringent than PD-L1 tumoral expression in order to improve the benefit-toxicity ratio. We deeply analyzed the tumor microenvironment of patients included in the IONESCO multicenter phase 2 trial (stage IB &gt; 4cm–IIIA, non N2 resectable NSCLC). Diagnostic biopsies and surgical resection specimen after 3 cycles of Durvalumab were available. We previously showed that the % of residual viable tumor cells (RVT) was associated with disease free survival (DFS) and overall survival (OS). PD-L1 tumor positive score was not correlated to RVT nor survival. Methods: 46 patients were included in the IONESCO trial. Among them, diagnostic biopsy (n = 32), surgical resection specimen post durvalumab (n = 39) and paired tumor samples (n = 31) were analyzed. Immune environment was assessed using 7 quantitative 7-plex immunofluorescence panels generating 349 different cellular phenotypes in 3 different compartments (whole tumor, intra cytokeratin and stroma), focusing on T and B lymphocytes, macrophages, immune checkpoint, NK cells, apoptosis, innate and adaptive immunity, dendritic cells. Densities of cells were quantified using Fluorescent Multiplex immunohistochemistry performed on Leica Bond RX, using OpalTM technology. A fisher's exact test or chi2 test was used for demographics variables and RVT. HRs and 95% CIs were estimated using a Cox model. NCT number: NCT03030131. Results: With median follow-up of 4.5 years, 16/31 patients had a disease recurrence. Histology, sex, stage and survival were significantly associated with intra-tumor densities of CD3, CD8, PD1, TIM3, and CD163 cells in the diagnostic biopsy, and with the delta of CD3, FoxP3, CD4, CD163PD1, TIM3, PDL1 on immune cells and TIM3PDL1 cells, between biopsy and surgical specimen. DFS was significantly associated with high density of CD8+TIM3+ in biopsies (HR = 0.25 [0.09-0.71], p = 0.0092) and with the high density of CD20+ cells in surgical specimen (HR = 0.36 [0.13-0.97], p = 0.04). The RVT was significantly associated with CK+Caspase3- cells and CK+PDL1+ cells in the surgical specimen. No biomarker was associated with OS. Conclusions: Multiparameter analysis of the immune NSCLC environment of patients treated with neoadjuvant anti-PDL1 allows identification of markers associated with clinical, pathological parameters and DFS. Our findings highlight the substantial impact of high CD8+TIM3+ cell density pre-durvalumab and of high CD20+ cell density post-durvalumab on DFS. These findings deserve to be assessed in patients treated with neoadjuvant combined immunotherapy chemotherapy. Clinical trial information: NCT03030131 .

Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing leukemia cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion.

Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approximately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. CD20 express on 30 to 50% of B-ALL, which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy. Adding Rituximab to chemotherapy protocol had been demonstrated to improve the outcome for CD20-positive ALL. However, rare study explored the influence of Rituximab combined with CAR-T therapy. We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T therapy, all of whom had failed multiple lines of therapy. Before CAR-T infusion, we administered Rituximab to 10 patients with high CD20 expression at a dose of 375mg/m2 for 1 day. Meanwhile, we selected 10 patients with the comparable features who underwent CAR-T treatment without Rituximab in the same period as the control group. In vitro, the surface molecule expression and killing of CAR-T post Rituximab-treated B-ALL cells co-incubated with CAR-T cells were detected by flow cytometry. The median follow-up of Rituximab and Control groups were 29.27 and 9.83 months. We found that adding Rituximab may confer a favorable prognosis compared with Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates both were longer in the Rituximab group (90% vs. 26.7%, p=0.0342; 41.7% vs. 25%, p=0.308). In vitro, we observed that Rituximab-treated tumour cells are more sensitive to CAR-T killing and a broad range of cytokines and chemokines were produced when Rituximab-treated Nalm-6 cells co-cultured with 19-22CAR-T cells, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). To investigate whether Rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells repeatedly in vitro with Rituximab-treated Nalm-6 to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of exhaustion molecules (LAG-3, PD-1, TIM-3) on CAR-T cells were significantly lower in the Rituximab group than in the Control group. Rituximab combined with CAR-T therapy is effective for improving the long-term prognosis of B-ALL patients who have failed multiple lines of therapy. In vitro, we observed that rituximab potentially improves CAR-T efficacy by sensitizing ALL to CART-mediated cytotoxicity and reducing CAR-T exhaustion.

Abstract LB129: Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies

Abstract CD38 and CD47 are co-expressed in various hematologic malignancies including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), T-cell ALL and B-cell chronic lymphocytic leukemia (CLL). We present, herein, several CD38 antibody - CD47 ligand trap bispecific antibodies (BsAbs) in an ADCC-enhanced IgG1 form that harbor high CD38 binding affinity, low CD47 binding affinity, along with negligible affinity to red blood cells (RBCs) and no induction of RBCs agglutination. Using flow cytometry and Biolayer interferometry (BLI), we confirmed that our BsAbs can simultaneously bind to both CD38 and CD47, and that binding to either antigen first does not prevent binding to the other antigen subsequently. On CD47+/CD38low Jurkat (T leukemic) cells, our BsAbs reveal less blocking effect on the CD47/SIRPα interaction than a monospecific CD47 ligand trap molecule based on which our BsAbs were designed; conversely, on CD38/CD47 double-positive Reh (pro-B ALL) cells, our BsAbs reveal 200-500-fold greater blocking effect on the CD47/SIRPα interaction than the same monospecific CD47 ligand trap molecule, indicating that the inhibition of CD47/SIRPα by our BsAbs is depended on the engagement of CD38 on tumor cells. On CD47+/CD38+ Daudi (B NHL) lymphoma cells and L-1236 (B Hodgkin) lymphoma cells, four of our BsAbs inhibited CD38 enzymatic activity, which is responsible for immune-suppressive adenosine production in tumor microenvironment. Our BsAbs presented potent dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against CD47+/CD38+ Raji (B NHL) and NCI-H929 (B plasmacytoma myeloma) cells, while revealing negligible ADCC activity against CD47+/CD38− cells, indicating low off-tumor toxicity. Likewise, our BsAbs also harbor potent antibody-dependent cellular phagocytosis (ADCP) activity against Raji NHL cells, varied degree of complement-dependent cytotoxicity (CDC) activity and direct apoptosis induction against Daudi NHL cells. In a mouse xenograft model harboring NCI-H929 multiple myeloma, our BsAbs produced more potent tumor growth inhibition, than monospecific anti-CD47 ligand trap, anti-CD38 (isatuximab) or combination of isatuximab and anti-CD47 ligand trap, at the same molecular dose per body weight. Notably, clone IMM5605-12C10 eradicated all tumors and achieved 100% complete response rate in a group of six mice. In summary, simultaneous targeting CD38 and CD47 using our CD38 antibody - CD47 ligand trap bispecific design in an ADCC-enhanced IgG1 form presents a novel multimodal approach for treating hematologic malignancies that are resistant to anti-CD38 antibodies. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Yanan Yang, Huiqin Guo, Dandan Liu, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies [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 LB129.