Surface CD44 Research Articles

Iberdomide Enhances Dara Mediated Cytotoxicity through Upregulation of CDC Activity and Elevated NK Cell Mediated ADCC

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with current treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent anti-tumor activity and immunomodulatory effects. Clinical studies utilizing the combination of IMiD agents and daratumumab (DARA), an anti-CD38 monoclonal antibody, have shown improved patient outcomes for MM. However, patients ultimately relapse demonstrating the need for novel therapies. Iberdomide (IBER) is a novel cereblon E3 ligase modulator. It promotes faster and deeper degradation of the substrate targets compared to IMiDs, resulting in enhanced anti-proliferative, pro-apoptotic and immune stimulatory activity. In early clinical studies, IBER has been shown to overcome resistance to IMiDs and in the ongoing Phase 3 EXCALIBER study (NCT04975997) is being explored for treatment of newly diagnosed (ND) and relapsed/refractory (RR) MM in combination with DARA and dexamethasone. Here, we investigate the potential mechanisms of synergy between IBER and DARA in preclinical models. Methods: Complement dependent cytotoxicity (CDC) of DARA was assessed with 15% human serum for human complement components and assays were performed by titrating either single agent or combinations in a tritiated thymidine assay. Antibody dependent cellular cytotoxicity (ADCC) was performed by co-culturing MM cells with varying expression levels of CD38 and effector cells and assessing target cell viability with annexin-v/topro-3 flow. A humanized PBMC adoptive transfer mouse model, utilizing human PBMC engraftment 14 days prior to implantation, was used to assess in vivo activity of IBER, DARA and IBER + DARA. Results: In CDC assays, we observed variable sensitivity of MM cells to complement-specific CDC in the presence of DARA (0.1-1 µg/ml), regardless of cell surface CD38 expression. Notably, IBER treatment resulted in upregulation of CD38 surface expression in 60% of MM cell lines tested. LP1 and MOLP8 displayed susceptibility to DARA-mediated CDC. DARA showed a dose-dependent increase in CDC activity in both cell lines. Notably, IBER+DARA also showed ~35% more synergistic CDC activity in both cell lines, than the single agents. In ADCC assays, we observed pretreatment of PBMC with IBER (vs. DMSO) significantly increased DARA mediated ADCC in H929 and LP-1 cells. To confirm that this effect was directed by NK cells, we cultured NK cells for 24 hr in supernatants from stimulated PBMC in presence of IBER prior to co-culture with MM cells. We noted that IBER-treated PBMC supernatant significantly enhanced the NK ADCC activity of DARA suggesting that IBER enhancement of cytokine secretion from PBMC increases NK cell mediated ADCC. Orthogonally, we pre-treated MM cells with IBER or DMSO prior to co-culture in ADCC assay. Notably, IBER treatment of MM cells, without treating immune cells, also increased DARA-mediated ADCC, indicating that IBER directly increases sensitivity to DARA-directed ADCC. To investigate the MOA of IBER-DARA synergy in ADCC, we analyzed gene expression and cell surface changes of a panel of NK-specific receptor ligands on MM cells following treatment with IBER. We observed IBER-induced upregulation of MICA mRNA levels (~2x) and surface expression (~40%) in MM target cells. We used RNAi to knock down Ikaros, an IBER substrate, which resulted in increased MICA expression (~3x) in MM cells. Furthermore, we observed that RNAi knock down of MICA in MM cells resulted in a significant decrease (60%, p<0.0001) in MM cell killing by NK cells, suggesting IBER-enhanced sensitivity of MM cells to NK-cell killing was in part, due to the upregulation of MICA through degradation of Ikaros. Lastly, we examined the anti-MM effects of IBER in a humanized xenograft model with a lenalidomide resistant cell line. Longitudinal analysis identified persistence of human PBMC engraftment and activation of immune subsets (NK cells and T-cells) that were increased upon IBER treatments, consistent with clinical observations. The combination of IBER/DARA resulted in ~22% tumor growth inhibition (TGI) in comparison to single agents, DARA and IBER and ~37% TGI in comparison to vehicle. Conclusion: Taken together, these data demonstrate that IBER and DARA work synergistically in preclinical models primarily through enhancement of both ADCC and CDC. This combination has the potential to improve outcomes in MM.

Resistance to CD38 Antigen Targeted Therapy in Multiple Myeloma May Result from Underrecognized Chromosome 4 Gene Deletions

Anti-CD38 monoclonal antibodies (mAbs), daratumumab (dara) and isatuximab (isa), are approved to treat multiple myeloma (MM) in the first line and relapsed/refractory settings. Resistance portends a poor prognosis, yet underlying mechanisms have not been well characterized. Despite generally high CD38 expression on most malignant plasma cells (PCs), antigen density may impact the clinical efficacy of targeted therapy. Unfortunately, therapeutic anti-CD38 mAbs interfere with detection and quantification of CD38 surface level by most conventional clinical flow cytometry assays, limiting characterization of CD38 antigen escape. Tumor cell evasion of antigen specific therapeutics can occur with target loss or downmodulation, a phenomenon conventionally referred to as antigen escape. To elucidate mechanisms of CD38 antigen escape, we evaluated MM patients treated with anti-CD38 mAbs who underwent a marrow evaluation between 1/2017 and 10/2022 at a single institution. Eighty-one of 161 (50%) patients had become clinically refractory over their course. Among 82 (51%) patients with ≥1 chromosomal genomic array test (CGAT) performed, 10 (12%) were found to harbor a CD38 deletion ( Table). With a CGAT coverage of 31%, the estimated prevalence of CD38 deletions was 39%. The median interval from diagnosis to CD38 deletion was calculated as 6.7 years (IQR 2.8 to 11.6) for CD38-deleted patients and estimated as 12.9 years (95% CI 12 to NA; Figure) for all patients. The presence of a CD38 deletion was associated with an increasing number of chromosomal abnormalities on concurrent FISH (OR 2.00, 95% CI 1.33 to 3.29, p=0.002). Specific FISH and flow cytometry findings occurred in conjunction with a CD38 deletion. In 50% of patients, flow cytometry detected a bimodal distribution of CD38 fluorescence intensity, reflecting two populations of malignant plasma cells with distinct CD38 surface levels. No patient showed a uniform malignant plasma cell population with a normal surface CD38 level. When the CD38 (4p15.32) deletion was identified in the context of a large 4p deletion spanning the FGFR3 locus, the FGFR3 (4p16) probe could detect the relevant loss of 4p. This was seen in 6 of 8 patients with a concurrent FGFR3 deletion by CGAT. The proportion of clones harboring a CD38 deletion could be quantified by FISH over time when testing was performed on a CD138-enriched sample. Low-level CD38 deletions (≤25%) did not confer resistance to dara-containing multi-drug regimens. In two cases where such deletions were present at diagnosis, disease control is ongoing in response to dara-Pd and dara-Rd. We suggest that acquired CD38 gene deletions appear to be common, the proportion of patients harboring them increases over time irrespective of prior exposure to CD38 mAbs, and their presence facilitates tumor antigen escape. We demonstrate that CGAT can identify CD38 deletions. Conventional clinical flow cytometry assays are unable to quantify the CD38 surface level due to interference from anti-CD38 mAbs. However, we propose that the finding of a bimodal distribution of CD38 fluorescence by flow cytometry could serve as an initial screen to identify at-risk patients warranting confirmatory CD38 mutational analysis. Following characterization of a deletion, FISH could be used to monitor the clonal level longitudinally. Our suggestion that the presence of a CD38 deletion in >25% of malignant PC clones predicts for reduced CD38 mAb efficacy requires prospective validation.

Xanthium strumarium seed extract boosts osteogenesis in human dental pulp stem cell model

BackgroundIn traditional medicine, Xanthium strumarium is used as an anti-inflammatory and anti-arthritic plant-based medicine. Human Dental Pulp Stem Cells (hDPSCs) are an ideal in vitro model for drug and bioactive compound screening. This study assessed the potential of X. strumarium aqueous extract on hDPSCs differentiation towards the osteogenic lineage. Materials and methodsHDPSCs were isolated and cultured by explant method and characterized by surface marker expression, Colony Forming units fibroblasts (CFU-F), Population Doubling time (PDT), and tri-lineage differentiation. X. strumarium aqueous seed extract (XSE) was prepared and its cytotoxic effect on hDPSCs was examined by MTT assay. The effect of XSE on hDPSC differentiation into osteocytes was investigated by biochemical staining and gene expression. ResultsThe hDPSCs were positive for CD73, CD90, and CD105 and negative for CD34, CD45, and HLA-DR surface markers. The cells had a colony-forming ability with a PDT of 44.91 h. The hDPSCs differentiated into osteocytes, chondrocytes, and adipocytes. The XSE concentration of 15 μg/ml had a significant increase in hDPSC viability. Alizarin Red S staining revealed that XSE treatment enhanced calcium accumulation and matrix mineralization in hDPSCs. XSE treatment also increased osteonectin and IL-6 transcript expression in osteogenesis-induced hDPSCs. ConclusionX. strumarium aqueous extract is a suitable candidate for bone repair because it promotes osteogenic differentiation in hDPSCs. Therefore this could be explored further in the treatment of bone disorders.

Triply Enhanced Immunotherapy via Dual Glycan Reforming Integrated with Perforation.

The enhancement of immunotherapy is an emerging direction to develop highly effective and practical cancer therapeutic methods. Here a triply enhanced immunotherapy drug (TEID) is designed for ingeniously integrating in situ dual glycan reforming with perforation on cell membrane. The TEID is composed of galactose and neuraminidase conjugated streptolysin O (SLO-Gal and SLO-NEU), which are encapsulated in a hyaluronic acid (HA) shell for targeted recognition to tumor tissue via cell surface CD44. After targeted delivery and HAase-mediated degradation in the tumor region, the TEID releases SLO-Gal and SLO-NEU, which can easily anchor Gal and NEU on the tumor cell membrane via the perforation of SLO to perform dual glycan reforming for the introduction of Gal and the cleavage of sialic acid. The former can activate immune cells to secret cytokines for immune-killing, and the latter can weaken the immune inhibition to improve the immunotherapeutic efficacy. Meanwhile, the perforation of SLO can promote the delivery of cytokines into the tumor cells to further enhance the efficacy. The designed triply enhanced immunotherapy strategy opens a significant and promising route to promote clinical immunotherapy of cancer.

The Effect of Wharton's Jelly-Derived Conditioned Medium on the In Vitro Maturation of Immature Oocytes, Embryo Development, and Genes Expression Involved in Apoptosis.

Oocyte cytoplasmic maturation is a crucial process during in vitro maturation (IVM), and finding an appropriate IVM medium that promotes oocyte competence is very critical in assisted reproductive technology (ART). The aim of this study was to investigate the effects of umbilical cord Wharton's jelly (WJ-MSCs)-derived conditioned media on the maturation of immature oocytes and their developmental potential in humans after IVM, as well as apoptotic gene expression. A total of 392 germinal vesicle (GV) oocytes were collected from 207 women aged 25-35 years and divided into two IVM groups: (1) control group, which was cultured in CleavTM medium, and (2) experimental group, which was cultured in supernatants of umbilical cord Wharton's jelly as a conditioned medium (CM). First, WJ-MSCs were isolated, and their purity was analyzed. The immunophenotypes of WJ-MSCs were analyzed by flow cytometry. The quantitative expression of BCL2, BAX, and BAG1 in matured oocytes and embryos was evaluated through quantitative real-time polymerase chain reaction (qRT-PCR). Our findings showed that WJ-MSCs have a high proliferating capacity. The purity of the isolated cells was further validated by immunophenotyping, which revealed that their surface antigen expression had phenotypical properties similar to WJ-MSCs. When compared to CD34 and CD45 surface markers, the enlarged cells were positive for CD90, CD105, and CD44. There were significant differences in cytoplasmic maturation of oocytes and embryo quality between the two groups. The mRNA expression levels of BCL-2, BAG1, and BAX in matured oocytes and embryos were also significantly different between the two groups. Therefore, WJ-MSCs medium indicated potential efficacy in terms of ameliorating oocyte maturation and in promoting the development and genes expression of BAX, BCL-2, and BAG1.

A machine-learning model that incorporates CD45 surface expression predicts hematopoietic progenitor cell recovery after freeze–thaw

Background aimsSufficient doses of viable CD34+ (vCD34) hematopoietic progenitor cells (HPCs) are crucial for engraftment. Additional-day apheresis collections can compensate for potential loss during cryopreservation but incur high cost and additional risk. To aid predicting such losses for clinical decision support, we developed a machine-learning model using variables obtainable on the day of collection. MethodsIn total, 370 consecutive autologous HPCs, apheresis-collected since 2014 at the Children's Hospital of Philadelphia, were retrospectively reviewed. Flow cytometry was used to assess vCD34% on fresh products and thawed quality control vials. The ratio of vCD34% thawed to fresh, which we call “post-thaw index,” was used as an outcome measure, with a “poor” post-thaw index defined as <70%. HPC CD45 normalized mean fluorescence intensity (MFI) was calculated by dividing CD45 MFI of HPCs to the CD45 MFI of lymphocytes in the same sample. We trained XGBoost, k-nearest neighbor and random forest models for the prediction and calibrated the best model to minimize falsely-reassuring predictions. ResultsIn total, 63 of 370 (17%) products had a poor post-thaw index. The best model was XGBoost, with an area under the receiver operator curve of 0.83 evaluated on an independent test data set. The most important predictor for a poor post-thaw index was the HPC CD45 normalized MFI. Transplants after 2015, based on the lower of the two vCD34% values, showed faster engraftment than older transplants, which were based on fresh vCD34% only (average 10.6 vs 11.7 days, P = 0.0006). ConclusionsTransplants taking into account post-thaw vCD34% improved engraftment time in our patients; however, it came at the cost of unnecessary multi-day collections. The results from applying our predictive algorithm retrospectively to our data suggest that more than one-third of additional-day collections could have been avoided. Our investigation also identified CD45 nMFI as a novel marker for assessing HPC health post-thaw.

Degradation of CD47‐SIRPα axis by pomalidomide potentiates CD20 antibody‐dependent cellular phagocytosis against B‐cell lymphoma

Introduction: Anti-CD20 monoclonal antibodies (mAbs) have revolutionized the treatment of B-cell lymphomas (BCL). The chemo-free combination with lenalidomide (Len) and rituximab (R2) as a first-line therapy has shown promising activity in indolent lymphomas. However, the efficacy of R2 regimen is yet unsatisfactory in aggressive settings, thereby novel synergistic strategy with next-generation CD20 mAbs and/or newer immunomodulatory drugs (IMiD) are urgently needed. Pomalidomide (Pom) is more powerful than Len in immunomodulatory properties including redirection of tumor-associated macrophages (TAM), which may confer a therapeutic opportunity to potentiate antibody-dependent cellular phagocytosis (ADCP) in elimination of lymphoma cells. This study aimed to evaluate the macrophage-based anti-lymphoma activities of Pom plus CD20 mAbs. Methods: BCL cell lines (SU-DHL-4, Raji and Daudi) were co-cultured with human monocyte-derived macrophages and treated with various combinations with IMiDs (Len or Pom) plus CD20 mAbs (rituximab or obinutuzumab). Phagocytosis and cell viability of lymphoma cells were tested by flow cytometry. Protein mass spectrometry and co-immunoprecipitation (co-IP) were performed to identify Pom-redirected neosubstrates of cereblon (CRBN). IMiD-responsive CrbnI391V mice were bred to Eμ-Myc lymphoma mice to evaluate the in vivo immunomodulatory effects of Pom on macrophages. Results: In vitro co-culture systems showed that Pom plus obinutuzumab elicited the strongest ADCP effects, resulting in robust phagocytosis of tumor cells by macrophages and markedly decreased tumor cell number, when compared to other combinations including Len plus rituximab (R2), Len plus obinutuzumab (GALEN) or Pom plus rituximab. Mechanistically, we identified CD47, a well-known negative regulator of macrophage phagocytosis, as a novel CRBN neosubstrate in BCL cells using mass spectrometry screening and co-IP validation. Interestingly, we found that only Pom rather than Len or thalidomide could efficiently induce degradation of total and surface CD47 in SU-DHL-4 cells. We further confirmed that knockdown of CRBN abrogated Pom-induced CD47 degradation, indicating the on-target effect. More importantly, based on the murine spontaneous lymphoma models (Eμ-Myc CrbnI391V mice), we found administration of Pom elicited a shift from M2 (CD206+) to M1 (CD86+) phenotype of tumor-associated macrophages and significant decrease of SIRPα+ populations in both M1 and M2 subtypes. Conclusion: Our study provides evidence for the superior effects of Pom plus obinutuzumab by harnessing the anti-lymphoma activity of TAMs, and suggests degradation of CD47-SIRPα axis as a novel mechanism underlying the drug synergy. Further preclinical and clinical investigations are needed to determine the in vivo synergistic efficacy and potential toxicity of this combined treatment. The research was funded by: This study was supported by the National Natural Science Foundation of China (No. 81470336 to KZ). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Immunotherapy No conflicts of interests pertinent to the abstract.

Extracellular matrix remodelling in obesity and metabolic disorders.

Obesity causes extracellular matrix (ECM) remodelling which can develop into serious pathology and fibrosis, having metabolic effects in insulin-sensitive tissues. The ECM components may be increased in response to overnutrition. This review will focus on specific obesity-associated molecular and pathophysiological mechanisms of ECM remodelling and the impact of specific interactions on tissue metabolism. In obesity, complex network of signalling molecules such as cytokines and growth factors have been implicated in fibrosis. Increased ECM deposition contributes to the pathogenesis of insulin resistance at least in part through activation of cell surface integrin receptors and CD44 signalling cascades. These cell surface receptors transmit signals to the cell adhesome which orchestrates an intracellular response that adapts to the extracellular environment. Matrix proteins, glycoproteins, and polysaccharides interact through ligand-specific cell surface receptors that interact with the cytosolic adhesion proteins to elicit specific actions. Cell adhesion proteins may have catalytic activity or serve as scaffolds. The vast number of cell surface receptors and the complexity of the cell adhesome have made study of their roles challenging in health and disease. Further complicating the role of ECM-cell receptor interactions is the variation between cell types. This review will focus on recent insights gained from studies of two highly conserved, ubiquitously axes and how they contribute to insulin resistance and metabolic dysfunction in obesity. These are the collagen-integrin receptor-IPP (ILK-PINCH-Parvin) axis and the hyaluronan-CD44 interaction. We speculate that targeting ECM components or their receptor-mediated cell signalling may provide novel insights into the treatment of obesity-associated cardiometabolic complications.

O015 CD34+ vascular stem cells play a key role in vein graft remodelling

Abstract Introduction Venous conduits that are grafted into arterial circulation undergo neointima remodelling, causing excessive hyperplasia that contributes to early graft failure which a major drawback of surgical vein grafting. Stem cells expressing the CD34 surface protein have been postulated to have a key role in the process, but their involvement has not been studied. Methods A segment of the inferior vena cava of wild-type mice was interposition grafted into mice in which CD34-expressing cells were located through their genetically induced expression of tdTomato red marker protein (CD34-CreERT2xRosa26-tdTomato mice). This enabled us to determine whether CD34+ derived cells found in the vein after grafting originated from the donor mouse or from the host. Grafts were explanted 4wks after surgery and analysed by immunohistology and single cell RNA sequencing to resolve cell-level gene expression. Cells from vein grafts were also cultured in vitro and proliferation/differentiation assays were carried out on purified CD34+ cells. Results 70% of cells in the vein graft neointima were derived from CD34+ graft lineage whereas there was no significant contribution of recipient-derived stem cells or bone marrow cells to the neointima. Vein graft neointima smooth muscle cells (SMC) are not stem cells derived. CD34+ stem cells differentiated predominantly into fibroblasts which induced vein SMC phenotype switching and proliferation. Conclusion The majority of CD34+ stem cells found in the neointima of veins, grafted into the arterial circulation were derived from the resident cell population. These cells

Generation of a potent anti-PD-L1-CD47 bispecific antibody with a strong therapeutic and safety profile for cancer immunotherapy

Cell surface molecules PD-L1 and CD47 are potent inhibitors of adaptive and innate anti-cancer immunity. We sought to generate a safe, therapeutic, bispecific antibody specifically targeting, and blocking both PD-L1 and CD47 inhibitory activity. Novel anti-PDL-1 and anti-CD47 antibodies with favorable inhibitory activity, were humanized and constructed into a unique bi-specific antibody intended for clinical use. Previous pre-clinical and clinical studies using anti-CD47 antibodies indicated anemia and thrombocytopenia as potential risks. QL401 is a PD-L1 x CD47 bispecific antibody engineered to reduce effect on red blood cells while retaining potent phagocytic activation of macrophages in vitro and delayed tumor growth in vivo. QL401 comprises three functional components: a PD-L1 binding Fab arm, a CD47 binding scFv arm, and a human IgG4 backbone. The PD-L1 binding arm provides both tumor targeting and blocking of PD-1 for reactivating T cells. The CD47 arm blocks the binding of SIRPα, while the IgG4 Fc retains Fc gamma receptor binding to provide a phagocytic signal. In preclinical efficacy studies, QL401 potently blocked SIRPα to promote phagocytosis of tumor cells with sub-nanomolar potency. In vivo efficacy studies in mouse xenograft tumor models showed QL401 to be comparable or superior to PD-L1 or CD47 monoclonal antibodies alone or in combination. In vitro safety evaluation of QL401 showed significantly reduced binding and phagocytosis of red blood cells, in contrast to CD47 monoclonal antibodies. In addition, QL401 did not induce hemagglutination. In non-human primates, QL401 was well tolerated up to 100 mg/kg without reduction of red blood cells or platelets below the normal range. QL401 is presently in a human phase I safety study.

CD24-Siglec interactions in inflammatory diseases.

CD24 is a small glycosylphosphatidylinositol (GPI)-anchored glycoprotein with broad expression in multiple cell types. Due to differential glycosylation, cell surface CD24 have been shown to interact with various receptors to mediate multiple physiological functions. Nearly 15 years ago, CD24 was shown to interact with Siglec G/10 to selectively inhibit inflammatory response to tissue injuries. Subsequent studies demonstrate that sialylated CD24 (SialoCD24) is a major endogenous ligand for CD33-family of Siglecs to protect the host against inflammatory and autoimmune diseases, metabolic disorders and most notably respiratory distress in COVID-19. The discoveries on CD24-Siglec interactions propelled active translational research to treat graft-vs-host diseases, cancer, COVID-19 and metabolic disorders. This mini-review provides a succinct summary on biological significance of CD24-Siglec pathway in regulation of inflammatory diseases with emphasis on clinical translation.

Profiling of Natural Killer Interactions With Cancer Cells Using Mass Cytometry

We developed a comprehensive method for functional assessment of the changes in immune populations and killing activity of peripheral blood mononuclear cells after cocultures with cancer cells using mass cytometry. In this study, a 43-marker mass cytometry panel was applied to a coculture of immune cells from healthy donors’ peripheral blood mononuclear cells with diverse cancer cell lines. DNA content combined with classical CD45 surface staining was used as gating parameters for cocultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg/DNAhigh). This strategy allows for universal discrimination of cancer cells from immune populations without the need for a specific cancer cell marker and simultaneous assessment of phenotypical changes in both populations. The use of mass cytometry allows for simultaneous detection of changes in natural killer, natural killer T cell, and T cell phenotypes and degranulation of immune populations upon target recognition, analysis of target cells for cytotoxic protein granzyme B content, and cancer cell death. These findings have broad applicability in research and clinical settings with the aim to phenotype and assess functional changes following not only NK-cancer cell interactions but also the effect of those interactions on other immune populations.

Elucidating the immunological landscape of acute myocardial infarction using an integrated single-cell enzyme and antibody quantification (iSEAQ) assay

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research Foundation Background Characterizing enzyme secretion with surface antigen expression of circulating leukocytes provides deep immunological insights that may benefit diagnostics and therapeutics of acute myocardial infarction (AMI). Current methods are limited by laborious sample preparation, lengthy assay time and limited analytical throughput. Purpose We aimed to develop a point-of-care platform for real-time, granular immune profiling of AMI during index hospitalization. Methods We developed a system for a 40-min assay termed integrated single-cell enzyme and antigen quantification (iSEAQ), consisting of an integrated microfluidic device for a streamlined process to recover and isolate single leukocytes from whole blood, and a customized 5-channel high-speed droplet fluorescence cytometer for sensitive quantification of key protein activity resembling live leukocyte activity in-vivo (Figure 1). Using a panel of 2 secreted enzymes (granzyme B (GzB) and neutrophil elastase (NE)) and 3 surface antigens (cluster of differentiation (CD) 66b, CD3 and CD31), we tracked the longitudinal shift of immune functionality of 9 patients admitted for acute ST-segment elevation myocardial infarction (STEMI) patients (median age 52 years, 100% males) at 3 timepoints during the index hospitalization: 1) on admission prior to coronary revascularization, 2) within 24 hours of revascularization, and 3) pre-discharge. The 5-channel fluorescence data were imported using a python script and pre-processed, before piping into Numpy, Pandas, Scipy and UMAP library for data preparation and classification. Seaborn and Matplotlib were used for all data visualization and plots. Data was analyzed using student two-tailed t-tests, with a significance level of &amp;lt;5%. Results Leveraging on its ability to dissect enzyme secretion by phenotype class, iSEAQ unraveled distinct contributions of CD31, NE and GzB from peripheral granulocytes, T lymphocytes and other cells (Figure 2). The majority immune response is expressed in CD66b+ granulocytes showing increased NE+ secretion and CD31 surface antigen expression denoted in Class 0 in Figure 2. Compared to samples from healthy donors and STEMI patients on admission, the NE+ and CD31+ expressions were significantly lower in pre-discharge samples of STEMI patients (p&amp;lt;0.05). iSEAQ also distinguished anterior from inferior STEMI, as evident from a significantly elevated NE+CD31-secretion for CD66b+ granulocytes in anterior STEMI (365±70 vs 83±35 fg, p &amp;lt; 0.001), showing preliminary promise in the ability of iSEAQ to risk stratify. Conclusion iSEAQ provided a comprehensive granular immune atlas of the longitudinal immune responses to STEMI during hospital admission. With its open design for assay panel using commercial biosensors, iSEAQ is a potentially powerful tool for taking functionality snapshots of immune system in many diseases.

Abstract LB340: Spur cell-delivered peptide antigen generates T cell immunity to eradicate mouse KRASG12D spontaneous lung cancer

Abstract Red blood cells (RBCs) have been widely used in delivering various drugs including cancer antigens. However, due to their inherent biocompatibility and low immunogenicity, RBC-based tumor antigen delivery often fails to initiate anti-tumor immunity and results in lower clinical efficacy of cancer vaccines. Here we report that tumor antigen-encapsulated RBCs can be rapidly transformed to spur cells which have much higher immunogenicity. Compared to normal RBCs, spur cells display a defused surface CD47 pattern with a lower affinity to SIRPα, and thus can be rapidly engulfed by activated macrophages and dendritic cells. The tumor antigen-loaded spur cells elicit high antigen responses both in vitro and in vivo. In combination with the SHP-1 inhibitor, spur cell-delivered KRASG12D antigenic peptides markedly suppress tumor growth in KRASG12D spontaneous mouse lung cancer model. In addition, a specific T cell population targeting the KRASG12D mutated peptide antigen is detected in cancer-survival mice. Our study thus has developed spur cell-delivered cancer neoantigen as an effective cancer vaccine. Citation Format: Shuang Qu, Jiafan Wang, Meng Jia, Hongwei Liang, Yuan Liu, Ke Zen. Spur cell-delivered peptide antigen generates T cell immunity to eradicate mouse KRASG12D spontaneous lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB340.

Abstract 4421: Targeting myeloid-derived suppressor cells with actinium-225 lintuzumab, a CD33 antibody radioconjugate to enhance antitumor immunity

Abstract Introduction: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid lineage cells with potent immunosuppressive activity found enriched in cancer patients. Crosstalk between MDSCs and the tumor microenvironment can promote tumor immune evasion and thus there is growing interest in developing therapeutic approaches to intervene in the MDSC suppressive function. Both monocytic-MDSC (M-MDSC) and granulocytic-MDSC (G-MDSC) subpopulations express the cell surface CD33 myeloid marker. For well-defined cell surface markers like CD33, there is considerable interest in the use of radionuclides as therapeutic payloads, particularly α-particle emitters such as actinium-225 (225Ac) since they deliver substantially higher decay energies over a much shorter distance than β-emitters, rendering them more suitable for precise, potent, and efficient target cell killing while minimizing toxicity to surrounding bystander cells. Actimab A, the anti-CD33 antibody lintuzumab armed with the 225Ac radioisotope (CD33 ARC), is currently being evaluated in R/R AML and has demonstrated significant anti-leukemic activity in Phase 1/2 clinical trials. We therefore hypothesized that MDSCs can be directly targeted by the CD33 ARC. Hence, we evaluated the therapeutic potential of the CD33 ARC to deplete MDSCs through preclinical studies in vitro and in vivo with humanized mouse model. Methods: CD33 ARC was generated by conjugating lintuzumab with p-SCN-Bn-DOTA and subsequently radiolabeled with 225Ac. Primary MDSCs were isolated from cancer patient peripheral blood or healthy donor PBMCs. The specific binding of CD33 ARC to CD33 positive MDSCs and the decreased viability of MDSCs in response to treatment in vitro was characterized by immunophenotyping using flow cytometry. The therapeutic efficacy of CD33 ARC to deplete MDSC in vivo was evaluated in human CD34 reconstituted humanized NOG-EXL mice. Results: CD33 positive MDSCs (M-MDSCs and G-MDSCs) were identified in human cancer peripheral blood samples, including colorectal and lung. Significantly more MDSCs were found in the peripheral blood of cancer patients in comparison to healthy donors. CD33 ARC treatment of human cancer MDSCs induced a potent dose-dependent reduction in MDSC viability leading to increased depletion of MDSCs. Furthermore, in the humanized NOG-EXL mouse model, CD33 ARC therapy demonstrated in vivo activity of CD33 ARC to deplete human MDSCs. Conclusions: In this study, we demonstrate CD33 ARC alpha targeted radiotherapy depletes human CD33 positive immune suppressing MDSCs present in multiple cancer types, to enhance antitumor immunity. These findings present a translatable strategy that supports further evaluation of 225Ac lintuzumab as a MDSC targeting agent to improve the efficacy of antitumor therapies. Citation Format: Amanda Chin, Mary Chen, Jason Li, Megan McCloskey, Caroline Jennings, Le-Cun Xu, Monideepa Roy, Denis Beckford, Helen Kotanides. Targeting myeloid-derived suppressor cells with actinium-225 lintuzumab, a CD33 antibody radioconjugate to enhance antitumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4421.

Abstract 3045: Role of CD10 negative stroma in the development of precursor lesion of endometriosis associated ovarian cancer

Abstract Introduction: Ovarian clear cell carcinoma (OCCC) is a rare, aggressive, chemo-resistant subtype of epithelial ovarian cancer, and is associated with a significantly worse five-year survival compared to other ovarian cancer types. This is in part due to its distinct cell of origin which is thought to arise within sites of endometriosis. Atypical endometriosis is considered a precursor lesion of endometriosis-associated ovarian cancers, including OCCC. However, the mechanism underlying the development of atypical endometriosis and subsequent malignant transformation is still unknown. We recently demonstrated the existence of a OCCC tumor-promoting mesenchymal stem cell (enMSCs), marked by the loss of CD10 expression, within the endometriotic microenvironment. Further, we demonstrated that CD10 negative enMSCs selectively enhance OCCC growth, but not high-grade serous ovarian cancer (HGSOC) growth which provides further evidence that the endometriotic microenvironment is uniquely supportive of OCCC tumorigenesis. Thus, we hypothesized that loss of stromal CD10 expression leads to the creation of the precursor microenvironment for OCCC. Methods: EnMSCs and endometriosis epithelial cells were isolated from endometriosis tissue. Tissues were collected from women who undergo surgery for benign endometriosis. Based on surface CD10 expression, enMSCs were divided into CD10 negative or positive populations. Endometriosis epithelium proliferation was assessed using in vitro assays. Long term organoid of CD10 negative enMSCs and endometriosis epithelium were implanted into NSG mice to assess tumor initiation capacity. Results: Our data showed that co-culture with CD10 negative enMSCs enhanced the proliferation of endometriosis epithelium. Further, CD10 negative enMSCs enhanced the sphere formation capacity (sphere number and sphere size) of endometriosis epithelial cells when compared to endometriosis epithelium co-cultured with CD10 positive enMSCs or cultured alone. Furthermore, long term growth of organoids containing CD10 negative enMSCs and endometriosis epithelium stimulated the development of cytologic atypia in the epithelial cells. Strikingly, these organoids, when injected into the fat pad of NSG mice, initiated localized tumors. In summary, our data highlighted the role of CD10 negative enMSCs in the development of cytologic atypia in endometriosis epithelium and inducing tumor formation in vivo. This work has the potential for significant clinical impact as identification of a stromal biomarker for OCCC development would have important implications in the clinical management of women with endometriosis, including determining the need for risk-reducing surgery. Further, this work has the potential to identify new targets to disrupt the unique microenvironment of OCCC, thus providing new therapeutic strategies for this deadly disease. Citation Format: Huda Issa Atiya, Lan Coffman. Role of CD10 negative stroma in the development of precursor lesion of endometriosis associated ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3045.

Better resolving of anti‐CD38 antibody interference with blood compatibility testing by using manual polybrene method compared with dithiothreitol‐pretreatment indirect antiglobulin test

AbstractBackgroundIt is advised to pretreat the reagent erythrocytes with Dithiothreitol (DTT) to denature the surface CD38 to prevent anti‐CD38 monoclonal antibodies (MoAb) from interfering with the blood compatibility test. Anti‐CD38 has little impact on the Polybrene test, but it is still unknown how sensitive it is to detect irregular antibodies and how effective it is when compared to the standard DTT‐based method.MethodsTwenty‐one patients receiving daratumumab (N = 13) and isatuximab (N = 8) had their serum collected. Standard anti‐sera (anti‐c, D, E, Fyb, Jka, M, Mia) with serial dilution were added to patients' serum. Antibody screening tests were performed simultaneously using the manual polybrene method (MP) and DTT‐pretreated, automatic indirect antiglobulin test (DTT‐IAT) to compare the detection sensitivity. These two methods' operating times and costs were also analyzed.ResultsBoth MP and DTT‐IAT can overcome the interference caused by anti‐CD38 MoAb. However, MP is more sensitive in detecting anti‐M and anti‐Mia and is comparable to DTT‐IAT in detecting other antibodies. In terms of cost and operating time, MP is also far superior to DTT‐IAT.ConclusionMP is a cost‐effective alternative to DTT‐IAT in resolving anti‐CD38 interference and is especially suitable for populations with a high prevalence of anti‐M and anti‐Mia. However, both methods have a well‐known drawback of low detection sensitivity for anti‐K, and K‐units should be provided to patients to prevent hemolytic transfusion reactions.

Duplex Responsive Nanoplatform with Cascade Targeting for Atherosclerosis Photoacoustic Diagnosis and Multichannel Combination Therapy.

The culprits of atherosclerosis are endothelial damage, local disorders of lipid metabolism, and progressive inflammation. Early atherosclerosis is typically difficult to diagnose in time due to the lack of obvious symptoms, thus missing the best period of treatment. In this work, a π-conjugated polymer (PMeTPP-MBT) based on 3,6-bis(4-methylthiophen-2-yl)-2,5-bis(2-octyldodecyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione is designed as a novel photoacoustic contrast agent. On this basis, an intelligent responsive theranostic nanoplatform (PA/ASePSD) combining astaxanthin and SS-31 peptide and loading with PMeTPP-MBT is developed. The high affinity between the dextran shell with the broken endothelial surface VCAM-1 and CD44 confers active targeting of PA/ASePSD to atherosclerotic lesions. High levels of ROS in the acidic plaque microenvironment act as an intelligent cascade switch to achieve controlled release of astaxanthin, SS-31 peptide, and PMeTPP-MBT for non-invasive photoacoustic diagnosis, as well as plaque inhibition mediated by anti-inflammation and multichannel regulation (including ABCA1, ABCG1, CD36, and LOX-1) of lipid metabolism. Both in vitro and in vivo evaluations confirm the impressive anti-atherosclerotic capability and the accurate photoacoustic diagnosis of PA/ASePSD nanoparticles, thus promising a candidate for early-stage atherosclerosis theranostics.

EMD originates from hyaluronan-induced homophilic interactions of CD44 variant-expressing MM cells under shear stress.

Extramedullary disease (EMD) is known to be associated with chemoresistance and poor prognosis in multiple myeloma (MM); however, the mechanisms of its development are not fully understood. Elucidating the mechanism of EMD development and its therapeutic targeting would greatly contribute to further improvement of treatment outcome in patients with MM. Here, we show that bone marrow stroma cell-derived hyaluronan (HA) elicits homophilic interactions of MM cells by binding to surface CD44, especially long-stretch variants, under physiological shear stress and generates cell clusters that might develop into EMD. We recapitulated the development of EMD via administration of HA in a syngeneic murine MM model in a CD44-dependent manner. HA-induced MM cell clusters exhibited the specific resistance to proteasome inhibitors (PIs) invitro and in murine models via γ-secretase-mediated cleavage of the intracellular domains of CD44, which in turn transactivated PI resistance-inducible genes. Treatment of HA-injected mice with anti-CD44 antibody or γ-secretase inhibitors readily suppressed the development of EMD from transplanted MM cells and significantly prolonged the survival of recipients by overcoming PI resistance. The HA-CD44 axis represents a novel pathway to trigger EMD development and could be a target of the prediction, prevention, and treatment of EMD in patients with MM.

Thrombospondin-1, CD47, and SIRPα display cell-specific molecular signatures in human islets and pancreata.

Thrombospondin-1 (TSP1) is a secreted protein minimally expressed in health but increased in disease and age. TSP1 binds to the cell membrane receptor CD47, which itself engages signal regulatory protein α (SIRPα), and the latter creates a checkpoint for immune activation. Individuals with cancer administered checkpoint-blocking molecules developed insulin-dependent diabetes. Relevant to this, CD47 blocking antibodies and SIRPα fusion proteins are in clinical trials. We characterized the molecular signature of TSP1, CD47, and SIRPα in human islets and pancreata. Fresh islets and pancreatic tissue from nondiabetic individuals were obtained. The expression of THBS1, CD47, and SIRPA was determined using single-cell mRNA sequencing, immunofluorescence microscopy, Western blot, and flow cytometry. Islets were exposed to diabetes-affiliated inflammatory cytokines and changes in protein expression were determined. CD47 mRNA was expressed in all islet cell types. THBS1 mRNA was restricted primarily to endothelial and mesenchymal cells, whereas SIRPA mRNA was found mostly in macrophages. Immunofluorescence staining showed CD47 protein expressed by β cells and present in the exocrine pancreas. TSP1 and SIRPα proteins were not seen in islets or the exocrine pancreas. Western blot and flow cytometry confirmed immunofluorescent expression patterns. Importantly, human islets produced substantial quantities of secreted TSP1. Human pancreatic exocrine and endocrine tissue expressed CD47, whereas fresh islets displayed cell surface CD47 and secreted TSP1 at baseline and in inflammation. These findings suggest unexpected effects on islets from agents that intersect TSP1-CD47-SIRPα.NEW & NOTEWORTHY CD47 is a cell surface receptor with two primary ligands, soluble thrombospondin-1 (TSP1) and cell surface signal regulatory protein alpha (SIRPα). Both interactions provide checkpoints for immune cell activity. We determined that fresh human islets display CD47 and secrete TSP1. However, human islet endocrine cells lack SIRPα. These gene signatures are likely important given the increasing use of CD47 and SIRPα blocking molecules in individuals with cancer.