Soluble IL-2 Research Articles

Changes in anthropometry, adiposity, and inflammation in Black and White women engaged in intentional weight loss.

We examined associations among changes in anthropometry, regional adiposity, and inflammatory markers in Black and White women participating in intentional weight loss. A total of 104 women with BMI ≥ 25 kg/m2 self-selected bariatric surgery (n = 66) or a diet and exercise program (n = 38). Anthropometric, dual-energy x-ray absorptiometry-quantified regional adiposity, and inflammatory markers (C-reactive protein [CRP], tumor necrosis factor α [TNF-α], soluble TNF receptor I [sTNFRI], sTNFRII, interleukin [IL]-6, and soluble IL-1 receptor antagonist) were measured at baseline and 6 months. Weight, BMI, visceral adipose tissue, and regional (android and gynoid) adiposity declined in the bariatric surgery group. Among bariatric surgery participants, Black women experienced declines of lesser magnitude in terms of weight and BMI than White women, but changes in regional adiposity and visceral adipose tissue did not differ. In the bariatric surgery group, decreases in weight and BMI were associated with decreases in CRP and IL-6 among White women, but not Black women. Decreases in weight, BMI, and android fat were associated with increases in TNF-α, sTNFRI, and sTNFRII among Black women, but not White women. Decreases in anthropometry and adiposity were observed among Black and White bariatric surgery participants; however, associations among changes in adiposity, anthropometry, and inflammation differed by race.

Regulation of plasma soluble receptors of TNF and IL-1 in patients with COVID-19 differs from that observed in sepsis

IL-1α/β and TNF are closely linked to the pathology of severe COVID-19 and sepsis. The soluble forms of their receptors, functioning as decoy receptors, exhibit inhibitory effects. However, little is known about their regulation in severe bacterial and viral infections, which we aimed to investigate in this study. The circulating soluble receptors of TNF (sTNFR1 and sTNFR2) and IL-1α/β (sIL-1R1, sIL-1R2) were evaluated in the plasma of patients with COVID-19, severe bacterial infections, and sepsis and compared with healthy controls. Additionally, IL1R1, IL1R2, TNFRSF1A, and TNFRSF1B expression was evaluated at the single cell level in PBMCs derived from COVID-19 or sepsis patients. Plasma concentrations of sIL-1R1, sTNFR1, and sTNFR2 were significantly higher in COVID-19 patients compared to healthy subjects. Notably, sIL-1R1 levels were particularly elevated in ICU COVID-19 patients, and transcriptome analysis indicated heightened IL1R1 expression in PBMCs from severe COVID-19 patients. In severe bacterial infections, only sTNFR1 and sTNFR2 exhibited increased levels compared to healthy controls. Sepsis patients had decreased sIL-1R1 plasma concentrations but elevated sIL-1R2, sTNFR1, and sTNFR2 levels compared to healthy individuals, reflecting the heightened expression due to the increased numbers of monocytes present in sepsis. Finally, elevated concentrations of sIL-1R2, sTNFR1, and sTNFR2 were moderately associated with reduced 28-day survival in sepsis patients. Our study reveals distinct regulation of plasma concentrations of soluble IL-1 receptors in COVID-19 and sepsis. Moreover, soluble TNF receptors 1 and 2 consistently rise in all conditions and show a positive correlation with disease severity in sepsis.

TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant.

High-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in HR-NBL patients; however, graft-versus-host-disease (GVHD) and disease progression have limited clinical application. Ex-vivo stimulated allogeneic natural killer (NK) cells represent a potential approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL. Ex-vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble IL-15/IL-15Rα alone or with irradiated CD137L/CD54+ AgN2a-4P (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-TIM-3. Lethally irradiated B6AJF1 Mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish their contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and overall survival. Co-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Ki-67 and NKG2D, NKp46, TRAIL and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared to IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared to NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomes, TRAIL and FasL expression observed. Analysis of RNA from 15-4P NK cells exposed to TIM-3 blockade showed gene expression of chemokines, NKG2D/DAP12 signaling, non-canonical NF-κb pathway and TRAIL signaling. Blockade of NKG2D, TRAIL or FasL on 15-4P NK cells abrogated cytotoxicity. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared to NK cells or anti-TIM-3 alone, without inducing GVHD. Depletion of NK cells, but not T cells, abrogated the GVT effect. Allo-HCT can be a platform for treating NBL using combination ex-vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD.

Circulating Markers of Necroptosis in Acute Pancreatitis

ObjectivesNecroptosis, a programmed inflammatory cell death, is involved in the pathogenesis of acute pancreatitis (AP). We compared levels of interleukin (IL)-33 (released upon necroptosis), sST2 (soluble IL-33 receptor), MLKL, RIPK1 and RIPK3 (necroptosis executioner proteins), and proinflammatory cytokines IL-6, TNF and IL-1β at various severity categories and stages of AP.MethodsPlasma from 20 patients with early mild AP (MAP) (symptom onset < 72 h), 7 with severe AP (SAP) without and 4 with persistent organ failure (OF) at sampling, 8 patients with late SAP and 20 healthy controls (HC) were studied by ELISAs.ResultsEarly sST2 and IL-6 levels predicted the development of SAP and were higher in both MAP and early and late SAP than in HC. RIPK3 levels were higher than in HC in the patients who had or would later have SAP. MLKL levels were associated with the presence of OFs, particularly in the late phase, but were also higher in MAP than in HC.ConclusionssST2, RIPK3 and IL-6 levels may have prognostic value in AP. Elevated MLKL levels are associated with OF in AP. Better understanding of necroptosis in AP pathophysiology is needed to evaluate whether inhibiting and targeting necroptosis is a potential therapeutic option in AP.

A Systematic Review of the Predictive and Diagnostic Uses of Neuroinflammation Biomarkers for Epileptogenesis.

A central role for neuroinflammation in epileptogenesis has recently been suggested by several investigations. This systematic review explores the role of inflammatory mediators in epileptogenesis, its association with seizure severity, and its correlation with drug-resistant epilepsy (DRE). The study analysed articles published in JCR journals from 2019 to 2024. The search strategy comprised the MESH, free terms of "Neuroinflammation", and selective searches for the following single biomarkers that had previously been selected from the relevant literature: "High mobility group box 1/HMGB1", "Toll-Like-Receptor 4/TLR-4", "Interleukin-1/IL-1", "Interleukin-6/IL-6", "Transforming growth factor beta/TGF-β", and "Tumour necrosis factor-alpha/TNF-α". These queries were all combined with the MESH terms "Epileptogenesis" and "Epilepsy". We found 243 articles related to epileptogenesis and neuroinflammation, with 356 articles from selective searches by biomarker type. After eliminating duplicates, 324 articles were evaluated, with 272 excluded and 55 evaluated by the authors. A total of 21 articles were included in the qualitative evaluation, including 18 case-control studies, 2 case series, and 1 prospective study. As conclusion, this systematic review provides acceptable support for five biomarkers, including TNF-α and some of its soluble receptors (sTNFr2), HMGB1, TLR-4, CCL2 and IL-33. Certain receptors, cytokines, and chemokines are examples of neuroinflammation-related biomarkers that may be crucial for the early diagnosis of refractory epilepsy or may be connected to the control of epileptic seizures. Their value will be better defined by future studies.

CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS. Murine NK cells were activated and expanded ex vivo with soluble IL-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, GVHD and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray. AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Treatment with combination CD155 and anti-DNAM-1 pretreated alloNK ameliorated survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS. These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT. Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against OS and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

Unraveling the non-fitness status of NK cells: Examining the NKp30 receptor and its isoforms distribution in HIV/HCV coinfected patients

BackgroundHIV/HCV coinfection is associated with a rapid progression to liver damage. Specifically, NK cell population dysregulation is of particular interest, as these cells have been shown to block HCV replication effectively and have an anti-fibrogenic activity. The NKp30 receptor is linked to tumor cell lysis and has a crucial role during viral infections. In the present study, we determined the subpopulations of NK cells based on CD56 and CD16 expression, NKp30 receptor expression, its isoforms A, B, and C, along with the cytotoxicity molecules in patients with HIV/HCV. Results: evidenced by the APRI and FIB-4 indices, the HCV-infected patients presented greater liver damage than the HIV and HIV/HCV groups. The HCV group presented a decreased expression of NKp30 isoform A, and NK cell frequency was not different between groups; however, CD56brigth subpopulation, NKp30 receptor, and CD247 adaptor chain were decreased in HIV/HCV patients; further, we described increased levels of soluble IL-8, IL-10, IL-12, and IL-23 in the serum of HIV/HCV patients. Conclusions: HCV and HIV/HCV patients have multiple parameters of non-fitness status in NK cells; awareness of these dysfunctional immunological parameters in HIV/HCV and HCV patients can elucidate possible novel therapeutics directed towards the improvement of NK cell fitness status, in order to improve their function against liver damage.

Polymer Micropatches as B-Cell Engagers.

B cells, despite their several unique functionalities, remain largely untapped for use as an adoptive cell therapy and are limited to in vitro use for antibody production. B cells can be easily sourced, they possess excellent lymphoid-homing capabilities, and they can act as antigen-presenting cells (APCs), offering an alternative to dendritic cells (DCs), which have shown limited efficacy in the clinical setting. Soluble factors such as IL-4 and anti-CD40 antibody can enhance the activation, survival, and antigen-presenting capabilities of B cells; however, it is difficult to attain sufficiently high concentrations of these biologics to stimulate B cells in vivo. Micropatches as Cell Engagers (MACE) are polymeric microparticles, surface functionalized with anti-CD40 and anti-IgM, which can attach to B cells and simultaneously engage multiple B-cell receptors (BCR) and CD40 receptors. Stimulation of these receptors through MACE, unlike free antibodies, enhanced the display of costimulatory molecules on the B-cell surface, increased B-cell viability, and improved antigen presentation by B cells to T cells in vitro. B-cell activation by MACE further synergized with soluble IL-4 and anti-CD40. MACE also elicited T-cell chemokine secretion by B cells. Upon intravenous adoptive transfer, MACE-bound B cells homed to the spleen and lymph nodes, key sites for antigen presentation to T cells. Adoptive transfer of MACE-B cells pulsed with the CD4+ and CD8+ epitopes of ovalbumin significantly delayed tumor progression in a murine subcutaneous EG7-OVA tumor model, demonstrating the functional benefit conferred to B cells by MACE.

Self-sufficient primary natural killer cells engineered to express T cell receptors and interleukin-15 exhibit improved effector function and persistence.

NK cells can be genetically engineered to express a transgenic T-cell receptor (TCR). This approach offers an alternative strategy to target heterogenous tumors, as NK:TCR cells can eradicate both tumor cells with high expression of HLA class I and antigen of interest or HLA class I negative tumors. Expansion and survival of NK cells relies on the presence of IL-15. Therefore, autonomous production of IL-15 by NK:TCR cells might improve functional persistence of NK cells. Here we present an optimized NK:TCR product harnessed with a construct encoding for soluble IL-15 (NK:TCR/IL-15), to support their proliferation, persistence and cytotoxic capabilities. Expression of tumor-specific TCRs in peripheral blood derived NK-cells was achieved following retroviral transduction. NK:TCR/IL-15 cells were compared with NK:TCR cells for autonomous cytokine production, proliferation and survival. NK:BOB1-TCR/IL-15 cells, expressing a HLA-B*07:02-restricted TCR against BOB1, a B-cell lineage specific transcription factor highly expressed in all B-cell malignancies, were compared with control NK:BOB1-TCR and NK:CMV-TCR/IL-15 cells for effector function against TCR antigen positive malignant B-cell lines in vitro and in vivo. Viral incorporation of the interleukin-15 gene into engineered NK:TCR cells was feasible and high expression of the TCR was maintained, resulting in pure NK:TCR/IL-15 cell products generated from peripheral blood of multiple donors. Self-sufficient secretion of IL-15 by NK:TCR cells enables engineered NK cells to proliferate in vitro without addition of extra cytokines. NK:TCR/IL-15 demonstrated a marked enhancement of TCR-mediated cytotoxicity as well as enhanced NK-mediated cytotoxicity resulting in improved persistence and performance of NK:BOB1-TCR/IL-15 cells in an orthotopic multiple myeloma mouse model. However, in contrast to prolonged anti-tumor reactivity by NK:BOB1-TCR/IL-15, we observed in one of the experiments an accumulation of NK:BOB1-TCR/IL-15 cells in several organs of treated mice, leading to unexpected death 30 days post-NK infusion. This study showed that NK:TCR/IL-15 cells secrete low levels of IL-15 and can proliferate in an environment lacking cytokines. Repeated in vitro and in vivo experiments confirmed the effectiveness and target specificity of our product, in which addition of IL-15 supports TCR- and NK-mediated cytotoxicity.

Abstract LB072: Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans

Abstract Introduction: We have previously shown the successful engineering of membrane-bound IL15 (mbIL15)-expressing tumor-infiltrating lymphocytes (TIL; cytoTIL15™ cells) from solid tumors such as melanoma, which are CD8+ enriched TIL exhibiting enhanced persistence and anti-tumor activity compared with unengineered TIL (SITC 2022, 2023; clinical candidate OBX-115: NCT05470283). The mbIL15 expressed by cytoTIL15 cells is regulated via a carbonic anhydrase 2 (CA2) drug-responsive domain, which allows expression to be induced upon delivery of the FDA-approved small molecule ligand acetazolamide (ACZ) and was designed to engage the IL2Rβ and IL2Rγ receptors in both cis and in trans. Using natural killer (NK) cells, which are potently responsive to soluble, trans-presented IL15, as an experimental model system, we examined the potential of mbIL15 on cytoTIL15 cells to potentiate activity in both cis and trans. Methods: cytoTIL15 cells and unengineered TIL were generated as described previously (SITC 2022, 2023). Cells were assessed for expression of mbIL15 and phosphorylation of downstream signaling molecules STAT5 and S6 by flow cytometry, and co-cultured with allogeneic healthy donor NK cells for 1-10 days with and without transwell inserts. Functional effects of cytoTIL15 cells were compared to those of soluble recombinant IL15. Supernatant was obtained from co-cultures for assessment of cytokine production. Results: cytoTIL15 cells demonstrated robust ACZ-dependent mbIL15 expression, and ACZ dosing regulated mbIL15 activity in cis by potentiating the phosphorylation of STAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.012) and S6 (geoMFI with 0 vs. 25 µM ACZ, p=0.016). Upon removal of ACZ, mbIL15 expression and pSTAT5 and pS6 returned to basal levels within 24 hours. mbIL15 expressed on the surface of cytoTIL15 cells could also activate cells in trans in an ACZ-dependent titratable manner. Specifically, co-culture of cytoTIL15 cells with NK cells led to an ACZ-dependent increase in pSTAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.038) in NK cells, which was not seen in co-cultures of unengineered TIL with NK cells. Additionally, NK cells showed a similar cytokine production profile when exposed to mbIL15 by cytoTIL15 cells as with soluble, trans-presented IL15. NK cell transactivation relied on direct cell contact with cytoTIL15 cells, as magnitudes of phosphorylation were decreased in co-cultures with NK and cytoTIL15 cells separated by a transwell membrane (pSTAT5 geoMFI p=0.008). Conclusions: cytoTIL15 cells demonstrate functional engagement of cytokine receptors via mbIL15 in both cis and in trans, including phosphorylation of STAT5 and S6. Taken together, these data highlight the potential for robust activity of cytoTIL15 cells through potentiation of ACZ-dependent mbIL15 activity in both cis and trans. Citation Format: Rachel Burga, Gauri Kulkarni, Zheng Ao, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB072.

ST2 and IL-33 polymorphisms and the development of childhood asthma: a prospective birth cohort study in Finnish children.

The ST2/IL-33 signaling pathway has an important role in the host inflammatory response. Here we aimed to study the association of ST2 and IL-33 polymorphisms with serum soluble (s) ST2 and IL-33 concentrations in healthy Finnish children and, in addition, their association with childhood asthma. In total, 146 children were followed from birth to the age 7 years for the development of asthma. Single-nucleotide polymorphisms (SNPs) in ST2 and IL-33 were determined, and associations of the SNP variants with serum levels of sST2 and IL-33 at age of 13 months and with recurrent wheezing and childhood asthma at 7 years of age were analyzed. Children with ST2 rs1041973 AC/AA genotypes had significantly lower level of serum sST2 (2453 pg/mL; IQR 2265) than those with CC genotype (5437 pg/mL; IQR 2575; p = < 0.0001). Similar difference was also observed with ST2 rs13408661. No differences were observed between subjects with studied IL-33 SNPs. Children who carried genetic variants of ST2 rs1041973 or rs13408661 seemed to have a higher risk of asthma. In contrast, children who carried genetic variants of IL-33 rs12551268 were less often diagnosed with asthma. Even though these SNPs seemed to associate with asthma, the differences were not statistically significant.

Preclinical Evaluation of Off-The-Shelf PD-L1+ Human Natural Killer Cells Secreting IL15 to Treat Non-Small Cell Lung Cancer.

We described previously a human natural killer (NK) cell population that upregulates PD-L1 expression upon recognizing and reacting to tumor cells or exposure to a combination of IL12, IL18, and IL15. Here, to investigate the safety and efficacy of tumor-reactive and cytokine-activated (TRACK) NK cells, human NK cells from umbilical cord blood were expanded, transduced with a retroviral vector encoding soluble (s) IL15, and further cytokine activated to induce PD-L1 expression. Our results show cryopreserved and thawed sIL15_TRACK NK cells had significantly improved cytotoxicity against non-small cell lung cancer (NSCLC) in vitro when compared with non-transduced (NT) NK cells, PD-L1+ NK cells lacking sIL15 expression (NT_TRACK NK), or NK cells expressing sIL15 without further cytokine activation (sIL15 NK cells). Intravenous injection of sIL15_TRACK NK cells into immunodeficient mice with NSCLC significantly slowed tumor growth and improved survival when compared with NT NK and sIL15 NK cells. The addition of the anti-PD-L1 atezolizumab further improved control of NSCLC growth by sIL15_TRACK NK cells in vivo. Moreover, a dose-dependent efficacy was assessed for sIL15_TRACK NK cells without observed toxicity. These experiments indicate that the administration of frozen, off-the-shelf allogeneic sIL15_TRACK NK cells is safe in preclinical models of human NSCLC and has potent antitumor activity without and with the administration of atezolizumab. A phase I clinical trial modeled after this preclinical study using sIL15_TRACK NK cells alone or with atezolizumab for relapsed or refractory NSCLC is currently underway (NCT05334329).

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor 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 1338.

Abstract 1322: A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated MM cell killing in vitro and in vivo

Abstract Introduction Multiple myeloma (MM) is the second most prevalent hematopoietic malignancy with an overall 5-year survival rate of 58%. B cell maturation antigen (BCMA) is selectively expressed on normal and malignant plasma cells, making it an attractive MM target. Anti-BCMA chimeric antigen receptor (CAR) T cell therapy and T cell engagers have shown promise as treatment options for certain MM patients; however, potential toxicities associated with these anti-BCMA therapies, such as cytokine release syndrome (CRS), have limited broader utilization. Much like T cells, natural killer (NK) cells are cytolytic and have demonstrated an innate capacity to reduce tumor burden while exhibiting a more favorable safety profile in clinical testing. To that end, we developed a cryopreserved allogeneic cell therapy comprised of genetically modified human umbilical cord blood-derived (CB) NK cells transduced with a gammaretroviral vector, which incorporates genes for an anti-BCMA CAR and soluble human interleukin-15 (sIL-15). Referred to hereafter as anti-BCMA CAR-NK, it exhibits both innate NK- and CAR-mediated killing in vitro and robust in vivo activity against established MM tumors. Methods CBNK cells were isolated from donor cord blood units, propagated using feeder cells, transduced with a gammaretroviral vector to express an anti-BCMA CAR and soluble human IL-15, and propagated further before harvest and cryopreservation. Donor-equivalent untransduced (UTD) NK cells were generated via the same process but without the transduction step. Cryopreserved anti-BCMA CAR-NK and UTD NK cells were used for in vitro studies, including short-term killing, as well as for evaluation of activity against established MM tumors in vivo using the MM.1S-Luc4 model. Results The anti-BCMA CAR was successfully expressed across all batches of anti-BCMA CAR-NK generated. Results from an in vitro cytotoxicity assay indicated that anti-BCMA CAR-NK kills BCMA expressing MM.1S-Luc4 tumor cells and secretes IFNγ, TNFα, and granzyme B at greater amounts compared to donor-equivalent UTD NK. Anti-BCMA CAR-NK and UTD NK cells also demonstrated equivalent cytotoxicity towards BCMA non-expressing cell lines, JJN3-Luc BCMA KO and NCI-H520, in an effector:target cancer cell dependent manner, highlighting the potential for treatment with anti-BCMA CAR-NK post-prior BCMA therapy. In in vivo studies, anti-BCMA CAR-NK exhibited robust anti-tumor activity in an MM.1S-Luc4 NSG mouse xenograft model, with no signs of anti-BCMA CAR-NK related body weight loss. Conclusion A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated killing in vitro and robust in vivo activity in MM tumor models. Preclinical data supports future clinical evaluation in relapsed/refractory MM patients who have received prior BCMA therapy and IND enabling studies are ongoing. Citation Format: LeeAnn Talarico, Christina Wong, Chunyan Pang, Taylor Hickman, Chenqi Hu, Amy Shaw, Emily Wisniewski, Shao-Chiang (Michael) Lai, Pranjal Sharma, Shaun Moore, Luan Nguyen, Kayla Rhuda, Saurin Patel, Paul Lin, Rafet Basar, Shawn Cogan, Kat Sofjan, Arun Ramamurthy, Aaron Handler, Kathryn Fraser, Yana Wang, Katayoun Rezvani, Michael D. Curley. A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated MM cell killing in vitro and in vivo [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 1322.

Abstract 1318: Targeting multiple myeloma with BCMA-CAR NK cells expressing a GPRC5D-NKG2D bispecific antibody

Abstract Recently developed therapeutics for multiple myeloma (MM) include targeted therapy, immunomodulatory drugs, and immunotherapy, the latter of which consists of B-cell maturation antigen (BCMA)-CAR cells, bispecific antibodies (BsAbs) to engage T cells. However, challenges including relapse and toxicity still exist. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), emerged as a novel target for MM therapy. Therefore, following our established system [Cancer Immunol Res PMID: 29769244; Leukemia, PMID:24067492], we generated a BsAb by fusing anti-GPCR5D single-chain variable fragment (scFv) and an anti-NKG2D scFv (GPRC5D-NKG2D BsAb) in a human IgG4 platform and expressed it from BCMA-CAR and soluble IL15 natural killer (NK) cells (s15.BsAb.BCMA-CAR NK) to address the current known limitations. Our data showed that s15.BsAb.BCMA-CAR NK cells secreted GPRC5D-NKG2D BsAb both in vitro and in vivo. To evaluate the function of GPRC5D- NKG2D BsAb expressed from NK cells in vivo, we treated MM tumor-bearing mice with the purified antibody. The data showed that GPRC5D-NKG2D BsAb demonstrated a significantly better survival effect than saline (P≤0.01). We tested s15.BsAb.BCMA-CAR NK cells for cytotoxicity against a homogenous tumor cell mode (BCMA + GPRC5D + ) and a heterogeneous MM tumor cell model, including 50% BCMA + GPRC5D + , 25% BCMA ─ GPRC5D + , and 25% BCMA + GPRC5D ─ cells. Real-time cell analysis (RTCA) data showed s15.BsAb.BCMA-CAR NK cells killed tumor cells in vitro better than both BCMA-CAR NK cells and empty vector-transduced NK cells in an antigen-dependent manner. To test the efficacy of s15.BsAb.BCMA-CAR NK cells in vivo, we established a heterogeneous MM tumor cell mouse model, by mixing BCMA + GPRC5D + OPM2 cells and BCMA + GPRC5D ─ OPM2 cells at the ratio of 2:1. MM mice were treated with “off-the-shelf” different construct NK cells. The results showed that s15.BsAb.BCMA-CAR NK cells significantly prolonged survival compared to other NK cells(s15.BsAb.BCMA-CAR vs. BCMA-CAR P = 0.0012). Our data demonstrated that this all-in-one, multifaceted modality provides 2 ready-to-use biologics simultaneously, CAR NK cells and BsAb, for MM treatment. Citation Format: Lei Tian, Victoria Chen, Tasha Barr, Zhiyao Li, Yuqing Chen, Jiaying Zhang, Michael A. Caligiuri, Jianhua Yu. Targeting multiple myeloma with BCMA-CAR NK cells expressing a GPRC5D-NKG2D bispecific antibody [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 1318.

Altered IL-7 signaling in CD4+ T cells from patients with visceral leishmaniasis.

CD4+ T cells play a central role in control of L. donovani infection, through IFN-γ production required for activation of macrophages and killing of intracellular parasites. Impaired control of parasites can in part be explained by hampered CD4+ T cells effector functions in visceral leishmaniasis (VL) patients. In a recent studies that defined transcriptional signatures for CD4+ T cells from active VL patients, we found that expression of the IL-7 receptor alpha chain (IL-7Rα; CD127) was downregulated, compared to CD4+ T cells from endemic controls (ECs). Since IL-7 signaling is critical for the survival and homeostatic maintenance of CD4+ T cells, we investigated this signaling pathway in VL patients, relative to ECs. CD4+ T cells were enriched from peripheral blood collected from VL patients and EC subjects and expression of IL7 and IL7RA mRNA was measured by real time qPCR. IL-7 signaling potential and surface expression of CD127 and CD132 on CD4+ T cell was analyzed by multicolor flow cytometry. Plasma levels of soluble IL-7 and sIL-7Rα were measured by ELISA. Transcriptional profiling data sets generated previously from our group showed lower IL7RA mRNA expression in VL CD4+ T cells as compared to EC. A significant reduction was, however not seen when assessing IL7RA mRNA by RT-qPCR. Yet, the levels of soluble IL-7Rα (sIL-7Rα) were reduced in plasma of VL patients compared to ECs. Furthermore, the levels of soluble IL-7 were higher in plasma from VL patients compared to ECs. Interestingly, expression of the IL-7Rα protein was higher on VL patient CD4+ T cells as compared to EC, with activated CD38+ CD4+ T cells showing higher surface expression of IL-7Rα compared to CD38- CD4+ T cells in VL patients. CD4+ T cells from VL patients had higher signaling potential baseline and after stimulation with recombinant human IL-7 (rhIL-7) compared to EC, as measured by phosphorylation of STAT5 (pSTAT5). Interestingly, it was the CD38 negative cells that had the highest level of pSTAT5 in VL patient CD4+ T cells after IL-7 stimulation. Thus, despite unaltered or potentially lowered IL7RA mRNA expression by CD4+ T cells from VL patients, the surface expression of the IL-7Rα was higher compared to EC and increased pSTAT5 was seen following exposure to rhIL-7. Accordingly, IL-7 signaling appears to be functional and even enhanced in VL CD4+ T cells and cannot explain the impaired effector function of VL CD4+ T cells. The enhanced plasma IL-7 may serve as part of homeostatic feedback mechanism regulating IL7RA expression in CD4+ T cells.

Immunomodulatory Effect of Hypericin-Mediated Photodynamic Therapy on Oral Cancer Cells.

In 2020, there were 377,713 new oral and lip cancer diagnoses and 177,757 deaths. Oral cancer is a malignancy of the head and neck region, and 90% of cases are squamous cell carcinomas (OSCCs). One of the alternative methods of treating pre-cancerous lesions and oral cancer is photodynamic therapy (PDT). In addition to the cytotoxic effect, an important mechanism of PDT action is the immunomodulatory effect. This study used the OSCC (SCC-25) cell line and the healthy gingival fibroblast (HGF-1) line. A compound of natural origin-hypericin (HY)-was used as the photosensitizer (PS). The HY concentrations of 0-1 µM were used. After two hours of incubation with PS, the cells were irradiated with light doses of 0-20 J/cm2. The MTT test determined sublethal doses of PDT. Cell supernatants subjected to sublethal PDT were assessed for interleukin 6 (IL-6), soluble IL-6 receptor alpha (sIL-6Ralfa), sIL-6Rbeta, IL-8, IL-10, IL-11 IL-20, IL-32, and Pentraxin-3 using the Bio-Plex ProTM Assay. The phototoxic effect was observed starting with a light dose of 5 J/cm2 and amplified with increasing HY concentration and a light dose. HY-PDT affected the SCC-25 cell secretion of sIL-6Rbeta, IL-20, and Pentraxin-3. HY alone increased IL-8 secretion. In the case of HGF-1, the effect of HY-PDT on the secretion of IL-8 and IL-32 was found.

Engineered Natural Killer Cells Expressing Chimeric Ilt Receptors (CIR) Effectively Target HLA-G Positive AML Tumor Cells

Background Human leukocyte antigen-G (HLA-G), the immune checkpoint of pregnancy, directs immunosuppression by binding to Ig-like transcript (ILT) receptors (ILT2 in lymphoid cells and ILT4 in myeloid cells) via immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domains. HLA-G is expressed in more than 50% of solid tumors and leukemias as a mechanism to evade immune attack. As a result, HLA-G is an excellent tumor-specific antigen since its expression in normal tissues is low except for cytotrophoblasts in the placenta where it protects the fetus from the mother's immune system. We present here a novel approach to target HLA-G-expressing Acute Myeloid Leukemia (AML) cells by utilizing the natural receptors of ILT2 and ILT4 coupled to positive intracellular signaling domains. Such chimeric ILT receptors, or CIRs drive activation of Natural Killer (NK) cells. Further, we have identified unique intracellular activation modules that function to robustly enhance cytotoxicity and expansion of NK cells upon engagement with tumor cells. Methods CD56 + NK cells were isolated from PBMCs, cultured with IL-15 and activated with a feeder-free cocktail of an immobilized cytokine and activating ligand. Activated NK cells were transduced with γ-retroviruses directing expression of CIR proteins (CIR.4-1BB.CD3ζ or CIR.X.Y where X and Y are candidate activation and coactivation moieties), soluble IL-15 and a ΔCD19 marker or CAR control constructs directed to HLA-G or CD33. After 8 days of expansion, CIR- or CAR-NK cells were cocultured with GFP ffluc-expressing AML target cell lines with or without transgenic HLA-G (G1, G2 and G5) isoforms. Cytotoxicity of CIR-NK cells was determined by reduction of GFP fluorescence in Incucyte or Celligo instruments or luciferase activity and supernatants were analyzed for IFN-γ and TNF-α release by ELISA. Transduced NK cells were labeled with nuclear-RFP for measurement of expansion in coculture. In comparative experiments, T cells from PBMCs were transduced to produce CIR-T cells by standard methods. Results CIR (ILT2 or ILT4.4-1BB.ζ) constructs were efficiently and stably expressed in T and NK cells (&amp;gt;80% ΔCD19 + 8- and 14-days post activation). Mock-transduced NK cells displayed innate killing activity against HLA-G negative AML cells (THP1 and KG1) that was not augmented by CIR constructs (mock = 6.16E6 ± 0.77E6 vs. CIR = 6.43E6 ± 0.37E6 in coculture against KG1 cells). In cocultures against THP1 and KG1 target cells expressing exogenous G1, G2 or G5 isoforms, CIR-NK cells or CIR-T cells displayed enhanced target cell killing in 2 and 7-day assays relative to mock-transduced effector cells (mock = 1.47E6 ± 0.49E6 vs. CIR = 0.57E6 ± 0.6 in coculture against KG1-HLA-G1). Further, while mock NK cells transduced with RFP alone could kill one-third of AML cells expressing endogenous HLA-G1 and G5 (Molm13 and Kasumi1), CIR-NK cells eliminated more than 90% of these AMLs and their activivation resulted in 4-fold higher IFN-γ secretion. Notably, robust cytotoxicity of CIR-NK cells was maintained even at 28 days post activation (mock = 36.95E6 ± 21.85E6 vs. CIR = 7.38E6 ± 1.92 in coculture against Kasumi1). To further enhance the functionality of the CIR constructs, a screen of novel costimulatory molecules to replace 4-1BB and/or CD3ζ resulted in several potent candidates ultilizing MyD88 signaling that greatly improved the cytotoxicity (&amp;gt; 80% tumor elimination), proliferation (&amp;gt; 2-fold expansion) and proinflammatory cytokine production (IFN-γ and TNF-α) of NK cells in cocultures. A subset is displayed in Figure 1. Conclusions The use of natural receptors ILT2 and ILT4 to target HLA-G takes advantage of the physiological pairing of the ILT receptors with the multiple immunosuppressive HLA-G isoforms thereby circumventing the possibility of antigen escape often observed in CAR-directed therapy. We demonstrated that engineered CIR-NK cells can convert HLA-G expression in AML from a potent inhibitor of leukocyte activity into a target for anti-tumor control.

API-192, an Allogeneic CD19/20 CAR-NKT Cell Product Derived from Cord Blood CD34+ HSPCs for the Treatment of B-Cell Malignancies

Autologous chimeric antigen receptor (CAR)-T cell therapies induce durable remissions and improve survival in patients with advanced B cell malignancies. However, challenges such as manufacturing variability, cost, and time to treatment highlight the need for an allogeneic, off-the-shelf approach that preserves the efficacy of autologous CAR-T. Additionally, antigen escape is one of the established mechanisms of resistance to current CAR-T therapies which target a single antigen. Classical natural killer T (NKT) cells express an invariant T cell receptor (iTCR) that is restricted to the conserved CD1d protein, making it a promising cell type for allogeneic therapies as they do not require TCR gene editing to avoid graft-vs-host (GvH) disease. Furthermore, NKT cells mediate dendritic cell cross-priming, target immunosuppressive myeloid cells/macrophages in the tumor microenvironment, and directly kill CD1d + tumor cells through the iTCR. Endogenous NKT cells are an extremely rare population in the blood (&amp;lt;1% of lymphocytes), posing manufacturing challenges. API-192 is a human cord blood CD34 + hematopoietic stem and progenitor cell-derived NKT cell product engineered to express dual CARs targeting CD19 and CD20 and armored with soluble IL-15 for improved expansion and persistence. The Appia Cells Utilized for Allogeneic (ACUA) platform enables the production of large numbers of product NKT cells co-expressing multiple transgenes at high purity (&amp;gt;90% iTCR + 19CAR + 20CAR +) with no purification step or gene editing. Phenotypically, API-192 cells are predominantly CD4 -CD8 - or CD4 -CD8 +, express major activating NK receptors, and harbor intracellular stores of cytotoxic molecules, resembling endogenous NKT cells. API-192 kills Raji and Nalm6 tumor cells in a CAR-specific and dose-dependent manner and can control multiple rounds of tumor challenge in vitro. API-192 secretes pro-inflammatory cytokines and proliferates in response to tumor antigen . In human tumor xenograft models in immunocompromised mice, API-192 robustly expands, suppresses tumor growth, and exhibits prolonged persistence, enabling control of a tumor rechallenge 70 days after initial dosing. API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less IFN-γ release by NK and T cells in mixed lymphocyte reaction (MLR) assays, indicative of a more favorable immunogenic profile. Compared to conventional CAR-T cells, API-192 also secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with the expression of a non-HLA restricted TCR and low GvH risk. These results demonstrate the feasibility, potency, and potential safety of API-192, warranting further development as a novel allogeneic therapy.

Expression of interleukin-1β and its receptor in human granulosa cells and their association with steroidogenesis

This study aimed to investigate whether interleukin 1β (IL-1β) and soluble IL-1 receptor 2 (sIL-1R2) are expressed in human granulosa cells (GCs) and relate to ovarian steroidogenesis. Ninety-six women undergoing in vitro fertilization (IVF) were recruited. RT-PCR and immunocytochemistry were used to detect mRNAs and proteins of IL-1β and IL-1R2, respectively. The steroidogenesis of primary cultured GCs was evaluated following treatment with either IL-1β alone or IL-1β and FSH in combination. There were positive correlations between serum IL-1β and serum progesterone (r = 0.220, p = 0.032) and follicular fluid (FF) estradiol (r = 0.242, p = 0.018). Additionally, serum and FF sIL-1R2 were negatively and positively correlated with FF estradiol (r = −0.376, p = 0.005) and FF progesterone (r = 0.434, p = 0.001), respectively. The mRNA and protein expression of IL-1β and IL-1R2 became evident in GCs. IL-1β alone significantly increased estradiol secretion from GCs, but in the presence of FSH, it could notably promote progesterone secretion in addition to estradiol. In conclusion, IL-1β and sIL-1R2 are expressed in human GCs and substantially contribute to ovarian steroidogenesis, suggesting that the IL-1β system may be a potential target for optimizing ovarian hyperstimulation and steroidogenesis in IVF cycles.