Raji Cells Research Articles

Bispecific killer engager for targeted depletion of PD-1 positive lymphocytes: A new avenue for autoimmune disease treatment

Bispecific killer cell engagers (BiKEs) are a powerful tool to incite the killing power of natural killer (NK) cells. Here, we posited that the BiKE technology could be utilized to deplete activated immune cells expressing programmed death-1 (PD-1+ cells), and hence treat autoimmune diseases since these cells drive the disorders. We designed and generated PD-1 BiKE that targets an activating NK cell receptor, CD16, and PD-1. PD-1 BiKE showed specific binding to PD-1+ cells and engaged CD16 simultaneously. PD-1 BiKE enhanced NK cell-mediated apoptosis and depletion of PD-1+ Raji cells, but not PD-1- Raji cells. Further, PD-1 BiKE induced apoptosis of primary PD-1+ T lymphocytes that are highly relevant to autoimmune disease progression. The BiKE depleted 42% of primary T cells that were stimulated in vitro. Importantly, those ablated primary T cells were activated cells. Meanwhile, naive T cells were spared by the BiKE treatment, supporting the crucial selectivity of PD-1 BiKE-directed cell depletion. Lastly, PD-1 BiKE is more effective than a conventional depleting antibody in the depletion of PD-1+ cells. The current work supports PD-1 BiKE is a selective, potent, and safe tool to deplete PD-1+ cells.

Preclinical Aspects of [89Zr]Zr-DFO-Rituximab: A High Potential Agent for Immuno-PET Imaging.

An early diagnosis of cancer can lead to choosing more effective treatment and increase the number of cancer survivors. In this study, the preparation and preclinical aspects of [89Zr]Zr-DFO-Rituximab, a high-potential agent for PET imaging of Non- Hodgkin Lymphoma (NHL), were evaluated. DFO was conjugated to rituximab monoclonal antibody (mAb), and DFO-rituximab was successfully labeled with zirconium-89 (89Zr) at optimized conditions. The stability of the complex was assessed in human blood serum and PBS buffer. Radioimmunoreactivity (RIA) of the radioimmunoconjugate (RIC) was evaluated on CD20-overexpressing Raji cell line and CHO cells. The biodistribution of the radiolabeled mAb was studied in normal and tumorbearing rodents. Finally, the absorbed dose in human organs was estimated. The radiolabeled compound was prepared with radiochemical purity (RCP) >99% (RTLC) and a specific activity of 180±1.8 GBq/g. The RCP of the final complex PBS buffer and human blood serum was higher than 95%, even after 48 h post incubation. The RIA assay demonstrated that more than 63% of the radiolabeled compound (40 ng/ml, 0.5 mL) was bound to 5×106 Raji cells. The biodistribution of the final product in tumor-bearing mice showed a high accumulation of the RIC in the tumor site in all intervals post-injection. Tumor/ non-target ratios were increased over time, and longer imaging time was suggested. The dosimetry data indicated that the liver received the most absorbed dose after the complex injection. [89Zr]Zr-DFO-Rituximab represents a significant advancement in the field of oncological imaging and offers a robust platform for both diagnostic and therapeutic applications in the management of B-cell malignancies.

Quantitative investigation of the effect of mechanical and geometrical factors of a laboratory-scale bioreactor using a vibrating agitator on mammalian cell culture indices.

Mammalian cell cultures in laboratories are performed in static and dynamic methods, and cell growth indices are higher in dynamic mode. In this study, a lab-scale stirred bioreactor using a vibrating disc and a suitable setup has been introduced for dynamic cell culture, which creates proper mixing at low shear stress. 15 experiments have been done by Raji cell in batch mode using Box-Behnken design to quantitatively investigate the effect of mechanical and geometrical factors of this bioreactor on cell culture indices. Three structural factors, including disc diameter, vibration amplitude, and the height of the disc placement have been selected as the main factors. Three cell growth indices including the specific growth rate, the maximum cell concentration, and productivity have been considered as biological responses. Resulting models predict the value of each index under different settings of the factors with good accuracy. Results show that the disc diameter has the greatest effect among the investigated factors. Also, the specific growth rate, the natural logarithm of the maximum cell concentration, and productivity are about 0.033(1/h), 13.2, and 5133(cells/hmL), respectively by using a 25 (mm) disc with a vibration amplitude of 2.5 up to 3 (mm), and a placement height of 40 up to 60 (mm).

Development and Characterization of A Novel SpyTagged Modular Nanobody as A Detection Platform for CD22-Positive Cells.

CD22, as a surface protein of B cells, is used in the diagnosis and target-specific immunotherapy of B-cell malignancies. SpyTag and SpyCatcher, on the other hand, are two covalently coupled proteins capable of developing a bi- or multi-specific modular protein. The aim of this study was to develop FITC-conjugated SpyCatcher-SpyTagged anti-CD22 Nanobody (FITC-SpyC-SpyT-CD22Nb) to recognize CD22 on the surface of malignant B cells. In this experimental study, the SpyTag-CD22Nb construct was subcloned into a pET22 vector and expressed in E. coli BL21 (DE3). After purification using His-tag affinity chromatography, the size of the eluted protein was confirmed on a Western blot. In addition, the SpyCatcher protein, subcloned into pET28, was expressed in E. coli BL21 (DE3), purified by His-tag affinity chromatography and subjected to FITC labeling. FITC-SpyCatcher and SpyTag-CD22Nb were coupled in a 1:1 molar ratio. The specific binding of the produced FITC-SpyC-SpyT-CD22Nb was tested using CD22+ Raji and CD22- K562 cell lines and was evaluated by flow cytometry. SpyTag-CD22Nb and SpyCatcher were successfully expressed in E. coli BL21 (DE3). The 1:1 molar ratio of SpyTag-CD22Nb and FITC-SpyCatcher successfully formed FITC-SpyC-SpyT-CD22Nb at room temperature. The flow cytometry results showed that FITC-SpyC-SpyT-CD22Nb specifically binds to the CD22+ Raji cells, while there is no binding to the CD22- K562 control cells. The novel FITC-SpyC-SpyT-CD22Nb produced in the present study is capable of detecting the surficial expression of CD22. According to our findings, FITC-SpyC-SpyT-CD22Nb is applicable for specific targeting of CD22 in a therapeutic manner, i.e., chimeric antigen receptor (CAR)-T cell therapy and antibody drug conjugates (ADCs).

Chevalierlin: A spirocyclic alkaloid from a hydrothermal vent associated fungus Aspergillus chevalieri TW132-65

A previously undescribed spirodiketopiperazine-indole alkaloid, chevalierlin (1), two pairs of previously undescribed dihydroisocoumarin enantiomers eurotiumides H–I (2–3), as well as six related known compounds (4–9) were isolated from the culture of a hydrothermal vent associated fungus Aspergillus chevalieri TW132-65. Their structures were unambiguously determined by NMR, mass spectrometry, and ECD calculations. Chevalierlin (1) exhibits moderate cytotoxic activities with IC50 values of 6.20 ± 0.05 μM and 7.68 ± 0.01 μM against Namalwa and Raji cell lines.

NK Cell Degranulation Triggered by Rituximab Identifies Potential Markers of Subpopulations with Enhanced Cytotoxicity toward Malignant B Cells.

A promising strategy in cancer immunotherapy is to restore or enhance the cytotoxicity of NK cells, among others, by activating the mechanism of antibody-dependent cellular cytotoxicity (ADCC). Monoclonal antibodies targeting tumor antigens, such as rituximab (targeting CD20), induce NK cell-mediated ADCC and have been used to treat B cell malignancies, such as non-Hodgkin lymphoma, but not always successfully. The aim of this study was to analyze the gene expression profile of the NK cells involved in the cytolytic response stimulated by rituximab. NK cells were co-cultured with rituximab-opsonized Raji cells. Sorting into responder and non-responder groups was based on the presence of CD107a, which is a degranulation marker. RNA-seq results showed that the KIT and TNFSF4 genes were strongly down-regulated in the degranulating population of NK cells (responders); this was further confirmed by qRT-PCR. Both genes encode surface proteins with cellular signaling abilities, namely c-KIT and the OX40 ligand. Consistent with our findings, c-KIT was previously reported to correlate inversely with cytokine production by activated NK cells. The significance of these findings for cancer immunotherapy seems essential, as the pharmacological inhibition of c-KIT and OX40L, or gene ablation, could be further tested for the enhancement of the anti-tumor activity of NK cells in response to rituximab.

A bioinspired supramolecular nanoprodrug for precision therapy of B-cell non-Hodgkin’s lymphoma

Fludarabine (FA) is still considered as a first-line chemotherapy drug for hematological tumors related to B lymphocytes. However, it is worth noting that the non-specific distribution and non-different cytotoxicity of FA may lead to irreversible consequences such as central nervous system damage such as blindness, coma, and even death. Therefore, it is very important to develop a system to targeting delivery FA. In preliminary studies, it was found that B lymphoma cells would specific highly expressing the sialic acid-binding immunoglobulin-like lectin 2 (known as CD22). Inspired by the specific recognition of sialic acid residues and CD22, we have developed a supramolecular prodrug based on polysialic acid, an endogenous biomacromolecule, achieving targeted-therapy of B-cell non-Hodgkin’s lymphoma (B-NHL). Specifically, the prepared hydrophobic reactive oxygen species-responsive FA dimeric prodrug (F2A) interacts with the TPSA, which polysialic acid were modified by the thymidine derivatives, through non-covalent intermolecular interactions similar to “Watson–Crick” base pairing, resulting in the formation of nanoscale supramolecular prodrug (F@TPSA). Cell experiments have confirmed that F@TPSA can be endocytosed by CD22+ B lymphoma cells including Raji and Ramos cells, and there is a significant difference of endocytosis in other leukocytes. Furthermore, in B-NHL mouse model, compared with FA, F@TPSA is determined to have a stronger tumor targeting and inhibitory effect. More importantly, the distribution of F@TPSA in vivo tends to be enriched in lymphoma tissue rather than nonspecific, thus reducing the leukopenia of FA. The targeted delivery system based on PSA provides a new prodrug modification strategy for targeted treatment of B-NHL.Graphical abstract

Immunological landscape of human lymphoid explants during measles virus infection.

In humans, lymph nodes are the primary site of measles virus (MeV) replication. To understand the immunological events that occur at this site, we infected human lymphoid tissue explants using a pathogenic strain of MeV that expresses GFP. We found that MeV infected 5%-15% of cells across donors. Using single-cell RNA-Seq and flow cytometry, we found that while most of the 29 cell populations identified in the lymphoid culture were susceptible to MeV, there was a broad preferential infection of B cells and reduced infection of T cells. Further subsetting of T cells revealed that this reduction may be driven by the decreased infection of naive T cells. Transcriptional changes in infected B cells were dominated by an interferon-stimulated gene (ISG) signature. To determine which of these ISGs were most substantial, we evaluated the proteome of MeV-infected Raji cells by mass spectrometry. We found that IFIT1, IFIT2, IFIT3, ISG15, CXCL10, MX2, and XAF1 proteins were the most highly induced and positively correlated with their expression in the transcriptome. These data provide insight into the immunological events that occur in lymph nodes during infection and may lead to the development of therapeutic interventions.

Advancing Tumor-Targeted Chemo-Immunotherapy: Development of the CAR-M-derived Exosome-Drug Conjugate.

Traditional antibody-drug conjugates (ADCs) mainly suppress tumor growth through either chemotherapy with cytotoxic payloads or immunotherapy with immuno-modulators. However, a single therapeutic modality may limit their exploration. Herein, we developed a new type of drug conjugate termed CAR-EDC (CAR-M-derived exosome-drug conjugate) by using CAR-exosomes from CAR-M cells as the targeting drug carrier that contains a high level of CXCL10. CAR-exosomes could significantly enhance the immunological activation and migratory capacity of T lymphocytes and promote their differentiation into CD8+ T cells. It also increased the proportion of M1 macrophages. The CAR-EDC, covalently loaded with SN-38, was internalized into Raji cells through endocytosis mediated by the CAR molecules. It exerted excellent antitumor activity in vivo by virtue of not only chemotherapy by SN38 but also immunotherapy by CXCL10-mediated antitumor immunity. Generally, this study provides an exosome-drug conjugate system with enhanced antitumor effects over traditional ADCs through the synergism of chemotherapy and immunotherapy.

Sequence-to-expression approach to identify etiological non-coding DNA variations in P53 and cMYC-driven diseases.

Disease risk prediction based on genomic sequence and transcriptional profile can improve disease screening and prevention. Despite identifying many disease-associated DNA variants, distinguishing deleterious non-coding DNA variations remains poor for most common diseases. In this study, we designed in vitro experiments to uncover the significance of occupancy and competitive binding between P53 and cMYC on common target genes. Analyzing publicly available ChIP-seq data for P53 and cMYC in embryonic stem cells showed that ~344-366 regions are co-occupied, and on average, two cis-overlapping motifs (CisOMs) per region were identified, suggesting that co-occupancy is evolutionarily conserved. Using U2OS and Raji cells untreated and treated with doxorubicin to increase P53 protein level while potentially reducing cMYC level, ChIP-seq analysis illustrated that around 16 to 922 genomic regions were co-occupied by P53 and cMYC, and substitutions of cMYC signals by P53 were detected post doxorubicin treatment. Around 187 expressed genes near co-occupied regions were altered at mRNA level according to RNA-seq data analysis. We utilized a computational motif-matching approach to illustrate that changes in predicted P53 binding affinity in CisOMs of co-occupied elements significantly correlate with alterations in reporter gene expression. We performed a similar analysis using SNPs mapped in CisOMs for P53 and cMYC from ChIP-seq data, and expression of target genes from GTEx portal. We found significant correlation between change in cMYC-motif binding affinity in CisOMs and altered expression. Our study brings us closer to developing a generally applicable approach to filter etiological non-coding variations associated with common diseases.

The Imidazacridine Derivative LPSF/AC-05 Induces Apoptosis, Cell Cycle Arrest, and Topoisomerase II Inhibition in Breast Cancer, Leukemia, and Lymphoma.

Cancer is the major cause of morbidity and mortality worldwide. Current treatments for both solid and hematological tumors are associated with severe adverse effects and drug resistance, necessitating the development of novel selective antineoplastic drugs. The present study describes the antitumor activity of the imidazacridine derivative 5-acridin-9-ylmethylidene-2-thioxoimidazolidin-4-one (LPSF/AC05) in breast cancer, leuke-mia, and lymphoma cells. Cytotoxicity assays were performed in PBMC and in breast cancer, leukemia, and lymphoma cell lines using the MTT method. Changes in cell cycle progression and apoptosis were assessed using flow cytometry. Moreover, topoisomerase II inhibition as-says were performed. LPSF/AC05 exhibited cytotoxicity in six of the nine cell lines tested. The best results for leukemia and lymphoma were observed in the Toledo, Jurkat, and Raji cell lines (IC50 = 27.18, 31.04, and 33.36 M, respectively). For breast cancer, the best re-sults were observed in the triple-negative cell line MDA-MB-231 (IC50 = 27.54 μM). The compound showed excellent selectivity, with no toxicity to normal human cells (IC50 > 100M; selectivity index > 3). Cell death was primarily induced by apoptosis in all cell lines. Furthermore, LPSF/AC05 treatmentinduced cell cycle arrest at the G0/G1 phase in leuke-mia/lymphoma and at the G2/M phase in breast cancer. Finally, topoisomerase II was inhibited. These results indicate the potential ap-plication of LPSF/AC05 in cancer therapy.

Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model.

Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact ofsevere acute respiratory syndrome coronavirus 2(SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.

Effect of rituximab on remissions without minimal residual disease and immunogenicity in patients with relapsed/refractory hairy cell leukemia receiving moxetumomab pasudotox.

e19015 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) was FDA-approved for relapsed/refractory hairy cell leukemia (HCL) but is now unavailable due to vial expiration until a company resumes development. The phase 3 complete remission (CR) rate was 41%, higher in patients with lower anti-drug antibodies (ADA). Minimal residual disease (MRD) eradication led to longer CR duration. HCL and the poorer prognosis variant HCLv strongly express CD22 and CD20. A clinical trial was done to determine if Rituximab could decrease immunogenicity by killing normal B-cells in HCL patients receiving Moxe and hasten MRD-free CR by reducing HCL tumor burden. Methods: To permit Rituximab enough time to reduce ADA and tumor burden, it was administered 3 days before cycle 1 day 1 at 375 mg/m2, and Moxe was given by 30-minute infusion days 1, 3 and 5. On subsequent 28-day cycles, patients received Moxe days 1, 3 and 5, and Rituximab prior to Moxe on day 1. Patients received up to 4 cycles past MRD-free CR, or up to 8 total cycles if MRD-free CR was achieved after cycle 4. The ADA assay determined percent neutralization by serum of the cytotoxicity of Moxe on CD22+ Raji cells. Results: After 13 patients received Moxe-Rituximab (MoxeR) without dose-limiting toxicity (DLT), meeting the phase 1 endpoint, 5 additional patients received Moxe with the biosimilar Ruxience (MoxeR). The first 3 patients received Moxe at 30 mcg/Kg/dose and subsequent patients 40 mcg/Kg/dose. All 18 were evaluable for toxicity and response. Although no DLT, one patient had transient grade 3 hemolytic uremic syndrome during cycle 3 without significant symptoms or need for therapy. Of the 18 patients, 15 (83%) responded, 14 (78%) achieved CR and 13 (72%) MRD-free CR by blood and bone marrow (BM) aspirate flow cytometry and BM biopsy immunohistochemistry. MRD-free CRs included one with HCLv. Even though Moxe vial expiration prevented enrollment of the 26 planned patients needed for a 1-sided p-value <0.025 compared to Moxe alone where 30 (47%) of 64 phase 1-3 patients achieved MRD-free CR, the 53% relative improvement with MoxeR achieved a 1-sided p-value of 0.05. Compared to 29 (48%) of 61 evaluable phase 1-3 patients who received Moxe alone with high ADA levels, 4 (29%) of 18 patients had high ADA levels to MoxeR (p=0.048). At 14-51 (median 35.3) months of follow-up, all but 2 of 13 MRD-free CRs are continuing, with relapse-free survival 14-45 (median 34.5) months. Conclusions: Despite enrolling slightly fewer patients than planned, MoxeR was safe and more effective than Moxe alone at achieving MRD-free CR, probably due to lower immunogenicity and faster reduction of HCL/HCLv tumor burden. Since non-Hodgkin’s lymphoma (NHL) cells from patients are sensitive to Moxe like HCL, MoxeR could be tested after NHL treatment to convert MRD+ to MRD-free CRs. Clinical trial information: NCT03805932 .

The CD6 interactome orchestrates ligand-independent T cell inhibitory signaling

BackgroundT-cell membrane scaffold proteins are pivotal in T cell function, acting as versatile signaling hubs. While CD6 forms a large intracellular signalosome, it is distinguished from typical scaffolds like LAT or PAG by possessing a substantial ectodomain that binds CD166, a well-characterized ligand expressed on most antigen-presenting cells (APC), through the third domain (d3) of the extracellular region. Although the intact form of CD6 is the most abundant in T cells, an isoform lacking d3 (CD6∆d3) is transiently expressed on activated T cells. Still, the precise character of the signaling transduced by CD6, whether costimulatory or inhibitory, and the influence of its ectodomain on these activities are unclear.MethodsWe expressed CD6 variants with extracellular deletions or cytosolic mutations in Jurkat cells containing eGFP reporters for NF-κB and NF-AT transcription factor activation. Cell activation was assessed by eGFP flow cytometry following Jurkat cell engagement with superantigen-presenting Raji cells. Using imaging flow cytometry, we evaluated the impact of the CD6-CD166 pair on cell adhesiveness during the antigen-dependent and -independent priming of T cells. We also examined the role of extracellular or cytosolic sequences on CD6 translocation to the immunological synapse, using immunofluorescence-based imaging.ResultsOur investigation dissecting the functions of the extracellular and cytosolic regions of CD6 revealed that CD6 was trafficked to the immunological synapse and exerted tonic inhibition wholly dependent on its cytosolic tail. Surprisingly, however, translocation to the synapse occurred independently of the extracellular d3 and of engagement to CD166. On the other hand, CD6 binding to CD166 significantly increased T cell:APC adhesion. However, this activity was most evident in the absence of APC priming with superantigen, and thus, in the absence of TCR engagement.ConclusionsOur study identifies CD6 as a novel ‘on/off’ scaffold-receptor capable of modulating responsiveness in two ways. Firstly, and independently of ligand binding, it establishes signaling thresholds through tonic inhibition, functioning as a membrane-bound scaffold. Secondly, CD6 has the capacity for alternative splicing-dependent variable ligand engagement, modulating its checkpoint-like activity.

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes.

L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.

Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation

Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

Design, Synthesis, and Investigation of the Pharmacokinetics and Anticancer Activities of Indenoisoquinoline Derivatives That Stabilize the G-Quadruplex in the MYC Promoter and Inhibit Topoisomerase I.

G-quadruplexes are noncanonical four-stranded DNA secondary structures. MYC is a master oncogene and the G-quadruplex formed in the MYC promoter functions as a transcriptional silencer and can be stabilized by small molecules. We have previously revealed a novel mechanism of action for indenoisoquinoline anticancer drugs, dual-downregulation of MYC and inhibition of topoisomerase I. Herein, we report the design and synthesis of novel 7-aza-8,9-methylenedioxyindenoisoquinolines based on desirable substituents and π-π stacking interactions. These compounds stabilize the MYC promoter G-quadruplex, significantly lower MYC levels in cancer cells, and inhibit topoisomerase I. MYC targeting was demonstrated by differential activities in Raji vs CA-46 cells and cytotoxicity in MYC-dependent cell lines. Cytotoxicities in the NCI-60 panel of human cancer cell lines were investigated. Favorable pharmacokinetics were established, and in vivo anticancer activities were demonstrated in xenograft mouse models. Furthermore, favorable brain penetration, brain pharmacokinetics, and anticancer activity in an orthotopic glioblastoma mouse model were demonstrated.

High yield killing of lymphoma cells by anti-CD22 CAR-NK cell therapy.

Chimeric antigen receptors (CARs) offer a promising new approach for targeting B cell malignancies through the immune system. Despite the proven effectiveness of CAR T cells targeting CD19 and CD22 in hematological malignancies, it is imperative to note that their production remains a highly complex process. Unlike T cells, NK cells eliminate targets in a non-antigen-specific manner while avoiding graft vs. host disease (GvHD). CAR-NK cells are considered safer than CAR-T cells because they have a shorter lifespan and produce less toxic cytokines. Due to their unlimited ability to proliferate in vitro, NK-92 cells can be used as a source for CAR-engineered NK cells. We found that CARs created from the m971 anti-CD22 mAb, which specifically targets a proximal CD22 epitope, were more effective at anti-leukemic activity compared to those made with other binding domains. To further enhance the anti-leukemic capacity of NK cells, we used lentiviral transduction to generate the m971-CD28-CD3ζ NK-92. CD22 is highly expressed in B cell lymphoma. To evaluate the potential of targeting CD22, Raji cells were selected as CD22-positive cells. Our study aimed to investigate CD22 as a potential target for CAR-NK-92 therapy in the treatment of B cell lymphoma. We first generated m971-CD28-CD3ζ NK-92 that expressed a CAR for binding CD22 in vitro. Flow cytometric analysis was used to evaluate the expression of CAR. The 7AAD determined the cytotoxicity of the m971-CD28-CD3ζ NK-92 towards target lymphoma cell lines by flow cytometry assay. The ELISA assay evaluated cytokine production in CAR NK-92 cells in response to target cells. The m971-CD28-CD3ζ NK-92 cells have successfully expressed the CD22-specific CAR. m971-CD28-CD3ζ NK-92 cells efficiently lysed CD22-expressing lymphoma cell lines and produced large amounts of cytokines such as IFN-γ and GM-CSF but a lower level of IL-6 after coculturing with target cells. Based on our results, it is evident that transferring m971-CD28-CD3ζ NK-92 cells could be a promising immunotherapy for B cell lymphoma.

Abstract LB074: Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies

Abstract Autologous chimeric antigen receptor (CAR)-T therapies have elicited high rates of durable complete responses in B cell lymphomas. Unfortunately, disease progression, manufacturing failures, and prohibitive costs highlight the need for an alternative source of cells that would provide a consistent and readily available therapy for patients. The “Appia Cells Utilized for Allogeneic” (ACUA) platform is an innovative process that differentiates cord blood-derived CD34+ hematopoietic stem and progenitor cells into large numbers of natural killer T (NKT) cells to support off-the-shelf treatment. NKT cells express an invariant T cell receptor (iTCR) and are naturally non-alloreactive, making them an ideal cell type for an allogeneic product. The ACUA platform circumvents the challenge of expanding NKT cells from the periphery where the cells comprise less than 1% of T cells. API-192 is an ACUA-derived, NKT cell product engineered to express dual CARs targeting CD19 and CD20 to prevent tumor antigen escape. At research scale, API-192 is a highly pure product (>90% iTCR+ 19CAR+ 20CAR+) that secretes soluble interleukin-15 for improved expansion and persistence. The transgenic iTCR is not expressed above physiologic levels and upon engagement, API-192 proliferates in response to and is cytotoxic against CD1d+ tumor cells in a ligand-dependent manner. In response to Raji and Nalm6 tumor cells, API-192 secretes pro-inflammatory cytokines, proliferates, and is cytotoxic in a dose- and CAR-dependent manner. API-192 can also control multiple rounds of tumor cell rechallenge in vitro. Using human tumor xenograft mouse models, we show that API-192 expands, controls tumor growth, and prolongs survival. Furthermore, API-192 persists in the periphery, enabling control of a tumor rechallenge at 70 days after initial dosing of effector cells. Indicative of a favorable immunogenic profile, API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less interferon (IFN)-γ release by NK and T cells in a mixed lymphocyte reaction (MLR) assay. To address a theoretical concern that endogenous TCRαβ rearrangement could occur during the ACUA process, we performed genomic DNA based TCR sequencing to quantify the level of TCRα or TCRβ rearrangement and preliminary data suggested negligible levels (<0.2%). Using a MLR assay, we demonstrate that compared to conventional CAR-T cells, API-192 secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with a low graft vs host risk. In an autonomous growth assay, API-192 expands at a low level and subsequently contracts, suggesting a lack of dysregulated growth. Collectively, these results demonstrate the favorable manufacturability, potency, and safety of API-192 and its potential as a novel allogeneic therapy. Citation Format: Lanny Gov, Jiaji Yu, Maha Qubain, Saige L. Pompura, Sean D. Allen, Michael A. Christopher, Jeff Wiezorek. Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB074.

Abstract LB129: Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies

Abstract CD38 and CD47 are co-expressed in various hematologic malignancies including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), T-cell ALL and B-cell chronic lymphocytic leukemia (CLL). We present, herein, several CD38 antibody - CD47 ligand trap bispecific antibodies (BsAbs) in an ADCC-enhanced IgG1 form that harbor high CD38 binding affinity, low CD47 binding affinity, along with negligible affinity to red blood cells (RBCs) and no induction of RBCs agglutination. Using flow cytometry and Biolayer interferometry (BLI), we confirmed that our BsAbs can simultaneously bind to both CD38 and CD47, and that binding to either antigen first does not prevent binding to the other antigen subsequently. On CD47+/CD38low Jurkat (T leukemic) cells, our BsAbs reveal less blocking effect on the CD47/SIRPα interaction than a monospecific CD47 ligand trap molecule based on which our BsAbs were designed; conversely, on CD38/CD47 double-positive Reh (pro-B ALL) cells, our BsAbs reveal 200-500-fold greater blocking effect on the CD47/SIRPα interaction than the same monospecific CD47 ligand trap molecule, indicating that the inhibition of CD47/SIRPα by our BsAbs is depended on the engagement of CD38 on tumor cells. On CD47+/CD38+ Daudi (B NHL) lymphoma cells and L-1236 (B Hodgkin) lymphoma cells, four of our BsAbs inhibited CD38 enzymatic activity, which is responsible for immune-suppressive adenosine production in tumor microenvironment. Our BsAbs presented potent dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against CD47+/CD38+ Raji (B NHL) and NCI-H929 (B plasmacytoma myeloma) cells, while revealing negligible ADCC activity against CD47+/CD38− cells, indicating low off-tumor toxicity. Likewise, our BsAbs also harbor potent antibody-dependent cellular phagocytosis (ADCP) activity against Raji NHL cells, varied degree of complement-dependent cytotoxicity (CDC) activity and direct apoptosis induction against Daudi NHL cells. In a mouse xenograft model harboring NCI-H929 multiple myeloma, our BsAbs produced more potent tumor growth inhibition, than monospecific anti-CD47 ligand trap, anti-CD38 (isatuximab) or combination of isatuximab and anti-CD47 ligand trap, at the same molecular dose per body weight. Notably, clone IMM5605-12C10 eradicated all tumors and achieved 100% complete response rate in a group of six mice. In summary, simultaneous targeting CD38 and CD47 using our CD38 antibody - CD47 ligand trap bispecific design in an ADCC-enhanced IgG1 form presents a novel multimodal approach for treating hematologic malignancies that are resistant to anti-CD38 antibodies. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Yanan Yang, Huiqin Guo, Dandan Liu, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB129.