Peripheral Blood Mononuclear Cells Of Healthy Donors Research Articles

Assessing Functional Similarity of Biosimilar ABP 654 and Ustekinumab in Samples from Patients with Crohn's Disease.

ABP 654 is the first FDA-approved interchangeable biosimilar for ustekinumab reference product (RP). To support the totality of evidence (TOE), in vitro pharmacology studies were conducted in peripheral blood mononuclear cells (PBMCs) from healthy human donors and Crohn's disease (CD) patients to evaluate IL-23 and IL-12 inhibition by ABP654 and ustekinumab RP relevant to the mechanism of action of chronic inflammation. ABP654 and ustekinumab RP were assessed using inhibition of IL-23 and IL-12-mediated IFN-γ release, signal transducer and activator of transcription (STAT)3 and STAT4 phosphorylation, and IL-17 release. IFN-γ levels were determined using homogenous time-resolved fluorescence (HTRF). STAT3 and STAT4 phosphorylation were measured by flow cytometry. IL-17 was measured using a Cisbio IL-17 detection kit. IC50 values were calculated to assess the relative potency of ABP 654 and ustekinumab RP. ABP654 and ustekinumab RP demonstrated similar inhibition and relative potency of IL-23 and IL-12-mediated IFN-γ release, and no difference in inhibition of IL-23/IL-12-mediated STAT3/STAT4 phosphorylation in healthy donor PBMCs, as evidenced by the overlapping standard deviation (SD). In CD PBMCs, ABP654 and ustekinumab RP also showed no difference in IC50 values for inhibition of IL-23/IL-12-mediated STAT3/STAT4 phosphorylation. ABP654 and ustekinumab RP showed no difference in IC50 values for IL-23-induced IL-17 release, in either healthy (ABP 654, 458.7±110.8 pM; ustekinumab (EU), 514.6±48.7 pM) or CD (ABP 654, 260.8±88.5 pM; ustekinumab (EU), 256.9±96.8 pM) donor cells with overlapping SD. These studies demonstrated similar inhibition of IL-23 and IL-12 function by ABP 654 and ustekinumab RP in both healthy and CD PBMCs. Overall, these assays support the conclusion that ABP654 and ustekinumab RP are functionally similar, thereby contributing to the TOE supporting a demonstration of biosimilarity.

Effects of coagulation factors on bone cells and consequences of their absence in haemophilia a patients

Haemophilia is associated with reduced bone mass and mineral density. Due to the rarity of the disease and the heterogeneity among the studies, the pathogenesis of bone loss is still under investigation. We studied the effects of coagulation factors on bone cells and characterized in a pilot study the osteoclastogenic potential of patients’ osteoclast precursors. To evaluate the effect of coagulation factors on osteoclasts, we treated Healthy Donor-Peripheral Blood Mononuclear Cells (HD-PBMC) with Factor VIII (FVIII), von Willebrand Factor (VWF), FVIII/VWF complex, activated Factor IX (FIXa), activated Factor X (FXa) and Thrombin (THB). FVIII, VWF, FVIII/VWF, FXa and THB treatments reduced osteoclast differentiation of HD-PBMC and VWF affected also bone resorption. Interestingly, PBMC isolated from patients with moderate/severe haemophilia showed an increased osteoclastogenic potential due to the alteration of osteoclast precursors. Moreover, increased expression of genes involved in osteoclast differentiation/activity was revealed in osteoclasts of an adult patient with moderate haemophilia. Control osteoblasts treated with the coagulation factors showed that FVIII and VWF reduced ALP positivity; the opposite effect was observed following THB treatment. Moreover, FVIII, VWF and FVIII/VWF reduced mineralization ability. These results could be important to understand how coagulation factors deficiency influences bone remodeling activity in haemophilia.

Synthesis and Biochemical Evaluation of Ethanoanthracenes and Related Compounds: Antiproliferative and Pro-Apoptotic Effects in Chronic Lymphocytic Leukemia (CLL).

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells, and it is the most frequent form of leukemia diagnosed in Western countries. It is characterized by the proliferation and accumulation of neoplastic B lymphocytes in the blood, lymph nodes, bone marrow and spleen. We report the synthesis and antiproliferative effects of a series of novel ethanoanthracene compounds in CLL cell lines. Structural modifications were achieved via the Diels-Alder reaction of 9-(2-nitrovinyl)anthracene and 3-(anthracen-9-yl)-1-arylprop-2-en-1-ones (anthracene chalcones) with dienophiles, including maleic anhydride and N-substituted maleimides, to afford a series of 9-(E)-(2-nitrovinyl)-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones, 9-(E)-3-oxo-3-phenylprop-1-en-1-yl)-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones and related compounds. Single-crystal X-ray analysis confirmed the structures of the novel ethanoanthracenes 23f, 23h, 24a, 24g, 25f and 27. The products were evaluated in HG-3 and PGA-1 CLL cell lines (representative of poor and good patient prognosis, respectively). The most potent compounds were identified as 20a, 20f, 23a and 25n with IC50 values in the ranges of 0.17-2.69 µM (HG-3) and 0.35-1.97 µM (PGA-1). The pro-apoptotic effects of the potent compounds 20a, 20f, 23a and 25n were demonstrated in CLL cell lines HG-3 (82-95%) and PGA-1 (87-97%) at 10 µM, with low toxicity (12-16%) observed in healthy-donor peripheral blood mononuclear cells (PBMCs) at concentrations representative of the compounds IC50 values for both the HG-3 and PGA-1 CLL cell lines. The antiproliferative effect of the selected compounds, 20a, 20f, 23a and 25n, was mediated through ROS flux with a marked increase in cell viability upon pretreatment with the antioxidant NAC. 25n also demonstrated sub-micromolar activity in the NCI 60 cancer cell line panel, with a mean GI50 value of 0.245 µM. This ethanoanthracene series of compounds offers potential for the further development of lead structures as novel chemotherapeutics to target CLL.

Abstract 4996: Analysis of the effect of eribulin on tumor immunity against triple-negative breast cancer

Abstract Purpose: Chemotherapeutic agents may induce immunomodulatory effects on immune cells and cancer cells in patients with triple negative breast cancer (TNBC). Recent studies have demonstrated that absolute lymphocyte count in peripheral blood can predict response to eribulin in patients with breast cancer, suggesting that eribulin have some favorable effects on immune cells. However, there have been no reports demonstrating the mechanisms of how eribulin affects the immune cells of patients with breast cancer. This study aimed to analyze the effect of eribulin on T cells in vitro and in vivo. Materials & Methods: Using human peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with TNBC, we investigated the effects of eribulin on proliferation of these cells, changes in the expression of surface markers, and antitumor effects on TNBC cells. Three TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-157) were used in this study. Human PBMCs were activated by CD3/CD28 stimulation in vitro for six days and analyzed for T-cell surface markers by flow cytometry. For three patients with TNBC who underwent eribulin treatment, blood was collected at baseline and on days 8, 22, and 64 after treatment, and PBMCs were isolated, and analyzed by flow cytometry. Results: The proliferation of activated CD8+ T cells from healthy donors and patients was facilitated when cultured with 1nM of eribulin compared to the control. Moreover, eribulin significantly decreased CD4/8 ratio in T cells (p< 0.05). In PBMCs from healthy donors, the frequency of CD45RA+ CD45RO- cells and CCR7+ cells in CD8+ T cells was significantly increased by eribulin treatment (p< 0.05). Furthermore, eribulin significantly increased CD127+ KLRG1- cells (memory precursor effector cells) and TCF1+ cells in CD8+ T cells (p< 0.01). Furthermore, the anti-tumor effect of activated T cells from PBMCs of healthy donors and patients with TNBC was enhanced when cultured with eribulin in all three TNBC cell lines compared to control, and IFNγ+ CD8+ T cells were significantly increased by eribulin treatment (p< 0.05). However, in TNBC patients treated with eribulin in clinical practice, there were no significant changes in the CD4/8 ratio, the frequency of CD45RA+ cells or CCR7+ cells in CD8+ T cells before and after treatment of eribulin. Conclusions: Our findings suggest that eribulin can facilitate the proliferation of CD8+ T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells in vitro. Further analysis of the effects of eribulin on TNBC cells and immune cells in vivo is required, and we continue to analyze immunomodulation in patients treated with eribulin. Citation Format: Shimizu Tadafumi, Takaaki Oba, Ken-ichi Ito. Analysis of the effect of eribulin on tumor immunity against triple-negative breast cancer [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 4996.

Abstract 44: A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells

Abstract Chimeric antigen receptor (CAR) T cell therapies for hematologic malignancies, while promising and efficacious, can also result in a variety of adverse effects. Of note, on-target, off-tumor toxicity is one adverse effect that continues to limit the safety and efficacy of CAR-T cells and has even led to life-threatening or fatal outcomes in patients (Morgan et al. 2010.). To address this, we have developed a unique, codon-based approach by replacing strategically selected codons within CAR non-antigen binding domains with their rare synonymous isoforms. Hoekema et al. (1987) reported the ability of synonymous rare codons to alter gene and protein expression in other model systems. In CAR-T cells, we aim to use this novel, codon-based approach to fine tune and modulate CAR-T cell expression and function to elicit full on-tumor activity while lessening off-tumor toxicity. In our approach, we replaced specific endogenous codons within the non-antigen binding domains of Hu8F4-CAR, a TCR-like 2nd generation CAR developed by our group which targets the leukemia-associated antigen PR1/HLA-A2 (Ma et al. 2016.). Briefly, we created variants of Hu8F4-CAR containing rare codon substitutions in two subtypes: intracellular-based (type 1) and combined extracellular- and intracellular-based (type 2). Each variant subtype (type 1 or 2) had either between 6 and 30 or 27 and 57 rare codons introduced, respectively. Expression profiling and functional assessment of the Hu8F4-CAR variants was assessed in the Jurkat mutant cell line (J.RT3-T3.5) or human primary healthy peripheral blood mononuclear cells (PBMCs) that were enriched for T cells, respectively. Expression profiling results show that surface expression is reduced in J.RT3-T3.5 cells when assessed by flow cytometry by 1-1.5-fold compared to unmodified Hu8F4-CAR. When evaluated by immunoblotting, novel lower molecular weight bands emerge only in rare codon-modified variants, suggesting that rare codons may have altered translational or post-translational processing leading to truncated CAR protein isoforms (30, 45, or 60kDa vs 73kDa for unmodified Hu8F4-CAR), which may contribute to reduced cell surface CAR expression. Furthermore, we have conducted in vitro functional testing of the rare codon-modified variants and show reduced activity against non-malignant off-tumor targets that express PR1 and/or HLA-A2 including healthy donor PBMCs, mobilized hematopoietic cells, and bone marrow. Importantly, in addition to reduced off-tumor activity against healthy myeloid cells, rare codon-modified variants preserve their activity against on-tumor target cells, including the U937-A2+ cell line and primary acute myeloid leukemia. Altogether, these results suggest that our approach can modulate CAR expression and functionality creating a novel methodology to reduce the likelihood of off-tumor adverse effects typically associated with CAR therapies. Citation Format: Rolando A. Vedia, Sijie Lu, Anne V. Philips, Anna Sergeeva, Lisa S. St John, Karen Clise-Dwyer, Gheath Alatrash, Jeffrey J. Molldrem. A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 44.

The Role of Macrophage Migration Inhibitory Factor (MIF) in Regulation of T-Cell Activity in Multiple Myeloma

Background: Multiple myeloma (MM) is the second most common hematological malignancy associated with the overproduction of light chain or monoclonal immunoglobulin. MM remains incurable, with high relapses and refractory disease rates after first-line treatment. Hence, novel therapeutic approaches are needed to improve immune responses to prevent relapses or drug resistance after initial therapies. Immune responses are shaped by several factors, including mutations, pro- and anti-inflammatory cytokines and chemokines, and activation of immune checkpoints in the tumor microenvironment (TME) (Gonzalez et al., 2018). Macrophage migration inhibitory factor (MIF) as a soluble pro-inflammatory cytokine has become one of the most puzzling regulators of innate and adaptive immune responses. In myeloma, MIF was found to be significantly higher in MM cells from relapsed patients than those responding to the treatment. MIF has been implicated in drug resistance and suggested as a biomarker for predicting MM patient responses to proteasome inhibitors (Wang et al., 2020). We previously reported that MIF is highly expressed in MM and regulates CD84 (SLAMF5) expression in MM TME. We showed that MIF secreted from MM cells regulates PD-L1 expression through CD84 on CD14 + monocytic cells in the TME. Inhibition of MIF significantly reduced CD84 and PD-L1 expressions on human CD14 + cells and on myeloid-derived suppressor cells of the mice bearing MM (Lewinsky and Gunes et al., 2021). However, whether MIF directly regulates T-cell activity in MM TME has yet to be explored. Methods and Results: The current study reports that MIF may play a pivotal role in T-cell exhaustion in MM. Ouranalyses in published MM transcriptomic databases show that MM patients with low MIF had significantly longer overall and post-progression survival, indicating the therapeutic potential of MIF in MM. We also noted upregulated MIF gene expression in relapsed or refractory MM (R/RMM) patients (n=2) compared to that in newly diagnosed MM patients (n=7) and healthy individuals (n=9) in our and publicly available MM single-cell RNA sequencing datasets. We detected a profound MIF expression in MM cells and myeloid cell fractions, including monocytes, macrophages, and dendritic cells from the patients' bone marrow (BM) specimens. We previously reported that human MM cell lines highly express MIF. In this study, we assessed the impact of MIF secreted by MM cells on the immune cells, specifically CD8 + T cells. Our results showed that inhibition of MIF reduced the elevated T-cell exhaustion markers, PD1 and CTLA-4, on CD8 + T cells in healthy donors (HDs) peripheral blood mononuclear cells (PBMCs) following the coculturing with MM1.S cells (n=3 *P < 0.05 ). We also found that MIF inhibition induced the T-cell activation marker, LAMP1, on CD8 + T cells in this coculturing setting (n=3, **P < 0.01). Furthermore, we noted MIF inhibition reduced PD1 expression on CD8 + T cells in HDs PBMCs (n=3, **P < 0.01), suggesting MIF may regulate the exhaustion profile of CD8 + T cells. MIF can conduct paracrine or autocrine signalings by binding to its receptors, including CD74 and CD44, and lead to intracellular signaling cascades. Hence, we assessed CD74 gene expression as the counterpart of MIF in R/RMM, MM, and healthy patients. We noted significantly upregulated CD74 expression in R/RMM patients compared to newly diagnosed MM patients and healthy individuals. We also noted higher CD74 expression in CD8 + T cells than in CD4 + T cells in R/RMM patients, suggesting that MIF/CD74 axis may be a potential target to enhance CD8 + T cell-mediated immunity in R/RMM. To further explore the impact of MIF inhibition on MM progression, we performed a pilot in vivo study by inoculating murine 5TGM1 cells into two groups of syngeneic C57BL/KaLwRijHsd mice. After two weeks, the mice were injected with either sterile PBS as a control or a MIF inhibitor. The results of this pilot study showed that MIF inhibition significantly decreased MM cells in BM and PB of myeloma-bearing mice and reduced T-cell exhaustion markers on CD8 + T cells compared to that in the control mice (n=3-4, *P < 0.05, **P < 0.01, ***P < 0.001). Conclusion: Overall, our preliminary data indicate that MIF may have a crucial role in T-cell activity by regulating T-cell exhaustion markers in MM. Further studies are needed to explore the therapeutic potential of MIF to reduce T-cell exhaustion to improve anti-tumor immunity in MM.

CD84 (SLAMF5) As a Potential Target for Immunomodulation in Cutaneous T-Cell Lymphoma (CTCL)

Background: Cutaneous T-cell lymphoma (CTCL) develops from clonally expanded, skin-homing CD4 + T cells within a background of chronic inflammation. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of CTCLs. Patients with advanced-stage MF/SS experience progressively worsening disease and decreased overall survival (Scarisbrick et al., 2015). Immune cells and immune checkpoints (ICs) are essential factors for tumor growth and the host immune response in CTCL. Tumor-associated macrophages (TAMs [M2-like]) play a key role in CTCL growth, and neoplastic T cells escape immune surveillance via the PD-L1/PD1 axis (Wu et al., 2014). However, the approach of ICs inhibition with monoclonal antibodies (mAbs) in CTCL has been challenged due to modest efficacy and remaining immunosuppression. Hence, novel therapeutic strategies are needed to improve anti-tumor immunity in CTCL. We previously characterized CD84 (SLAMF5) as one of the key players in the tumor microenvironment (TME) by regulating multiple ICs, including PD-L1 and PD1, and modulating myeloid cell fractions and T cells in our preclinical MM models. Our CD84 targeting strategy using anti-CD84 mAb significantly reduced PD1 on effector CD8 + T cells and PD-L1 on the tumor and myeloid cells, decreased tumor burden, and increased the survival of the mice with myeloma (Lewinsky and Gunes et al., 2021). The role of CD84 in ICs regulation and anti-tumor immunity in CTCL remains to be explored. Methods and Results: In this study, we evaluated the role of CD84 in CTCL and, for the first time, reported CD84 as a potential target to reduce immunosuppression through the PD-L1/PD1 axis in CTCL. We found upregulated CD84 CD274 (PD-L1) , and PDCD1 (PD1) gene expressions in CTCL patients (n=5) compared to healthy individuals (n=4). We also noted higher CD84 geneexpression in SS patients (n=8) than in MF patients (n=6). We next evaluated CD84 expression on the formalin-fixed paraffin-embedded (FFPE) tumor sections derived from SS patients' skin lesions. We noted that CD84 is highly expressed in the TME and co-expressed in Ki67 + Sézary cells. We further evaluated the CD84 expression in immune cell clusters in SS patients and healthy individuals using publicly available single-cell RNA sequencing (scRNA-seq) datasets. We noted a profound CD84 expression in CD4 + T cells and CD163 + M2-like macrophages in SS patients. We also noted a strong correlation between CD84 and CD274 (p=0.0086) and CD84 and PDCD1 (p=0.0002) in SS patients. We next assessed the expressions of CD84, PD-L1 on CD163 + M2-like macrophages, and PD1 on T cell subsets in a coculturing setting with human SS cell line, Hut78, to determine the impact of Sézary cells on these cell compartments in the TME. We observed a significant increase in the percentages of CD163 + M2-like macrophages and CD4 + T cells and elevated CD84, PD-L1, and PD1 expressions on these cells following the coculturing of peripheral blood mononuclear cells (PBMCs) derived from healthy donors (HDs) with Hut78 cells (n=3, *P < 0.05, **P < 0.01). While the percentages of CD8 + T cells were slightly reduced, CD84 and PD1 expressions were elevated in these cells following the coculturing with Hut78 cells (n=3, *P < 0.05, ***P < 0.001). Dysregulation of STAT proteins, particularly STAT3, has been shown in MF/SS (Olszewska et al., 2020). To explore whether Sézary cells regulate STAT3 in CD163 + M2-like macrophages, we assessed pSTAT3 levels in CD163 + M2-like macrophages following the coculturing of HDs PBMCs with Hut78 cells. We found that Hut78 cellssignificantly increased intracellular pSTAT3 levels inCD163 + M2-like macrophages (n=3, *P < 0.05). Furthermore, we noted the inhibition of STAT signalingusing a pan-STAT inhibitor decreased PD-L1 and PD1 expression onCD163 + M2-like macrophages and T-cell subsets, respectively. Notably, we found the stimulation of CD84 signaling with an agonist anti-CD84 mAb enhanced pSTAT3 levels in CD163 + M2-like macrophages, suggesting the STAT pathway might regulate PD-L1 expression in immunosuppressive CD163 + M2-like macrophages through CD84 signaling in SS. Conclusion: Our preliminary data from this exploratory study indicate that CD84 may be a potential target to reduce immunosuppression in CTCL. Further studies are needed to explore the functions of CD84 as an immune receptor in CTCL TME and its therapeutic potential in CTCL.

Single cell transcriptomics reveal the heterogeneities of TCR Vα7.2+CD161+ and TCR Vα7.2+CD161− T cells in human peripheral blood

PurposeT cell receptor (TCR) usually determines the specificity and unique function of T cells. Recently, the unconventional T cells with a unique TCR have attracted great attentions because of their clinical importance. TCR Vα7.2+ cells, that consist of the CD161+ mucosal associated invariant T (MAIT) cells and CD161− non-MAIT T cells, have been reported to play crucial roles in immune defenses. However, their characterizations in human blood are still obscure. This study aims to investigate the signatures and functions of circulating TCR Vα7.2+CD161+ MAIT and TCR Vα7.2+CD161− cells under steady state.MethodsThe TCR Vα7.2+CD161+ and TCR Vα7.2+CD161− cells were separately sorted from healthy donor peripheral blood mononuclear cells (PBMCs) and send for single cell RNA sequencing (scRNA-seq). Flow cytometry analysis was used to verify the findings obtained from scRNA-seq analysis.ResultsOur findings demonstrated that there are more TCR Vα7.2+CD161+ cells than TCR Vα7.2+CD161− cells in healthy donor PBMCs and revealed the differences between them. Under steady state, 4 TCR Vα7.2+CD161+ MAIT clusters existed in peripheral blood. Pseudotime analysis further implied the development trajectory of these MAIT cells, which was ordered from CCR7+ resting cluster to LGALS3+ transitional cluster, followed by KLRG1+ cluster and ending with CX3CR1+ terminally differentiated cytotoxic cluster. In addition, our results revealed that TCR Vα7.2+CD161− cells consist of different kind of conventional T cells. These TCR Vα7.2+CD161− non-MAIT cells showed a higher level of Granzyme B expression and upregulated genes associated with cytotoxicity, which implicated their roles in immune defense.ConclusionOur findings advanced the understandings of the evolution of circulating MAIT cells. We also preliminarily defined the TCR Vα7.2+CD161− PBMCs as a combination of versatile CD4+ and CD8+ populations with cytotoxicity.

Mechanisms of resistance to bispecific T-cell engager therapy for ovarian cancer.

5571 Background: Patients with recurrent ovarian cancer (OC) invariably develop multidrug resistance and have poor prognoses. Bispecific T-cell engager (BiTE) immunotherapy represents an innovative therapeutic modality where cytotoxic T-cells are activated and recruited to tumor cells using a predefined tumor-associated antigen. Clinical trials evaluating BiTEs in OC are currently in progress with promising results, and exploration of potential mechanisms of resistance to BiTEs could provide meaningful insight into disease biology and combination strategies. Methods: After obtaining informed consent, experiments were performed on patient samples collected under an IRB-approved tissue-banking protocol. Samples were collected before patients started clinical trials and at disease progression. Serum, peripheral blood mononuclear cells (PBMCs), and malignant ascites were collected when available for translational analysis. Immune cell subsets were evaluated for activation and exhaustion via flow cytometry, and tumor cells were subjected to western blot analysis and IHC. Ex vivo cytotoxicity assays were performed using BITEs, patient tumor samples, OC cell lines (immortalized), and patient-derived and healthy donor PBMCs. Patient serum samples were analyzed by multiplex ELISA to determine cytokine and chemokine signatures. Results: Serum and PBMC samples from 10 patients and ascites from 2 patients were used for these analyses. Cells derived from patient ascites after progression on BiTEs were primarily comprised of tumor cells and myeloid-derived suppressor cells (MDSCs). These tumor cells had lost expression of the target antigen recognized by the BITE and upregulated markers as N-cadherin and vimentin with concordant downregulation of E-cadherin consistent with epithelial to mesenchymal transition (EMT). Ascites immune subset analysis showed a predominance of CD11B+, CD163+, and PD-L1+ cells. Compared to PBMCs from healthy donors, patient PBMCs showed higher frequencies of CD4+ TIM3+ LAG3+ cells consistent with T-cell dysfunction. Compared with healthy donors, serum from patients revealed higher levels of myeloid recruiting and activating chemokines and cytokines (CCL3, CXCR3, CXCL10, and IL18) and low immunostimulatory cytokines (IL-7 and IFN-α). Coculture of patient tumor cells with BiTEs and T-cells ex vivo showed diminished cytotoxicity, consistent with the loss of target antigen expression. Patient PBMCs retained cytotoxicity against OC cell lines ex vivo, suggesting preserved cytotoxic capacity. Finally, T-cells cocultured in the presence of patient serum displayed suppressed cytotoxicity against OC cell lines. Conclusions: Resistance to BiTE therapy is multifactorial, primarily involving tumor-associated antigen loss, EMT, and an immunosuppressive tumor microenvironment. Combination treatment approaches to mitigate these factors might increase response rates.

Abstract 2799: Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology

Abstract Transforming growth factor-beta (TGF-ß) is a master regulatory cytokine with pleotropic effects important in tumor development. TGF-ß1 binds TGF-ß receptor 2, which recruits and phosphorylates TGF-ß receptor 1 (TGFBR1). TGFBR1 in turn phosphorylates SMAD2 and SMAD3. Phosphorylated SMAD2/3 (pSMAD2/3) complexes with SMAD4 to translocate to the nucleus and enact transcriptional programs. In late-stage tumors, TGF-ß signaling is oncogenic, promoting growth, invasion, immune suppression, tissue remodeling, and epithelial to mesenchymal transition (EMT). In an effort to counter the oncogenic effects of TGF-ß signaling, Sumitomo Pharma Oncology, Inc. is developing TP-6379, an investigational, orally available, small molecule, selective TGFBR1 inhibitor. TP-6379 has a biochemical IC50 of 1.07 nM and has been shown to reduce pSMAD2 in Rhabdomyosarcoma cells with an IC50 of 108 nM. A dose dependent increase of TP-6379 was observed in plasma and tumors of A549 tumor bearing athymic nude mice (10, 50, 75, and 100 mg/kg PO). In another study using the same mouse model, pSMAD2 has shown an inverse correlation with pharmacokinetics showing > 90% inhibition for up to 4 hours (75 mg/kg PO). Pharmacodynamic (PD) biomarkers are critical for determining biological activity, but procuring serial tumor biopsies for PD analysis is challenging for many tumor types. We hypothesize that pSMAD2 can be measured in non-tumor tissues as a surrogate for activity within the tumor. Surrogate tissues, such as peripheral blood mononuclear cells (PBMCs), skin punches, and even circulating tumor cells (CTCs) are much less invasive and can be sampled over multiple timepoints. We observed a measurable decrease of PD biomarkers pSMAD2/3 in PBMCs and skin after treatment with TP-6379 using a bead-based immunoassay (Luminex). Healthy human donor PBMCs treated ex vivo for 2 hours with up to 1 µM TP-6379 showed a 96% reduction in pSMAD2/3 signal. Although TP-6379 was still active in reducing pSMAD2 in PBMCs when treated in whole blood for 2 hours, it required around 30-fold more drug to achieve a similar effect, possibly due to serum protein binding. Skin samples from in vivo dosed tumor bearing mice also show significant inhibition of pSMAD2/3, which correlated with significant pSMAD2/3 inhibition in tumors from both EMT6 and 4T1 syngeneic triple-negative breast cancer mouse tumor models. We confirmed that the Luminex assay is sensitive enough to reliably measure pSMAD2/3 in healthy human doner skin punches down to 3 mm in diameter. Lastly, CTCs from breast cancer patients’ whole blood treated ex vivo for 24 hours with as low as 1 µM TP-6379 showed a decrease in pSMAD2 and EMT marker SNAI1 by as much as 96% and 86%, respectively, when measured by immunofluorescence. In summary, our research has shown that pSMAD2/3 and EMT marker SNAI1 are valid PD biomarkers for TP-6379 therapy. We propose that these biomarkers would be best measured clinically using PBMCs, CTCs and/or skin punches. Citation Format: Richard E. Heinz, Curtis A. Allred, David A. Kircher, Yuta Matsumura, Bettina Franz, Zhizhou Ye, Jinny Lee, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui, Steven L. Warner. Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology [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 2799.

Characterization of "Off-Target" Immune Modulation Induced by Live Attenuated Yellow Fever Vaccine.

Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19.

Severe COVID-19 is characterized by cytokine storm, an excessive production of proinflammatory cytokines that contributes to acute lung damage and death. Dexamethasone is routinely used to treat severe COVID-19 and has been shown to reduce patient mortality. However, the mechanisms underlying the beneficial effects of dexamethasone are poorly understood. We conducted transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild disease, and patients with severe COVID-19 with and without dexamethasone treatment. We then treated healthy donor PBMCs in vitro with dexamethasone and investigated the effects of dexamethasone treatment ion channel abundance (by RT-qPCR and flow cytometry) and function (by electrophysiology, Ca2+ influx measurements and cytokine release) in T cells. We observed that dexamethasone treatment in severe COVID-19 inhibited pro-inflammatory and immune exhaustion pathways, circulating cytotoxic and Th1 cells, interferon (IFN) signaling, genes involved in cytokine storm, and Ca2+ signaling. Ca2+ influx is regulated by Kv1.3 potassium channels, but their role in COVID-19 pathogenesis remains elusive. Kv1.3 mRNA was increased in PBMCs of severe COVID-19 patients, and was significantly reduced in the dexamethasone-treated group. In agreement with these findings, in vitro treatment of healthy donor PBMCs with dexamethasone reduced Kv1.3 abundance in T cells and CD56dimNK cells. Furthermore, functional studies showed that dexamethasone treatment significantly reduced Kv1.3 activity, Ca2+ influx and IFN-g production in T cells. Our findings suggest that dexamethasone attenuates inflammatory cytokine release via Kv1.3 suppression, and this mechanism contributes to dexamethasone-mediated immunosuppression in severe COVID-19.

Zinc aspartate induces proliferation of resting and antigen-stimulated human PBMC under high-density cell culture condition

BackgroundZinc, one of the most important essential trace elements in the human body, regulates a wide range of cellular functions of immune cells, such as proliferation, differentiation and survival. Zinc deficiency affects both the innate and adaptive immune system. Zinc supplementation was discussed as possible therapy for infectious diseases and T cell-mediated autoimmune diseases. However, the influence of commercial zinc preparations on proliferation and cytokine production of resting and antigen-stimulated peripheral blood mononuclear cells (PBMC) has not yet been completely investigated. MethodsHere, we examined whether zinc aspartate (Unizink®), an approved drug to treat zinc deficiency in patients, induces proliferation, cytokine production, and induction of apoptosis/caspase 3/7 activity of resting PBMC under high-density cell culture condition. In addition, we performed antigen-specific proliferation experiments, where PBMCs of healthy donors vaccinated against Influenza A (H1N1) and/or SARS-CoV-2 were stimulated with Influenza A (H1N1) peptides or SARS-CoV-2 peptides as well as the Mixed Lymphocyte Culture (MLC) in the presence of increasing concentrations of zinc aspartate. ResultsWe observed a dose-dependent enhancement of proliferation and induction of cytokine production (IFN-γ, IL-5, GM-CSF and CXCL10) of resting PBMC in presence of zinc aspartate. The number of cells with active caspase 3/7 and, consecutively, the amount of cells undergoing apoptosis steadily decreased in presence of zinc aspartate. Moreover, zinc aspartate was capable of stimulating antigen-specific PBMC proliferation using MLC or influenza A (H1N1) and SARS-CoV-2 peptides in both a dose-dependent and a donor-specific manner. In the absence of zinc aspartate, we clearly could discriminate two groups of responders: low and high responders to antigenic stimulation. The addition of increasing concentration of zinc aspartate significantly stimulated the proliferation of PBMC from low responders, but not from high responders. ConclusionTaken together, our results suggest that zinc aspartate induces the proliferation of resting and antigen-stimulated PBMCs under high-density cell culture conditions. Thus, zinc might represent a supportive treatment in patients suffering from infectious diseases.

The mechanisms of anti-inflammatory action of enisamium iodide

The role of cyclooxygenases (COX-1 and/or COX-2), transcription nuclear factor NF-κB, anti-inflammatory cytokines - TGF1b, IL-4, IL-10 and pro-inflammatory cytokines IL-1, IL-6 were studied to substantiate the expediency of antiviral agent enisamium iodide (Nobazit) using to regulate key inflammatory components in acute respiratory infections, IL-8, TNF-alpha in the realization of the pharmacological activity of this drug. Gene expression was determined by real-time RT-PCR, the concentration of interleukins was determined by ELISA, and the viability of peripheral blood mononuclear cells (PBMC) was assessed by the MTT spectrophotometric method. The chemiluminescence method was used to assess PBMC oxidant activity. Enisamium iodide (10 μM) reduced mRNA levels of COX-1, COX-2, NF-κB, TGF1b, IL-1, IL-6 in stimulated PBMC of healthy donors by an average of 48% (p≤0.05). At 5 times higher concentration, 50 μM, enisamium iodide suppressed the expression of these genes by an average of 43% (p≤0.05). At a concentration of 100 μM, enisamium iodide reduced the expression of COX-2, TGF1b, IL-1, IL-6 by an average of 47% (p≤0.05). At a concentration of 10 μM, enisamium iodide stimulated the secretion of IL-10 by mononuclear cells by 1.2 times, p≤0.05. The tested drug at a concentration of 50 μM did not affected on the concentration of IL-1, IL-4, IL-8 and TNF-alpha, but significantly stimulated the production of IL-10 by 1.5 times, p≤0.05. The chemiluminescence method revealed that enisamium iodide in the entire concentration range (10-100 μM) does not reduce the viability of macrophages, but inhibits their oxidative activity (maximum value of CL intensity) by an average of 55% (p≤0.05). The anti-inflammatory effect of enisamium iodide at a concentration of 10 μM may be associated with inhibition of the expression of COX-1, 2, NF-κB, IL-1, IL-6, TGF1b and an increase in the expression and production of IL-10. An additional contribution to the anti-inflammatory activity of enisamium iodide is made by its antioxidant and antiradical activity. The absence of the effect of enisamium iodide (10-100 µM) on the viability of PBMC indicates its safety for the cells of the immune system and the expediency of using it to suppress inflammatory reactions in acute respiratory infections, restore the quality of life of patients and the possibility of using Nobazit as an effective agent for treatment of these infections of various etiologies.

Ex Vivo Treatment with Allogenic Mesenchymal Stem Cells of a Healthy Donor on Peripheral Blood Mononuclear Cells of Patients with Severe Alopecia Areata: Targeting Dysregulated T Cells and the Acquisition of Immunotolerance.

Alopecia areata (AA) is an autoimmune condition related to the collapse of the immune privilege of hair follicles. Certain AA populations present severe clinical manifestations, such as total scalp hair or body hair loss and a treatment refractory property. The aim of this study was to assess the effects of allogenic human mesenchymal stem cells (hMSCs) from healthy donors on the peripheral blood mononuclear cells (PBMCs) of severe AA patients, with a focus on the change in the cell fraction of Th1, Th17, and Treg cells and immunomodulatory functions. PBMCs of 10 AA patients and eight healthy controls were collected. Levels of Th17, Th1, and Treg subsets were determined via flow cytometry at baseline, activation status, and after co-culturing with hMSCs. All participants were severe AA patients with SALT > 50 and with a long disease duration. While the baseline Th1 and Treg levels of AA patients were comparable to those of healthy controls, their Th17 levels were significantly lower than those of the controls. When stimulated, the levels of CD4+IFN-γ+ T cells of the AA patients rose sharply compared to the baseline, which was not the case in those of healthy controls. The cell fraction of CD4+Foxp3+ regulatory T cells also abruptly increased in AA patients only. Co-culturing with allogenic hMSCs in activated AA PBMCs slightly suppressed the activation levels of CD4+INF-γ+ T cells, whereas it significantly induced the differentiation of CD4+Foxp3+ regulatory T cells. However, these changes were not prominent in the PBMCs of health controls. To examine the pathomechanisms, PBMCs of healthy donors were treated with IFN-γ to induce AA-like environment and then treated with allogenic grants and compared with ruxolitinib as a positive treatment control. hMSC treatment was shown to significantly inhibit the mRNA levels of proinflammatory cytokines, such as IFN-γ, TNF-α, IL-1α, IL-2R, IL-15, and IL-18, and chemokines, such as CCR7 and CCR10, in IFN-treated PBMCs. Interestingly, hMSCs suppressed the activation of JAK/STAT signaling by IFN in PBMCs with an effect that was comparable to that of ruxolitinib. Furthermore, the hMSC treatment showed stronger efficacy in inducing Foxp3, IL-10, and TGF-β mRNA transcription than ruxolitinib in IFN-treated PBMCs. This study suggests that allogenic hMSC treatments have therapeutic potential to induce immune tolerance and anti-inflammatory effects in severe AA patients.

Arginase-2-specific cytotoxic T cells specifically recognize functional regulatory T cells

BackgroundHigh expression of the metabolic enzyme arginase-2 (ARG2) by cancer cells, regulatory immune cells, or cells of the tumor stroma can reduce the availability of arginine (L-Arg) in the tumor...

Autologous humanized mouse models to study combination and single-agent immunotherapy for colorectal cancer patient-derived xenografts.

Designing studies of immunotherapy is limited due to a lack of pre-clinical models that reliably predict effective immunotherapy responses. To address this gap, we developed humanized mouse models of colorectal cancer (CRC) incorporating patient-derived xenografts (PDX) with human peripheral blood mononuclear cells (PBMC). Humanized mice with CRC PDXs were generated via engraftment of autologous (isolated from the same patients as the PDXs) or allogeneic (isolated from healthy donors) PBMCs. Human T cells were detected in mouse blood, tissues, and infiltrated the implanted PDXs. The inclusion of anti-PD-1 therapy revealed that tumor responses in autologous but not allogeneic models were more comparable to that of patients. An overall non-specific graft-vs-tumor effect occurred in allogeneic models and negatively correlated with that seen in patients. In contrast, autologous humanized mice more accurately correlated with treatment outcomes by engaging pre-existing tumor specific T-cell populations. As autologous T cells appear to be the major drivers of tumor response thus, autologous humanized mice may serve as models at predicting treatment outcomes in pre-clinical settings for therapies reliant on pre-existing tumor specific T-cell populations.

Evaluation of the local and circulating expression level of cannabinoid receptor 2 in patients with lung cancer

BackgroundAccumulating evidence suggests that cannabinoid receptor 2 (CB2R) plays determining and critical role in the pathophysiology of cancers and can influence the underlying mechanism of tumor cell proliferation and fate. To examine the expression pattern of this receptor in tumor tissue of patients with lung cancer and its relationship with disease exacerbation, our study has the advantage of simultaneously examining this receptor in peripheral blood mononuclear cells (PBMC) to determine the local and circulating pattern of this receptor in these patients. MethodsThe CB2R gene expression level was evaluated in 280 adenocarcinomas non-small lung cancer tumors, lung normal tissues and PBMC of patients and healthy donors through Real-Time PCR and the correlation of CB2R expression with the patient's clinical and pathological features were considered. ResultsBased on data, CB2R is considerably overexpressed in lung tumor tissue and PBMC of patients with lung cancer with a considerable increase in tumors with higher stage, metastatic feature and lung tumors with lymph node metastasis indicating the possible involvement of CB2R in lung tumor pathogenesis. The simultaneous increase in the CB2R expression at the tumor site and peripheral blood of patients can provide evidence for the possibility of examining this receptor as a non-invasive prognostic biomarker; while requiring more mechanistic evidence. ConclusionCollectively, CB2R overexpressed locally and in circulation in patients with lung cancer and was associated with tumor stage, metastasis and severity and may serve as a possible therapeutic target in lung cancer.

Distinct immune stimulatory effects of anti-human VISTA antibodies are determined by Fc-receptor interaction.

VISTA (PD-1H) is an immune regulatory molecule considered part of the next wave of immuno-oncology targets. VISTA is an immunoglobulin (Ig) superfamily cell surface molecule mainly expressed on myeloid cells, and to some extent on NK cells and T cells. In previous preclinical studies, some VISTA-targeting antibodies provided immune inhibitory signals, while other antibodies triggered immune stimulatory signals. Importantly, for therapeutic antibodies, the isotype backbone can have a strong impact on antibody function. To elucidate the mode of action of immune stimulatory anti-VISTA antibodies, we studied three different anti-human VISTA antibody clones, each on three different IgG isotypes currently used for therapeutic antibodies: unaltered IgG1 (IgG1-WT), IgG1-KO (IgG1-LL234,235AA-variant with reduced Fc-effector function), and IgG4-Pro (IgG4- S228P-variant with stabilized hinge region). Antibody functionality was analysed in mixed leukocyte reaction (MLR) of human peripheral blood mononuclear cells (PBMCs), as a model system for ongoing immune reactions, on unstimulated human PBMCs, as a model system for a resting immune system, and also on acute myeloid leukemia (AML) patient samples to evaluate anti-VISTA antibody effects on primary tumor material. The functions of three anti-human VISTA antibodies were determined by their IgG isotype backbones. An MLR of healthy donor PBMCs was effectively augmented by anti-VISTA-IgG4-Pro and anti-VISTA-IgG1-WT antibodies, as indicated by increased levels of cytokines, T cell activation markers and T cell proliferation. However, in a culture of unstimulated PBMCs of single healthy donors, only anti-VISTA-IgG1-WT antibodies increased the activation marker HLA-DR on resting myeloid cells, and chemokine levels. Interestingly, interactions with different Fc-receptors were required for these effects, namely CD64 for augmentation of MLR, and CD16 for activation of resting myeloid cells. Furthermore, anti-VISTA-IgG1-KO antibodies had nearly no impact in any model system. Similarly, in AML patient samples, anti-VISTA-antibody on IgG4-Pro backbone, but not on IgG1-KO backbone, increased interactions, as a novel readout of activity, between immune cells and CD34+ AML cancer cells. In conclusion, the immune stimulatory effects of antagonistic anti-VISTA antibodies are defined by the antibody isotype and interaction with different Fc-gamma-receptors, highlighting the importance of understanding these interactions when designing immune stimulatory antibody therapeutics for immuno-oncology applications.

P634: MHC-BASED LARGE-SCALE SCREENING FOR ANTI-TUMOR T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA REVEALS CD8+ T CELLS WITH SPECIFICITY AGAINST THE CLONOTYPIC B-CELL RECEPTOR IMMUNOGLOBULIN

Background: Chronic lymphocytic leukemia (CLL) is currently incurable, indicating a need for novel strategies towards disease eradication, including reinvigoration of anti-tumor immune responses. T cells in CLL appear selected by restricted antigens, with evidence suggesting that the selecting epitopes may lie within the clonotypic B-cell receptor immunoglobulins (BcR IG). Aims: We previously performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. Here, we performed major histocompatibility complex (MHC)-based large-scale screening to identify autologous CD8+ T cells recognizing the predicted neoepitopes. Methods: We evaluated 653 peptides derived from the clonotypic BcR IG of 25 CLL patients (median 21 predicted neoepitopes/patient, across 13 MHC-I alleles). Considering the MHC-I typing of each patient, we constructed patient-specific peptide-MHC multimers labeled with a unique DNA barcode plus a fluorochrome (PE). We generated MHC-specific multimer libraries which we then mixed with respective autologous T-cell enriched peripheral blood mononuclear cells (PBMCs). Duplicate samples/patient were analyzed. In addition, known viral peptide-MHC multimers labeled with a different fluorochrome (APC) as well as three MHC-matched healthy donor PBMCs were used as controls. PE- and APC-positive multimer-binding CD8+ T cells were sorted for each cell sample; subsequently, DNA barcode amplification and sequencing was performed. Sequencing data were processed (Barracoda software) to obtain the number of clonally reduced barcode reads assigned to a given sample and peptide-MHC specificity. The number of clonally reduced reads for a given pMHC specificity was used to estimate the frequency of T cells specific for a given epitope, based on the average number of T cell receptor–MHC multimer interactions detected in the total MHC multimer-binding T cell pool in a given cell sample. Peptides with a log fold change >2 were considered as significant [log fold change: (number of DNA barcode reads associated with a specific pMHC)/3x (number of total barcode reads derived from the same sample)]. Results: Overall, 3 peptide-MHC multimers were recognized by CD8+ T cells: (i) VTVADTAVYY (peptide A) and (ii) INLNPSLKRR (peptide B), both within the context of the A03*01 allele, and (iii) YSFTSYWINW (peptide C) within the context of the A24*02 allele. Peptide A derived from the IGHV4-34 FR3 region of a somatically hypermutated clonotypic BcR IG, containing a single A to V somatic hypermutation (SHM) at position 96. Peptide B derived from the IGHV4-39 CDR2-FR3 junction of a somatically hypermutated clonotypic BcR IG, containing 3 SHMs (T to I at position 65, Y to L at position 67 and S to R at position 74). Peptide C derived from the IGHV5-10-1 CDR1-FR2 junction of a clonotypic BcR IG assigned to stereotyped subset #1, containing the sole SHM of the IG (S to N at position 40). The immunogenicity of these peptides was further corroborated by the fact that they were recognized not only by the autologous T cells of the patient from whom the peptide derived, but also by T cells of other patients as well as a healthy donor sharing the respective MHC allele. Summary/Conclusion: In conclusion, we offer proof of concept that the targeted SHM which shapes the CLL BcR IG repertoire may produce idiotypic targets for T cell-based therapy or peptide vaccine design. Characterization of the epitope-binding T cells is currently underway by our group, aiming to provide further insight on how these cells could be recruited into effective anti-tumor responses.