CD19-expressing Cells Research Articles

P0150 A precision medicine approach for carriers of defined IL2RA polymorphism: JAK inhibition blocks exaggerated IL-2 signalling in vitro, with no difference in in vivo clinical response rates in a small clinical cohort

Abstract Background The rs61839660 (660) polymorphism lies within the IL2RA intronic enhancer and is a causal variant in Crohn’s disease, with a heterozygote frequency of 16.8%. IL-2Ra (CD25) is the high-affinity receptor for IL-2, with binding activating JAK-STAT signalling. We have demonstrated that risk (T) allele carriers exhibit higher CD4+ T cell and natural killer (NK) cell CD25 expression. In response to IL-2, ‘T’ allele carriers show enhanced JAK-STAT signalling, greater CD4+ effector T cell (Teff) pro-inflammatory cytokine release, and enhanced NK cytotoxicity compared to wild-type (CC) subjects.1 We hypothesised that this drives inflammation, and that ‘T’ allele carriers would be more likely to respond to Janus kinase inhibition (JAKi). Tofacitinib (TF) is a JAKi licensed for the treatment of UC. We examined the ability of JAKi to block IL-2-induced JAK-STAT signalling in 660 risk allele carriers in vitro, and explored clinical response to TF in a genotyped UC cohort, with the aim of understanding whether JAKi presents a personalised therapeutic approach in this population. Methods CD patients of each genotype were recruited in collaboration with the NIHR IBD Bioresource.2 We extracted PBMCs and flow-sorted CD4+ Teff and CD56dim NK cells. Cells were cultured for 18h in the presence/absence of 20nM TF, stimulated with IL-2, fixed, and stained for intracellular pSTAT5. TF-treated patients were identified, and provided a blood or saliva sample. Genotyping was performed by qPCR. Data were obtained from patient records, with clinical response defined as a reduction in the Simple Clinical Colitis Activity Index or Partial Mayo Score of >/= 3. Results pSTAT5 levels were elevated in TT subjects’ Teff compared to CC/CT subjects in response to 10IU/ml IL-2 (Fig 1a), with a reduction in Teff pSTAT5 MFI in the presence of TF. In NK cells, pSTAT5 MFI was increased in TT compared to CC donors in response to 100IU/ml IL-2 (Fig 1g). The pSTAT5 response to IL-2 was non-significantly reduced in the presence of TF in both CC and TT subjects’ NK cells. In the real-world UC cohort, there was no significant difference in clinical response to TF between genotypes (Fig 2a), nor in the proportion of patients who had a disease flare on dose reduction or requiring colectomy. There was no difference in TF treatment persistence between genotypes (Fig 2b). Conclusion TF diminishes the in vitro pSTAT5 response to IL-2 in CD4+ Teff and NK cells in carriers of the 660 risk allele. In a UC population, there was no difference in clinical response to TF between genotype cohorts. Our data are limited by the small study population, with larger studies needed to confirm whether the use of JAKi may represent a personalised therapeutic approach in 660 risk allele carriers.

Influence of graphene oxide nanoparticles on functional activity of Jurkat cell line

Graphene oxide offers properties that make it a promising material for numerous biomedical applications, including various cancer treatment methods. Nanomaterials may overcome certain limitations of conventional chemotherapy. In cancer research, cell lines like Jurkat (T cell acute lymphoblastic leukemia cells) serve as models for investigating tumor treatment strategies. Previous studies have explored the effect of pegylated graphene oxide nanoparticles on some parameters of Jurkat cells, such as metabolism and apoptosis. This study aims to investigate how pegylated graphene oxide nanoparticles, varying in size, surface functionalization, and concentration, influence the functional activity of Jurkat cells, including cell viability, IL-2 production, and CD69 expression, both spontaneously, and following external activation. Jurkat cells were cultured with different types of graphene oxide nanoparticles (100-200 nm and 1-5 ìm; functionalized with linear and branched polyethylene glycol (PEG)) at concentrations of 5 ìg/ mL and 25 ìg/ mL for 24 hours. For assessment of cell parameters under stimulation with different types of particles, phytohemagglutinin (PHA, 50 ìg/mL) was used. Subsequently, the cells were stained with Zombie Aqua and CD69-APC antibody, followed by flow cytometry analysis (CytoFlex S) to determine the percentage of viable cells and CD69-expressing cells. The presence of IL-2 in cell culture supernatants was quantified using ELISA tests. It was observed that nanoparticles at low concentrations did not induce cytotoxic effects; cell viability improved after PHA stimulation. Small particles (100-200 nm) coated with linear PEG induced IL-2 production and CD69 expression. However, 1-5 ìm graphene oxide modified with branched PEG at a concentration of 25 ìg/mL led to a decrease in CD69 expression following PHA-stimulation. It was shown for the first time that pegylated graphene oxide nanoparticles affect the functional activity of Jurkat cells. The influence of particles is dependent on the size, concentration, surface functionalization of graphene oxide, and activation by PHA.

Pharmacokinetics, Pharmacodynamics, Bioavailability, and Immunogenicity of Obexelimab Following Subcutaneous Administration in Healthy Japanese and Non-Japanese Volunteers.

Obexelimab is an investigational, bifunctional, non-depleting, humanized monoclonal antibody that binds CD19 and FcγRIIb to inhibit B cells, plasmablasts, and CD19-expressing plasma cells. In clinical trials, intravenous (IV) administration of obexelimab has been well-tolerated, and demonstrated clinical activity in patients with rheumatoid arthritis, systemic lupus erythematosus, and immunoglobulin G4-related disease. This study was performed to evaluate the pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of obexelimab following subcutaneous (SC) administration, and compare PK/PD profiles between healthy Japanese and non-Japanese volunteers. This was a Phase I, open-label, parallel group, multiple-dose study. Participants were randomized to five cohorts to receive three doses of obexelimab as 125mg SC every 14days (q14d), 250mg SC q14d, 375mg SC q14d, 250mg IV q14d, and 125mg SC every 7days, then monitored during a 28-day safety follow-up period. PK/PD assessments were performed after the first and third doses. A total of 50 healthy volunteers (25 Japanese and 25 non-Japanese) were enrolled and distributed evenly between dose cohorts. All SC dosing regimens were well-tolerated. Dose-proportional PK was observed following SC doses with a bioavailability of approximately 60%. No clinically meaningful differences in PK parameters were found between healthy Japanese and non-Japanese participants. Antidrug antibodies (ADA) were detected in 6/50 (12%) participants after dosing. ADA had no or minimal impact on PK in all six ADA positive participants. Near-complete CD19 receptor occupancy and an absolute B-cell count nadir of approximately 50% baseline levels were maintained for the duration of the study in both populations. Obexelimab SC administration demonstrated favorable bioavailability, was well-tolerated, and showed no clinically meaningful ethnic differences in PK/PD. These results support further clinical development of SC obexelimab to treat B-cell mediated autoimmune diseases. NCT02867098.

HIV-1 Elite Controllers Are Characterized by Elevated Levels of CD69-Expressing Natural Killer Cells.

HIV type 1 ((human immunodeficiency virus) HIV-1) elite controllers (ECs) are a rare subset of people living with HIV-1 (PLWH) who control viral replication in the absence of antiretroviral treatment (ART) and may provide a model for a functional cure. We investigated the role of natural killer (NK) cells in HIV-1 ECs from South Africa. Phenotypic (CD69, CD38, CD57, PD-1), functional (CD107a, IFN-γ (inferferon gamma)), and nutrient transporter profiles (glucose transporter 1, CD98) of NK cells from ECs (n = 20), viremic progressors (VPs; n = 19), PLWH on ART (n = 20), and people without HIV-1 (PWOH; n = 21) were analyzed using flow cytometry. The Kruskal-Wallis test and followed by the Mann-Whitney U test were used to determine differences among the study groups. The Spearman rank correlation coefficient was used to determine significant associations. Compared with the other study groups, the percentage of CD69-expressing NK cells was higher in ECs, whereas the percentage of CD38-expressing NK cells was higher in VPs. Percentages of CD69 + CD38 - NK cells were elevated in ECs compared with VPs ( P = 0.003), but were not different to PLWH on ART and PWOH. Differentiation, exhaustion, and metabolic profiles were not different in ECs compared with PLWH on ART and PWOH; however, NK cell function was lower than in PWOH. These findings demonstrate that NK cells from ECs have an activated, mature profile with low levels of immune exhaustion and a reduced metabolic phenotype suggesting functional competence. This insight could inform the development of novel immunotherapeutic strategies for treating HIV-1.

Daratumumab in the Management of Red Cell Aplasia Following Allogeneic Hematopoietic Stem Cell Transplantation.

Pure red cell aplasia (PRCA) is a rare but significant complication following major ABO-incompatible allogeneic hematopoietic stem cell transplantation (HSCT). The persistence of recipient B lymphocytes producing anti-donor isohemagglutinins leads to reticulocytopenia and anemia, often resulting in transfusion dependence. Current treatment options for post-HSCT PRCA are limited and frequently yield suboptimal responses, complicating patient management. Herein, we report three cases of post-HSCT PRCA successfully managed with daratumumab, a monoclonal antibody targeting CD38-expressing plasma cells. All patients demonstrated rapid reticulocyte recovery and transfusion independence after daratumumab treatment, despite prior treatment failures. These findings suggest that daratumumab may provide a more effective therapeutic approach, with a favorable safety profile compared to traditional therapies. Given its demonstrated efficacy and safety, daratumumab warrants consideration as a first-line treatment for post-HSCT PRCA, potentially improving patient quality of life and reducing transfusion-related complications. Further studies should explore optimal dosing and long-term outcomes.

A Pilot Study of the CD38 Antagonist Daratumumab in Patients with Metastatic Renal Cell Carcinoma or Muscle-Invasive Bladder Cancer.

We performed a pilot study of daratumumab (an mAb directed against CD38) in muscle-invasive bladder cancer (MIBC) and treatment-refractory metastatic renal cell carcinoma (mRCC). Patients with MIBC underwent baseline transurethral resection of the bladder tumor followed by four weekly doses of daratumumab prior to cystectomy. Patients with mRCC underwent baseline and sequential biopsies after eight weekly doses. The primary endpoint was safety. The secondary endpoints were pathologic complete response rate for the MIBC cohort and objective response rate and progression-free survival for the mRCC cohort. Exploratory analyses included immune monitoring and overall survival. A Bayesian sequential monitoring design for toxicity was used for excessive toxicity. In both the MIBC (n = 8) and mRCC (n = 8) cohorts, no toxicity events were encountered. In the MIBC cohort, one patient experienced pathologic complete response rate. In the mRCC cohort, no objective responses were reported, and the median progression-free survival was 1.5 months (95% confidence interval, 1.1-1.8 months). Immune monitoring found significant reductions in NK cells in circulation in both cohorts after treatment. In the tissue analysis, IHC found evidence of diminished CD38 presence in mRCC with treatment, whereas the baseline levels in MIBC were low. Treatment with daratumumab was safe. No signal of efficacy was detected in mRCC, and conclusions on the activity in MIBC were limited. Evidence of daratumumab targeting CD38 was detected in circulating immune cells and within the tumor microenvironment of mRCC and MIBC. In this prospective clinical trial of daratumumab, treatment in patients with MIBC and mRCC was safe. Limited efficacy was observed. Treatment with daratumumab resulted in CD38-expressing immune cell subsets to be targeted both in circulation and within the tumor microenvironment.

Peptide-based CE-SELEX enables convenient isolation of aptamers specifically recognizing CD20-expressing cells

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with Kd estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds.

Near-infrared duocarmycin photorelease from a Treg-targeted antibody-drug conjugate improves efficacy of PD-1 blockade in syngeneic murine tumor models

ABSTRACT Regulatory T cells (Tregs) play a crucial role in mediating immunosuppression in the tumor microenvironment. Furthermore, Tregs contribute to the lack of efficacy and hyperprogressive disease upon Programmed cell death protein 1 (PD-1) blockade immunotherapy. Thus, Tregs are considered a promising therapeutic target, especially when combined with PD-1 blockade. However, systemic depletion of Tregs causes severe autoimmune adverse events, which poses a serious challenge to Treg-directed therapy. Here, we developed a novel treatment to locally and predominantly damage Tregs by near-infrared duocarmycin photorelease (NIR-DPR). In this technology, we prepared anti-CD25 F(ab’)2 conjugates, which site-specifically uncage duocarmycin in CD25-expressing cells upon exposure to NIR light. In vitro, CD25-targeted NIR-DPR significantly increased apoptosis of CD25-expressing HT2-A5E cells. When tumors were irradiated with NIR light in vivo, intratumoral CD25+ Treg populations decreased and Ki-67 and Interleukin-10 expression was suppressed, indicating impaired functioning of intratumoral CD25+ Tregs. CD25-targeted NIR-DPR suppressed tumor growth and improved survival in syngeneic murine tumor models. Of note, CD25-targeted NIR-DPR synergistically enhanced the efficacy of PD-1 blockade, especially in tumors with higher CD8+/Treg PD-1 ratios. Furthermore, the combination therapy induced significant anti-cancer immunity including maturation of dendritic cells, extensive intratumoral infiltration of cytotoxic CD8+ T cells, and increased differentiation into CD8+ memory T cells. Altogether, CD25-targeted NIR-DPR locally and predominantly targets Tregs in the tumor microenvironment and synergistically improves the efficacy of PD-1 blockade, suggesting that this combination therapy can be a rational anti-cancer combination immunotherapy.

MEK inhibitor PD0325901 upregulates CD34 expression in endothelial cells via inhibition of ERK phosphorylation

IntroductionCD34-positive endothelial progenitor cells (EPCs) promote angiogenesis and are a promising tool for regenerative cell therapy of ischemic diseases. However, the number and quality of CD34-positive cells decrease owing to various external and internal factors; thus, an efficient method is needed to establish CD34-positive EPCs. The generation of functional cells by reprogramming, that is, manipulating cell fate via gene transfer and/or treatment with chemical compounds, has recently been reported. Therefore, we aimed to generate CD34-positive cells by the reprogramming of endothelial cells (ECs). MethodsBased on previous reports, seven candidate chemical compounds were selected to reprogram human umbilical vein ECs (HUVECs) to CD34-positive cells. Following stimulation with the chemical compounds, the expression of CD34 was evaluated using quantitative PCR, flow cytometry, and immunocytochemistry. ResultsHUVECs treated with the compounds exhibited increased CD34 expression. We cultured cells in alternate media lacking one of the seven compounds and found no CD34 expression in cells treated with PD0325901-free media, suggesting that PD0325901—a MEK inhibitor—mainly contributed to the increase in CD34 expression. We found that 98% of cells were CD34-positive after PD0325901 treatment alone for 7 d. Western blotting revealed that the phosphorylation of ERK was suppressed in PD0325901-treated cells. No upregulation of CD34 was observed in fibroblast cell lines, even after PD0325901 treatment. These results suggested that PD0325901 induces CD34-positive cells by inhibiting ERK phosphorylation in ECs. ConclusionsCD34 expression was strongly induced in ECs by treatment with the MEK inhibitor PD0325901 in vitro. Our study provides a useful reference for the establishment of CD34-positive EPCs and will contribute to the development of regenerative therapies, especially for ischemic diseases.

PMB-CT01 (BAFFR-CAR T cell) therapy to examine preliminary safety and clinical responses in patients with B-cell malignancies who are ineligible for or failed CD19-directed therapy, including CD19-negative disease.

6534 Background: CD19-targeted therapies have shown remarkable efficacy in patients with B-cell malignancies. However, relapse with CD19 loss as a mechanism of tumor escape is common and represents a serious challenge. Patients who are ineligible for or have failed prior CD19-directed therapy have limited salvage options and a very poor prognosis. The B-cell activating factor receptor (BAFF-R) is functionally expressed in B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin lymphomas (NHL), including in patients with CD19-negative relapse (Qin et al., Sci Transl Med. 2019). As a critical regulator of B-cell function and survival, BAFF-R may be less prone to downregulation by tumors as a mechanism of antigen escape. Methods: We are conducting two phase 1 clinical trials of BAFFR-CAR T cells in patients with relapsed/refractory (r/r) B-ALL (NCT04690595) and NHL (NCT05370430). Primary endpoints are dose limiting toxicities (DLTs) and all other toxicities. Secondary endpoints include response rate, minimal residual disease (MRD) negative rate, PFS and OS. Response is evaluated using European LeukemiaNet criteria for B-ALL and Lugano 2014 for NHL. Results: As of Feb. 1st, 2024, 1 B-ALL and 3 NHL patients have completed treatment and post-treatment evaluations. Each received 50M BAFFR CAR T cells (starting dose level in both trials). The B-ALL patient had CD19/CD20/CD22-negative disease, and had received prior blinatumomab. NHL patients #1 and #2 both had CD19-expressing mantle cell lymphoma (MCL) and had received prior CD19 CAR T cells. Patient #2 had also received a CD3/CD20 bispecific antibody. NHL patient #3 had CD19/CD20-negative T cell/histiocyte-rich large B-cell lymphoma (THRBCL) and had not received prior CD19 CAR T cells. There were no DLTs, all patients had Gr. 1 cytokine release syndrome, and 2/3 NHL patients had Gr. 1 immune effector cell-associated neurotoxicity syndrome. Robust CAR T cell expansion was seen in all patients with a peak on days 12-14 post-infusion. The B-ALL and the 2 MCL patients reached MRD-negative complete response (CR) at 1 month post-treatment. The THRBCL patient had partial response (PR) at 1 month that converted to CR at 3 months. The B-ALL patient successfully transitioned to allogeneic HCT while in CR at 3 months post-infusion. Responses are ongoing in all NHL patients at 14, 10, and 8 months for patients #1, #2 and #3, respectively. Additional patients have been enrolled at the next dose level (200M CAR T cells). Results for these patients will be presented at the meeting. Conclusions: With a 100% CR rate at 3 months in the first 4 patients and durable responses at dose level 1, BAFFR-CAR T cells are a promising therapeutic option for patients with r/r B-cell malignancies who are ineligible or failed prior CD19-targeted therapy, including with CD19 antigen loss. Clinical trial information: NCT04690595 , NCT05370430 .

Aflatoxin B1 exposure deteriorates immune abnormalities in a BTBR T+ Itpr3tf/J mouse model of autism by increasing inflammatory mediators' production in CD19-expressing cells

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by deficiencies in communication, repetitive and stereotyped behavioral patterns, and difficulties in reciprocal social engagement. The presence of immunological dysfunction in ASD has been well established. Aflatoxin B1 (AFB1) is a prevalent mycotoxin found in food and feed, causing immune toxicity and hepatotoxicity. AFB1 is significantly elevated in several regions around the globe. Existing research indicates that prolonged exposure to AFB1 results in neurological problems. The BTBR T+ Itpr3tf/J (BTBR) mice, which were used as an autism model, exhibit the primary behavioral traits that define ASD, such as repeated, stereotyped behaviors and impaired social interactions. The main objective of this work was to assess the toxic impact of AFB1 in BTBR mice. This work aimed to examine the effects of AFB1 on the expression of Notch-1, IL-6, MCP-1, iNOS, GM-CSF, and NF-κB p65 by CD19+ B cells in the spleen of the BTBR using flow cytometry. We also verified the impact of AFB1 exposure on the mRNA expression levels of Notch-1, IL-6, MCP-1, iNOS, GM-CSF, and NF-κB p65 in the brain of BTBR mice using real-time PCR. The findings of our study showed that the mice treated with AFB1 in the BTBR group exhibited a substantial increase in the presence of CD19+Notch-1+, CD19+IL-6+, CD19+MCP-1+, CD19+iNOS+, CD19+GM-CSF+, and CD19+NF-κB p65+ compared to the mice in the BTBR group that were treated with saline. Our findings also confirmed that administering AFB1 to BTBR mice leads to elevated mRNA expression levels of Notch-1, IL-6, MCP-1, iNOS, GM-CSF, and NF-κB p65 in the brain, in comparison to BTBR mice treated with saline. The data highlight that exposure to AFB1 worsens immunological abnormalities by increasing the expression of inflammatory mediators in BTBR mice.

Abstract 1334: Evaluation of NK cell combination therapies in mouse models of cancer

Abstract Adoptive immunotherapy with natural killer (NK) cells has emerged as a promising innovative approach against hematological malignancies and solid tumors. To enhance the functional aspects of NK cells and to achieve stronger, sustained anti-tumor responses, combination strategies are under development. They are focused on optimization of NK cell potency, persistence and tumor targeting using monoclonal antibodies, multi-specific NK cell engagers and immune modulators like cytokines. SAR445419 is a universal off-the-shelf allogeneic human NK cell (hNK) product made using Sanofi’s proprietary platform with potential broad application across hematologic and solid tumor indications. We evaluated the therapeutic potential of these NK cells in combination with internal assets and standard of care treatments in preclinical mouse models. The studies were performed in immunodeficient NOG mouse strains transgenic for hIL-15, supporting long-term engraftment of functional hNK cells. We demonstrated the therapeutic advantage of NK cell combination with an anti-CD20 monoclonal antibody (mAb) in a disseminated lymphoma model. We also reported prolonged lifespan of leukemia-bearing animals treated with NK cells and pegenzileukin, a PEGylated non-alpha rhIL-2 variant engineered to preferentially bind to the IL-2 βγ receptors and promote NK cell function. The anti-tumor response correlated with pegenzileukin-induced increase in NK cell numbers in the blood. A combination of CD38KO NK cells with an anti-CD38 mAb was also investigated in a model of multiple myeloma (MM). FcγR-KO immunodeficient mice were used in these studies to limit the confounding contribution of residual murine immune cells engaging with the Fc-competent therapeutic antibody and to specifically detect hNK-cell mediated activity. The comparison of CD38KO versus WT NK cell numbers in the blood of animals treated with an anti-CD38 mAb showed a significant decrease in CD38-expressing NK cell count that was mitigated by the genetic deletion of CD38. Obtained results suggest that CD38KO NK cells are resistant to anti-CD38 mAb-mediated fratricide and provide a rationale for the combination of CD38KO NK cells with anti-an CD38 mAb against MM. Overall, these preclinical in vivo studies have a pivotal role in the evaluation of NK cell combination therapies against cancer and reveal their therapeutic potential. Citation Format: Katarzyna Franciszkiewicz, Pauline Rettman, Beatrice Gauthier, Laurence Noblet, Celine Nicolazzi, Andre Kunert, Rob Igarashi, Loreley Calvet, Valeria Fantin, Sukhvinder Sidhu. Evaluation of NK cell combination therapies in mouse models of 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 1334.

The ecto-enzyme CD38 modulates CD4T cell immunometabolic responses and participates in HIV pathogenesis.

Despite abundant evidence correlating T cell CD38 expression and HIV infection pathogenesis, its role as a CD4T cell immunometabolic regulator remains unclear. We find that CD38's extracellular glycohydrolase activity restricts metabolic reprogramming after T cell receptor (TCR)-engaging stimulation in Jurkat T CD4 cells, together with functional responses, while reducing intracellular nicotinamide adenine dinucleotide and nicotinamide mononucleotide concentrations. Selective elimination of CD38's ectoenzyme function licenses them to decrease the oxygen consumption rate/extracellular acidification rate ratio upon TCR signaling and to increase cycling, proliferation, survival, and CD40L induction. Pharmacological inhibition of ecto-CD38 catalytic activity in TM cells from chronic HIV-infected patients rescued TCR-triggered responses, including differentiation and effector functions, while reverting abnormally increased basal glycolysis, cycling, and spontaneous proinflammatory cytokine production. Additionally, ecto-CD38 blockage normalized basal and TCR-induced mitochondrial morphofunctionality, while increasing respiratory capacity in cells from HIV+ patients and healthy individuals. Ectoenzyme CD38's immunometabolic restriction of TCR-involving stimulation is relevant to CD4T cell biology and to the deleterious effects of CD38 overexpression in HIV disease.

Molecular assessment of intratumoral immune cell subsets and potential mechanisms of resistance to odronextamab, a CD20×CD3 bispecific antibody, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma

BackgroundPatients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL) have a significant need for effective treatment options. Odronextamab is an Fc-silenced, human, CD20×CD3 bispecific antibody that targets CD20-expressing cells via T-cell-mediated...

Targeting CD20-expressing malignant melanoma cells augments BRAF inhibitor killing.

Mutant BRAF targeted therapies remain a standard of care for the treatment of metastatic malignant melanoma (MM); however, high initial response rates are tempered by the persistence of residual MM cells that eventually lead to disease recurrence and mortality. As MM recurrence during targeted therapy can present with the simultaneous occurrence of multiple tumour nodules at the original body sites, we hypothesized the presence of an intrinsically resistant MM cell subpopulation. To identify an MM cell subpopulation that is intrinsically resistant to targeted therapy and possibly responsible for MM recurrence. Using melanoma cell lines, we defined culture conditions for the reproducible three-dimensional growth of melanospheres to investigate putative cancer stem cell populations. We undertook RNA sequencing and bioinformatic analysis to characterize cell populations between adherent and nonadherent culture, and cells expressing or not expressing CD20. Furthermore, we defined an in vitro assay to evaluate the killing of melanoma cancer stem cells as a therapeutic test using combination therapies targeting driver mutation and CD20. We described the culture conditions that promote MM cells to form melanospheres with a reproducible colony-forming efficiency rate of 0.3-1.3%. RNA sequencing of melanosphere vs. conventional MM cell cultures (n = 6), irrespective of the BRAF mutation status, showed that melanosphere formation was associated with growth and differentiation transcriptional signatures resembling MM tumours. Importantly, melanosphere formation also led to the emergence of a CD20+ MM cell subpopulation, similar to that observed in primary human MM tumours. CD20+ MM cells were resistant to BRAF inhibitor therapy and, consistent with this finding, demonstrated a Forkhead box protein M1 transcriptomic profile (n = 6). Combining BRAF inhibitor and anti-CD20 antibody treatment led to the additional killing of previously resistant CD20+ BRAF mutant MM cells. In patients with MM that harbour a CD20+ subpopulation, combined therapy with BRAF inhibitor and anti-CD20 antibody could potentially kill residual MM cells and prevent disease recurrence.

Evaluation of the potential impact on pharmacokinetics of various cytochrome P450 substrates of increasing IL-6 levels following administration of the T-cell bispecific engager glofitamab.

Glofitamab is a novel T cell bispecific antibody developed for treatment of relapsed-refractory diffuse large B cell lymphoma and other non-Hodgkin's lymphoma indications. By simultaneously binding human CD20-expressing tumor cells and CD3 on T cells, glofitamab induces tumor cell lysis, in addition to T-cell activation, proliferation, and cytokine release. Here, we describe physiologically-based pharmacokinetic (PBPK) modeling performed to assess the impact of glofitamab-associated transient increases in interleukin 6 (IL-6) on the pharmacokinetics of several cytochrome P450 (CYP) substrates. By refinement of a previously described IL-6 model and inclusion of invitro CYP suppression data for CYP3A4, CYP1A2, and 2C9, a PBPK model was established in Simcyp to capture the induced IL-6 levels seen when glofitamab is administered at the intended dose and dosing regimen. Following model qualification, the PBPK model was used to predict the potential impact of CYP suppression on exposures of various CYP probe substrates. PBPK analysis predicted that, in the worst-case, the transient elevation of IL-6 would increase exposures of CYP3A4, CYP2C9, and CYP1A2 substrates by less than or equal to twofold. Increases for CYP3A4, CYP2C9, and CYP1A2 substrates were projected to be 1.75, 1.19, and 1.09-fold following the first administration and 2.08, 1.28, and 1.49-fold following repeated administrations. It is recommended that there are no restrictions on concomitant treatment with any other drugs. Consideration may be given for potential drug-drug interaction during the first cycle in patients who are receiving concomitant CYP substrates with a narrow therapeutic index via monitoring for toxicity or for drug concentrations.

Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells.

Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.

SGN-35C: A Novel CD30-Directed Antibody-Drug Conjugate for the Treatment of Lymphomas

SGN-35C is an antibody drug conjugate composed of an anti-CD30 antibody conjugated to a camptothecin-derived topoisomerase 1 (TOP1) inhibitor payload. SGN-35C was designed to leverage the antibody backbone from brentuximab vedotin (BV) and the novel mechanism of action of camptothecin-derived ADCs. Here, we evaluated the mechanism of action, cytotoxicity, and safety in in vitro and in vivo models. To characterize the mechanism of action of SGN-35C in vitro and in vivo experiments were performed in CD30-positive anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma (HL) cell lines, and versions of these cell lines that are resistant to BV. SGN-35C binding, internalization, induction of DNA damage, cell cycle analysis, and in vitro cytotoxicity were evaluated. To assess if payload released in CD30-positive cells could kill adjacent CD30-negative cells, an in vitro bystander assay of CD30-positive and CD30-negative co-cultures was developed. The in vivo antitumor activity of SGN-35C was evaluated in animals bearing subcutaneous lymphoma models. SGN-35C antitumor activity was evaluated in a xenograft model of heterogeneous CD30 expression consisting of a mixture of a BV-resistant Karpas-299 cell line mixed with parental Karpas-299 cells to assess bystander activity in vivo. The tolerability of SGN-35C was assessed in a non-human primate model. SGN-35C binding to CD30-positive cells was similar to the unconjugated anti-CD30 antibody and demonstrated internalization into CD30-expressing cells. Treatment of cells with SGN-35C for 24 hours led to an increase of the DNA damage marker phospho-γH2A.X, which induced an increase in the fraction of cells in S-phase, consistent with the mechanism of camptothecin-based TOP1 inhibitors. Cytotoxicity was observed in ALCL and HL cell lines treated with SGN-35C, including an ALCL cell line resistant to BV. In a mixture of CD30-positive and CD30-negative cells, SGN-35C treatment induced cytotoxicity of the CD30-negative cells, demonstrating that SGN-35C can elicit bystander activity. A single dose of SGN-35C led to significant tumor growth inhibition in animals bearing lymphoma xenografts, including a BV-resistant model and a model of heterogeneous CD30 expression, demonstrating that SGN-35C can elicit bystander activity in vivo. SGN-35C demonstrates a tolerability profile in non-human primates that is consistent with camptothecins. A Phase 1, first-in-human study is planned to evaluate the safety and anti-tumor activity of SGN-35C in lymphoid malignancies.

Brentuximab Vedotin Combined with Chemotherapy in the Treatment of Pediatric Patients with R/R Hodgkin Lymphoma: Results of a Real World Setting Study

INTRODUCTION: Hodgkin Lymphoma (HL) is a familiar malignant lymphoma of the lymphatic system that accounts for 10% of all lymphoma diagnoses and 5% of lymphoma-related deaths. According to statistics, the incidence of HL in children and adolescents (0-19 years old) in China is about 1.78/million, and an average of 569 new cases are estimated annually from 2019 to 2021 . Brentuximab vedotin (BV) is an antibody-drug conjugates (ADC) that selectively delivers monomethyl auristatin E, an antimicrotubule agent, into CD30-expressing cells. Most of the data on the efficacy and safety of BV came from prospective and retrospective studies in adults or foreign children, and there are insufficient data in Chinese children. The purpose of this study is to explore the efficacy and safety of BV Combined with chemotherapy in Chinese pediatric patients with refractory and/or relapsed Hodgkin's lymphoma. METHODS: Retrospective analysis of clinical data of 9 children with refractory and/or relapsed Hodgkin's lymphoma from October 2021 to May 2023 at Beijing Children's Hospital, Capital Medical University. Patients were classified according to: clinical stage(Ann Arbor stage), huge tumor, huge tumor in the mediastinum, symptoms of B, and whether multiple lymph nodes were involved. Among them, the low-risk group was IA, ⅡA without giant tumor. The middle risk group was IB and ⅢA without huge tumor. High-risk groups were patients with stage ⅡB, ⅢB, Ⅳ or (each stage) with large tumors, or children with > 4 lymph node regions. All patients received BV in combination with chemotherapy for at least 2 cycles, and patients were assessed for remission response according to the Revised Response Criteria for Malignant Lymphoma at the end of every 2 cycles and followed up until July 20, 2023. RESULTS: Totally 32 cycles of chemotherapy were recorded, including 36 doses of BV administration(Table 1). The overall response rate of the children after 2 cycles of treatment was 100%, and the complete response rate was 88.89%. After remission, 2 cases (P1, P2) were treated with BV single drug 1.8mg/kg, Q3W for 6 months, and 3 cases (P3, P4, P7) were treated with radiotherapy. Two patients (P5, P6) received sequential chemotherapy, and two patients (P8, P9) were followed by programmed cell death 1(PD-1) antibody maintenance treatment for 6 months. After receiving PD-1 antibody for 6 months, 1 patient (P9) was transferred to BV monotherapy for maintenance, with 18 times planned. 2 cases (P1, P2) were then treated with autologous stem cell transplantation. None of the children developed disease progression during treatment. All patients successfully completed all planned dosage of BV, and serious adverse events were dominated by grade 3 or higher hematocrit, none of which interfered with normal treatment. Interpretation. BV had demonstrated the better efficacy and tolerable safety profile in the treatment of refractory and/or relapsed Hodgkin's lymphoma in children. CONCLUSIONS: BV has been approved by National Medical Products Administration in China since 2020, the clinical cases are mostly from adult lymphoma patients, and children data of in China is rare. In this study, we retrospectively reviewed and analyzed the data of BV combined chemotherapy in the treatment of refractory and relapsed Hodgkin lymphoma from single center, and found that the treatment response was rapid, continuousand safe. However, this study is a single-center retrospective study with a small sample size So We are conducting a prospective study of BV in first-line Hodgkin lymphoma children patients in China and aiming to generate more data for this specific population.

Advancing CAR-T Therapy in Acute Lymphoblastic Leukemia: Multi-Omic Analyses of CD19-Directed CAR-T Cells Enabled By an Ex Vivo Co-Culture Platform

Background: Acute Lymphoblastic Leukemia (ALL) has witnessed remarkable progress in the treatment of patients with Chimeric Antigen Receptor T-cell (CAR-T) therapies, leading to high response rates and durable remissions. However, challenges persist for patients who do not respond to CAR-T treatment, experience relapse, or encounter adverse events such as cytokine release syndrome. Overcoming these challenges necessitates a deeper understanding of mechanisms and the development of advanced technologies and assays. This study focuses on harnessing the potential of an innovative microfluidic platform for high-throughput cell-based assays, enabling multi-omics and functional analysis using a minimal amount of primary patient cells. By utilizing this platform, we aim to gain insight into the dynamics of primary patient material by evaluating responses to aid in selection of promising CAR-T enhancements or combination therapies designed to overcome mechanisms of CAR-T failure or relapse to improve clinical outcomes. Methods: For the cytotoxicity assay, we utilized autologous or allogeneic pairs of CD19-directed CAR-T cells and T-cell negative fractions from ALL patients' blood apheresis samples, containing CD19-expressing cancer cells. A negative control was established using untransduced T-cells. To ensure high cell viability and purity, the cells underwent careful processing and were labeled with long-lived dyes for tracking. Multiple cell suspensions with precise Effector to Target to Negative Fraction ratios were prepared. Only a few thousand cells from each coculture suspension were seeded in microfluidic culture chambers, which required no fluidic control system. Experiments were conducted for up to 96 hours with media replacement every 24 hours. At each endpoint, media was collected for cytokine quantification, and cells were stained for live/dead counting as well as quantification of intracellular or surface marker expression by imaging. After image acquisition, cells were collected for RNA extraction and gene expression analysis. Results: Results demonstrated that the developed co-culture platform allows precise control over culture conditions, ensuring culture of accurate ratios of highly complex cell populations. The viability of primary cells in the device guaranteed functional relevance of the cytotoxicity assays for up to a 96-hour timepoint. Strong CAR-T-mediated cell killing was observed, with a dose-dependent effect depending on the co-culture's relative CAR-T content. Less than 50% cell survival was observed in cocultures with Effector to Target ratio 1:1, as compared to the untransduced T-cell control. Furthermore, strong CAR-T activation was observed with over 60% of CART expressing CD25 and CD69 surface markers, as measured by imaging. A multi-color staining panel allowed for precise monitoring of changes over time within the assay of immune cell populations, including cancer cells, monocytes/macrophages, and NK cells, among others. Cytokine analysis revealed an increased secretion of IL-2, TNFa, IFNg, and Granzyme B in coculture with CAR-T cells compared to untransduced T-cells, with a dose-dependent effect based on the CAR-T cell ratio. Finally, RNA isolated from remaining cells at the end of the assay was of high quality with yields sufficient to enable transcriptome analysis for each treatment condition. Discussion: This study contributes to the development of innovative multiplexed high throughput strategies for in vitro functionality assessments in cell therapy, including combination studies and deeper mechanistic understandings. The platform's ability to generate fully integrated, multi-omics readings from complex allogeneic and autologous cocultures of CAR-T and patient cells offers a new frontier in early discovery and translational cancer research. This opens new possibilities for drug discovery, drug screening, and disease modeling, ultimately advancing our understanding of complex biological processes and accelerating therapeutic development. Future directions include exploring mechanisms of resistance and sensitivity, including donor-specific effector cell functionality, combination studies, impacts of immunosuppressive cell types, patient heterogeneity of target antigen expression or other co-stimulatory molecules, and evaluation of product potency or fitness of incoming apheresis material.