CD19-expressing Cells Research Articles

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.

HIV-1 Elite Controllers are Characterised by Elevated Levels of CD69-Expressing Natural Killer Cells.

Human immunodeficiency virus type 1 (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 therapy (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), and nutrient transporter profiles (glucose transporter 1, CD98) of NK cells from ECs (n=20), viraemic progressors (VPs; n=19), people living with HIV-1 (PLWH) on ART (n=20), and people without HIV-1 (PWOH; n=21) were analysed using flow cytometry. The Kruskal-Wallis test followed by the Mann-Whitney U test were used to determine differences among the study groups. The Spearman's rank correlation coefficient was used to determine significant associations. Compared to 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 to 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.

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.

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.

Exploring BiTE-Integrated CAR T-Cell Therapy to Overcome Tumor Antigen Escape and Reinforce CAR-T Therapy in Mantle Cell Lymphoma

Background T cell engagers (BiTEs) are bispecific T-cell engaging antibodies that redirect T cells to target antigen-expressing tumor cells. Combining a second antigen-targeted bispecific T cell engager (BiTE) with CAR T cells or incorporating bispecific engaging antibody-secreting T cells (engager-T cells) is expected to augment T-cell activation and proliferative capacity, and antitumor cytotoxicity, even towards tumor cells expressing tumor antigens heterogeneously. Here, we report a BiTE-integrated T-cell therapeutic strategy to overcome tumor antigen escape and reinforce the efficacy of CAR-T therapy in mantle cell lymphoma (MCL). Methods CD20-directed bispecific T-cell engager (CD20-BiTE) was constructed with a publicly available VHH fragment from an anti-CD20 single-domain antibody, and anti-CD3 scFv from OKT3 monoclonal antibody. CD19-directed CAR construct is comprised of anti-CD19 scFv from publicly available FMC63 monoclonal antibody, CD8 transmembrane-, 4-1BB costimulatory-, and CD3ζ signaling-domains. Human primary pan-T cells were isolated from PBMC derived from normal human donors (MD Anderson Blood Bank) or patients with MCL. Activated T cells were used for effector functional assay or transduced with lentivirus to produce CAR T or engager-T cells. Effector function of bispecific antibodies and T cells was determined via T-cell activation, cytokine release, and T-cell-dependent cytotoxicity assay in a co-culture model. Results Treatment with CD20-BiTE enhances the effector function of naïve T cells: the BiTE-Fc fusion protein was ectopically expressed in Expi293 and purified by immobilized Protein-A. The binding specificity of scFv(s) of CD20-BiTE was confirmed to bind strongly and specifically to CD20-expressing cells. In vitro functional analyses demonstrated that co-culture of naïve T cells with CD20-exressing cells in the presence of CD20-BiTE induced antigen-specific activation of the T cells (increase in the expression of CD25 and CD69). In the presence of naïve T cells and antigen-positive MCL cells, CD20-BiTE enhanced the secretion of cytokines (IFNγ and TNFα, > 2-fold increase) and CD8 T-cell cytotoxic molecule granzyme B by T cells, in a BiTE-dependent and CD20-specific manner. The enhanced effector function translated into improved antitumor cytotoxicity, as activated T cells treated with CD20-BiTE exerted 43- 72% increase in the killing efficacy of JeKo-1 BTK-KD cells. CD20-BiTE-combined CAR T cells endowed with enhanced antitumor activity overcome tumor antigen escape and resistance to current CD19-CAR T cell therapy: when primary T cells and CD19-CAR T cells were co-cultured with MCL cells, addition of CD20-BiTE potentiated T cell-dependent cytotoxicity ( p < 0.001). Since both CD19-dependent and -independent CAR T cell-resistance often develop during adoptive cell therapy in MCL, we sought to overcome the resistance in MCL by combining a second antigen-targeted CD20-BiTE with CAR T cells or incorporating CD20-BiTE secreting T cells (engager-T cells). Remarkably, when combined with CD19-CAR T cells, CD20-BiTE mediates significant increase in the antitumor cytotoxicity, compared to T cells alone ( p ≤ 0,001 and p < 0.001 for 2nM and 5nM of the BiTE, respectively), in a CD20-specific fashion, in both MCL cells with diminished CD19 expression, and CD19-CAR T-resistant primary MCL cells. Importantly, in a transwell assay, CD19-CAR T cells demonstrated restored potent killing (>3-fold increase over CAR T cells alone) of the CD19-low or CAR T therapy resistant tumor cells, but only when the engager-T cells were present in the insert, suggesting the engaging molecules were able to redirect CD19-CAR T cells to CD20-positive tumor cells regardless of their CD19 expression. The anti-lymphoma efficacy of engager-CAR T cell therapy will be further evaluated in our well-established unique PDX model for MCL. In summary, we have successfully generated a CD20-directed bispecific T-cellengaging antibody and CD20-BiTE engager-T cells that could be combined with CAR T cell therapy, which was demonstrated effective in overcoming tumor antigen escape and resistance to current CD19-CAR T cell therapy in MCL.

A Phase 1, Open-Label, Dose Escalation and Dose Expansion Study of CLN-978 (CD19XCD3XHSA) in Patients with Relapsed/Refractory (R/R) B-Cell Non-Hodgkin Lymphoma (B-NHL)

Background: Patients with B-cell non-Hodgkin lymphoma (B-NHL) who are refractory to/or have relapsed (R/R) after treatment with anti-CD20 monoclonal antibodies, either alone or in combination with standard-of-care chemotherapy, have a poor prognosis. Response rates tend to decrease and duration of response shortens with each successive line. CD19 represents a significant target for the development of novel immunotherapies for the treatment of patients with R/R B-NHL, many of whom develop disease that is refractory to CD20-containing regimens. CD19-directed therapies approved for B-NHL include an Fc-enhanced anti-CD19 monoclonal antibody, a CD19-directed antibody-drug conjugate, and several CD19-directed CAR-T cell products. While CAR-T approaches are an important alternative for some B-NHL subtypes, many patients still relapse, and challenges remain with toxicity and accessibility in the community setting. Consequently, substantial unmet needs remain for effective treatments with broad applicability across B-NHL subtypes and practice settings. CLN-978 is a fully human, half-life extended, single-chain T cell engaging construct targeting CD19 and CD3, with a human serum albumin (HSA) binding moiety for half-life extension. Preclinically, CLN-978 effectively redirects T cells to kill CD19-expressing malignant cells and has demonstrated potent in vitro and in vivo efficacy against cancer cells expressing very low levels of human CD19. Study Design and Methods: This is a phase 1, open-label, multi-center, first-in-human, dose escalation, and dose expansion study of CLN-978 in patients with R/R B-NHL. Adult patients (≥ 18 years of age) with one of the following CD19+ B-cell histologies: diffuse large B-cell lymphoma - de novo or transformed, high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, or marginal zone lymphoma (nodal, extranodal, or mucosa-associated) for whom there is no available approved standard therapy will be recruited. Other key eligibility criteria include: ≥ 2 lines of prior chemotherapy, Eastern Cooperative Oncology Group Performance Status 0-2. Key exclusion criteria include prior treatment with allogeneic hematopoietic stem cell transplantation or any other investigational CD19 x CD3 T cell engager. Prior CAR-T therapy, unconjugated or radio-conjugated CD19 mAb, and CD19 antibody-drug conjugate are allowed. The study consists of 2 parts: 1) a dose escalation phase commencing with accelerated titration in single-patient cohorts followed by a conventional 3+3 dose escalation design to identify the recommended phase 2 dose (RP2D) and 2) a dose expansion phase to further characterize the safety and preliminary efficacy of CLN-978 in disease-specific cohorts at the RP2D. CLN-978 will be subcutaneously (SC)-administered once weekly (QW) in 28-day cycles until progressive disease, intolerable toxicity, or a maximum of 24 cycles of treatment. Initial administration of CLN-978, inclusive of potential priming doses and the first target dose, will be administered on an inpatient basis. Subsequent doses may be given in the outpatient setting. Based on emerging data, less frequent administration schedules may be implemented in Cycle 4 and beyond as approved by the Safety Review Committee. Safety, pharmacokinetic (PK), pharmacodynamic, and preliminary efficacy assessments will guide the selection of the dose and schedule for further evaluation. The primary endpoint is safety. Key secondary endpoints include PK, anti-drug antibodies, overall response rate, duration of response, time to response, time to subsequent anti-lymphoma therapy, progression-free survival, and overall survival. Approximately 90 patients (30 in dose escalation and 60 in dose expansion) will be enrolled at approximately 6 sites in the U.S., reflecting both community and academic centers. The study is currently enrolling (NCT05879744).

Targeted Integration of a Chimeric Antigen Receptor in Key Selected Loci in Primary Natural Killer Cells

Despite promising preclinical studies and early clinical data (Liu et al., NEJM, 2020), chimeric antigen receptor natural killer cells (CAR NK) are not yet on par with CAR T cells for the treatment of malignant diseases. Much effort has gone into developing NK-optimized CAR architectures (Li et al., Cell Stem Cell, 2018) and to increasing the persistence of CAR NK cells. We hypothesized that CAR NK cells could also benefit from the targeted integration of a CAR transgene, as compared with semi-random integration obtained using gammaretroviral vectors. Precise targeting has proven highly advantageous for TRAC CAR T cells owing to the optimal regulation and homogeneous expression conferred by the endogenous TCR alpha promoter (Eyquem et al., Nature, 2017). We first optimized an editing protocol for primary NK cells that uses electroporation of a CRISPR/Cas9 ribonucleoprotein followed by transduction with AAV6 bearing a homology-directed repair template. With this protocol we were able to knock out (KO) genes (including CD38, B2M, and different NK receptors), knock in transgenes (GFP-fusion protein, truncated EGFR, CAR), or both, at high efficiency. There was only moderate cell toxicity, and proliferation and cytotoxicity were conserved. Because of the high constitutive expression of B2M, we initially chose to focus on this locus and targeted a 1928z CAR transgene for regulation under the endogenous B2M promoter; our template design also rescues the B2M gene upon knock-in, enabling intermediate B2M expression (Figure 1). This strategy conferred high expression of the CAR and significantly more homogeneity as compared with gammaretroviral delivery (Figure 1). Furthermore, the rescued B2M expression protected B2M CAR NK cells from fratricide killing, whereas the B2M KO NK cells and the CAR NK cells with disrupted B2M expression were susceptible. B2M CAR NK cells also displayed higher in vitro cytotoxicity as compared with WT NK cells against two different CD19-expressing tumor cell lines (NALM6, SupB15). We tested B2M CAR NK cells in an NSG mouse xenograft model with an intraperitoneally injected human ovarian cancer cell line engineered to express CD19 (CD19 + SKOV3). Unexpectedly, compared to retroviral CAR-NK cells, B2M CAR NK cells conferred less tumor control (assessed by bioluminescence imaging) and less of a survival benefit. Therefore, we screened a panel of other loci for CAR targeting: five for NK activating receptors ( CD16A, CD56, DNAM1, NKp30, NKp46) and four for NK inhibitory receptors ( CD161, LILRB1, NKG2A, TIGIT). For activating receptors, the transgene was designed to use the endogenous promoter and to maintain expression of the targeted locus. For inhibitory receptors, the transgene was placed under the control of an EF1a exogenous promoter and designed to disrupt expression of the targeted locus. We compared these constructs and the initial B2M CAR NK design for CAR expression (by flow cytometry) and in vitro functionality (cytotoxicity assays) and observed wide variation in knock-in efficiency and CAR expression (Figure 2). For the top constructs based on knock-in efficiency, we conducted in vitro cytotoxicity assays against NALM6 and SupB15 cell lines. We found that when normalized for CAR percentage using the respective KO conditions, all tested knock-in loci performed better than B2M CAR NK cells, despite the lower CAR expression. Our current focus is on validating these top loci for the targeted integration of a 1928z CAR using in vivo models for comparison with B2M CAR NK and retroviral CAR NK. In summary, we have identified advantageous in vitro characteristics for the targeted integration of a CAR transgene in primary NK cells and screened 10 loci for CAR expression and in vitro cytotoxicity against tumor target cells. Confirmatory in vivo studies are ongoing and will be presented.

Signaling Role of Viral Protein Motif and Its Application in CAR T Cell Therapy

Adoptive cell therapy using Chimeric Antigen Receptor (CAR) T cells, such as Tisagenlecleucel and Axicabtagene Ciloleucel, has revolutionized cancer immunotherapy and become a game-changer in cancer care. However, several challenges, including durable remission and limited expansion after infusion, persist. Recent work from the Riddell lab (PMID: 34429382) demonstrated that T Cell Receptors (TCRs) are more sensitive than CARs due to the presence of T cell signaling proteins like CD3-, CD3ε, and CD3-, which facilitate the recruitment of Lck to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs). Although CARs were designed to mimic TCR signaling, they still lack the full sensitivity. In this study, we hypothesized that enhancing tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) presented on CD3 in CAR T cells amplifies the CAR-mediated signaling as well as improves sensitivity of CAR co-stimulatory signaling. To achieve this, we developed a novel co-stimulation system where anti-CD19 CAR T cells co-expressed Tip37, a 37-amino acid segment of the Herpesvirus saimiri (HVS) tyrosine-protein kinase-interacting protein (Tip). This specific segment interacts exclusively with the major T cell kinase, Lck, effectively regulating its kinase activation. The critical region for this interaction lies within 37 amino acids (146-182) in the central part of Tip, which contains two motifs: the SH3 binding motif (SH3B; 174-182) and the C-terminal Src-related kinase homology motif (CSKH; 146-155). The 37 amino acid peptide fragments alone form a stable complex with Lck. Lck is crucial in the initial stages of T cell activation, not only when triggered by the native TCR stimulation but also in response to CAR-antigen stimulation. Following antigen-induced T cell activation, Lck initiates the phosphorylation of tyrosines in the ITAMs present in CD3ζ domains. This leads to the recruitment of ZAP70 kinases to the ITAMs of CD3ζ, facilitating the formation of signaling protein complexes. The results of our in vitro experiments using the immortalized T cell line, Jurkat cells, demonstrate that the inclusion of the Tip37 component in the CAR leads to the formation of the Lck-CAR complex. This, in turn, significantly enhances the phosphorylation of ITAMs in CARs and triggers downstream signaling. Importantly, the CAR T cells with the Tip37 co-stimulatory domain do not secrete cytokines when in an unstimulated state. This suggests that the heightened Lck phosphorylation does not lead to the auto-activation of T cells. As a result, CAR T cells engineered with Tip37 can be considered safe and hold promise for future applications. We engineered CAR19 T cells co-expressing Tip (Tip-CART19 cells) with a co-stimulatory domain of 4-1BB and CD3 (Tip-CART19 cells). As a control, we generated CART19 cells lacking the TIP domain (WT CART19 cells). We confirmed that Tip expression induced enhanced CD3 phosphorylation in CART19 T cells. Additionally, we observed that a significantly augmented outgrowth of CD8+ cells engineered with the TIP-containing CAR compared with canonical CAR T cells. Further, TIP-CAR T cells displayed increased expression of CD38 and Ki67. Interestingly, we also noted an expansion of CCR7+CD45RA- central memory subsets in CD4+ CART19 cells and CCR7-CD45RA- effector memory subsets in CD8+ CART19 cells. Finally, as evidenced by apparent changes in phenotype, Tip-CART19 cells exhibited improved cytotoxic ability and significant outgrowth of CART19 cells with in vitro repeated CAR stimulation using CD19-expressing tumor cells, as previously described in our study (Fraietta et al., Nature 2018). This study highlights that amplification of the constitutive Lck recruitment and amplified CD3 phosphorylation in CART19 cells via the introduction of Tip37 domain results in enhanced anti-leukemia cytotoxic ability and preservation of proliferative function in CART19 cells. Our discovery introduces a novel approach for amplifying CAR signaling to improve CAR T cell therapy.