CD38 Expression Levels Research Articles

Specific targeting of Multiple Myeloma by Dual Split-signaling Chimeric Antigen Receptors T-cells directed against CD38 and CD138.

The success of BCMA-specific chimeric antigen receptor (CAR) T-cells illustrates the potential of this novel therapy for multiple myeloma (MM). Nonetheless, broadening CAR T-cell therapy beyond BCMA requires inventive strategies as there are only a few MM- or plasma cell-specific target antigens. We investigated the feasibility of achieving MM specificity by dual-split CD38/CD138 CAR targeting, whereby the stimulatory and costimulatory signals for T-cell activation are split into two separate stimulatory (sCAR) and costimulatory CARs (cCAR). Using various combinations of CD38 and CD138 sCARs and cCARs with different affinities, we generated several dual-split CAR T-cells and analyzed them for MM-specific effector functions in vitro. The best-functioning CAR T-cells were tested in vivo in a murine xenograft model. We found optimal designs of both CD38sCAR/CD138cCAR and CD138sCAR/CD38cCAR combinations, that effectively lysed MM cells, but spared single CD38 or CD138 positive healthy hematopoietic cells. While the CD38sCAR/CD138cCAR T-cells achieved MM-specific activity solely due to the low affinity of the CD38sCARs, the MM-specific cytotoxicity, cytokine release, and proliferation of CD138sCAR/CD38cCAR T‑cells were established through a true combinatorial stimulatory and costimulatory effect. The most optimal combination comprised a low affinity CD138sCAR combined with a high affinity CD38cCAR. These CD138sCAR/CD38cCAR T-cells also showed dual-antigen specific anti-MM effects in vivo. Importantly, they were also effective against MM cells from daratumumab pre-treated patients with decreased CD38 expression levels. We demonstrate the possibility to specifically target MM cells, even after CD38 targeted therapy, with carefully-designed dual-split CARs directed against CD38 and CD138.

The Use of Novel Antibodies to Characterize B cells in Health and Disease

IntroductionCD38, a glycoprotein ectoenzyme found on the surface of majority of hematopoietic cells, can exist in monomeric or multimeric conformations and enzymatic activity correlates with multimerization. During human B cell differentiation, CD38 expression levels are low on naïve and memory B cells, intermediate on transitional cells, and high on antibody-secreting plasmablasts and plasma cells. Conventional anti-CD38 (C-CD38) antibodies, recognize all CD38 formations and are not selective for identification of plasmablasts, thus requiring the use of additional antibodies including CD27. In contrast, a novel sea lamprey derived monoclonal VLRB antibody, tetVLRB MM3 (V-CD38), recognizes an epitope likely located within the catalytic site of active multimeric CD38 and therefore might be more specific to plasmablasts, which express active CD38. ObjectivesCharacterize the B cells recognized by V-CD38 in the peripheral blood of individuals with or without inborn errors of immunity (IEI). MethodsB cell subsets were characterized as described in figure 1 using conventional antibodies to CD3, CD19, IgM, CD27, C-CD38, and by V-CD38 generated by a collaborator. Results were presented as means with standard deviations (SD). Correlations were calculated using the Pearson’s Correlation Coefficient formula. ResultsTo validate our identification of plasmablasts with V-CD38, samples from patients with X-linked agammaglobulinemia lacking B cells, and tonsils rich in plasmablasts were used as negative and positive controls, respectively. Analysis of samples from 16 individuals revealed nearly identical (r = 0.99, p <.00001) proportions of CD27hi plasmablasts recognized by V-CD38 (0.51%+/-0.74%) and C-CD38 (0.49%+/-0.70%), suggesting that all cells expressed multimeric CD38. In contrast, only 45.28% ±16.19% of transitional IgMhighCD27-C-CD38int B cells were also recognized by V-CD38, (2.52%+/–3.62% and 1.15%+/–1.75%, respectively), a finding that was consistent across samples (r = 0.98, p <.00001). Preliminary studies in three IEI patients demonstrated markedly reduced plasmablasts (0.035%+/–0.026%), class-switched memory B cells (1.25% +/–1.52%) and transitional cells (1.25%+/–1.06%) compared to healthy controls (0.59%+/–0.74%, 15.29%+/–8.95%, and 2.81%+/–3.96% respectively). [Display omitted] ConclusionsWhile C-CD38 and V-CD38 antibodies can identify plasmablasts in peripheral blood, V-CD38 allows more selective detection of CD38 on plasmablasts. Some circulating transitional cells also express multimeric CD38. Additional studies will determine whether V-CD38 can better identify patients suffering from abnormal plasmablast formation and impaired production of antibodies.

Potential role of EBV and Toll-like receptor 9 ligand in patients with systemic lupus erythematosus.

SLE is a multisystem autoimmune disease characterized by multiple immunological abnormalities including production of autoantibodies. While the etiology of SLE is largely unknown, it is generally accepted that both genetic and environmental factors contribute to disease risk and immune dysregulation. Production of IFN-α is important for protecting the host against infections; however, over stimulation of innate immune pathways can induce autoimmune disease. Environmental factors, particularly Epstein-Barr virus (EBV), have been proposed to play an important role in SLE disease. Improper engagement of Toll-like receptor (TLR) pathways by endogenous or exogenous ligands may lead to the initiation of autoimmune responses and tissue injury. EBV is shown to be a potent stimulant of IFN-α by TLR signaling cascades. Given the highlighted role of IFN-α in SLE pathogenesis and potential role of EBV infection in this disease, the present study is aimed at exploring the in vitro effects of EBV infection and CPG (either alone or in combination) on IFN-α. We also examined the expression level of CD20 and BDCA-4 and CD123 in PBMCs in 32 SLE patients and 32 healthy controls. Our results showed PBMCs treated with CPG-induced higher levels of IFN-α and TLR-9 gene expression fold change compared to cells treated with either EBV or EBV-CPG. Moreover, PBMCs treated with CPG produced significantly higher IFN-α concentration in supernatant compared to cells treated with EBV but not EBV-CPG. Our results further highlight the potential role of EBV infection and TLRs in SLE patients although more studies are warranted to ascertain the global imprint that EBV infection can have on immune signature in patients with SLE.

Abstract 1751: Prognostic value of CD20/CD73 ratio in sensitive and relapsed/refractory diffuse large B-cell lymphoma cell lines

Abstract Purpose: Developing novel therapy strategies to treat relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) is critically needed. Previous studies in our group found despite dysregulation of apoptotic pathway in resistant DLBCL, upregulation in glycose metabolism and energetic ATP production were also found in r/r DLBCL. Therefore, decreasing or blockage ATP production may provide a novel method to overcome resistance in lymphoma. ATP/ADP is hydrolyzed into AMP by NTPDases, and AMP into adenosine and phosphate by ecto-5’-nucleotidase, known as CD73. CD73 is the rate limiting enzyme anchored the surface of tumor and immune cell. The expression level of CD73 on the tumor was reported significantly associated with inferior prognosis in variety of cancers. Moreover, expression level of CD73 on immune cell has been shown immunosuppression in multiple cancers. Targeting CD73 by monoclonal antibody or small inhibitor currently underwent multiple clinical trials. However, the role of CD73 in r/r DLBCL and the relationship between CD73 and CD20 have not been explored. Methods: Germinal center B-cell like, activated B-cell like lymphoma, Burkitt’s lymphoma and rituximab resistant cell lines generated in our lab were used. Surface expression of CD73 and CD20 were determined by staining anti-CD73 FITC and anti-CD20 Per-cy5.5 antibodies and running flow cytometry. The expression levels of CD73 and CD20 were detected by media fluorescence index (MFI). The CD20/CD73 ratio was calculated by CD73 MFI/CD20 MFI. Result: Previously our group found surface CD20 levels exhibited a linear correlation with rituximab induced cellular mediated cytotoxicity. The CD20 MFI of germinal center B-cell like, activated B-cell like lymphoma, Burkitt’s lymphoma and rituximab resistant cell lines were: DHL4 8652, DOHH2 6472, TMD8 3981, RL 2849, Raji 2011, U2932 604, RL 4RH 599 and Raji 4RH 576. The higher MFI of CD20, the more sensitive to rituximab treatment. Here we discovered that the surface level of CD73 had an inverse correlation with CD20 level in these cell lines. In detail, CD73 MFI were: DHL4 464, DOHH2 387, TMD8 363, RL 299, Raji 611, U2932 486, RL 4RH 631 and Raji 4RH 702. The CD20/CD73 ratio of cell lines from high to low were DOHH2, DHL4, TMD8, RL, Raji, U2932, Raji 4RH and RL 4RH, respectively. This CD20/CD73 ratio in each of cell lines was found more straight forward to represent the sensitivity of rituximab or chemo drug treatment in vitro condition. Conclusion: Our study was first time to reveal the association between CD20/CD73 ratio and immune-chemo response in lymphoma cell lines, especially in resistant lymphoma in vitro condition. This may give us prognostic value of CD20/CD73 ratio in predicting lymphoma response to immune/chemotherapy in clinical setting. Further study to find CD20 and CD73 association in the clinical setting and mechanism of action need to be warranted. Citation Format: Juan J. Gu, Cory Mavis, Jessica Wang, Taylor Mandeville, Francisco Hernandez-Ilizaliturri. Prognostic value of CD20/CD73 ratio in sensitive and relapsed/refractory diffuse large B-cell lymphoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1751.

CD30 expression is frequently decreased in relapsed classic Hodgkin lymphoma after anti-CD30 CAR T-cell therapy.

Chimeric antigen receptor (CAR) T-cells anti-CD30 is an innovative therapeutic option that has been used to treat cases of refractory/relapsed (R/R) classic Hodgkin lymphoma (CHL). Limited data are available regarding the CD30 expression status of patients who relapsed after this therapy. This is the first study to show decreased CD30 expression in R/R CHL in patients (n = 5) who underwent CAR T-cell therapy in our institution between 2018 and 2022. Although conventional immunohistochemical assays showed decreased CD30 expression in neoplastic cells in all cases (8/8) the tyramide amplification assay and RNAScope in situ hybridisation detected CD30 expression at different levels in 100% (n = 8/8) and 75% (n = 3/4), respectively. Hence, our findings document that certain levels of CD30 expression are retained by the neoplastic cells. This is not only of biological interest but also diagnostically important, as detection of CD30 is an essential factor in establishing a diagnosis of CHL.

All-trans retinoic acid improves NSD2-mediated RARα phase separation and efficacy of anti-CD38 CAR T-cell therapy in multiple myeloma

BackgroundImmunotherapies targeting CD38 have demonstrated salient efficacy in relapsed/refractory multiple myeloma (MM). However, loss of CD38 antigen and outgrowth of CD38 negative plasma cells have emerged as a major obstacle...

Lymphocytes from B-acute lymphoblastic leukemia patients present differential regulation of the adenosinergic axis depending on risk stratification

PurposeAlthough risk-stratified chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a significant number of cases. The identification of new therapeutic targets as well as prognostic and diagnostic biomarkers can improve B-ALL patients' clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients’ lymphocyte subpopulations.MethodsPeripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of {mathrm{CD}38}^{+}{/mathrm{CD}73}^{+}, and {mathrm{CD}39}^{+}{/mathrm{CD}73}^{+} in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC).ResultsComparing B-ALL patients to health donors, we observed an increase of CD4 + and CD8 + T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+ frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1β, and ADO concentrations, together with an increase in AMP in B-ALL patients' plasma.ConclusionAs immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of different cellular subsets. We observed a pro-tumor immunity expression profile in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL.

Comparison of dynamic changes in the peripheral CD8+ T cells function and differentiation in ESCC patients treated with radiotherapy combined with anti-PD-1 antibody or concurrent chemoradiotherapy.

The systematic immune status of cancer patients undergoing immunotherapy is little known. We prospectively identified the function and differentiation traits of peripheral CD8+ T cells based on our phase 1b clinical trial (NCT03222440) of radiotherapy combined with camrelizumab in patients with locally advanced esophageal squamous cell carcinoma (ESCC) and compared it with concurrent chemoradiotherapy (CCRT). 19 and 18 patients were included in the cohort of radiotherapy plus camrelizumab and cohort of CCRT treatment. By using flow cytometry, we evaluated the expression levels of PD-1, Eomes, T-bet and IFN-γ (function), CD38 and HLA-DR (activation), and differentiation subsets classified according to the expression levels of CD45RA and CD62L in peripheral CD8+ T cells before and during treatment. Effective binding of anti-PD-1 antibody camrelizumab with PD-1 on CD8+ T cells was detected during treatment. Both two treatments elevated the expression levels of activation molecules CD38 and HLA-DR on CD8+ T cells. PD-1+CD8+ T cells had more activation features than PD-1-CD8+ T cells in two groups and the treatments did not alter these differences. The two treatments activated both PD-1+ and PD-1- CD8+ T cells. PD-1+CD8+ T cells had less Naïve and TEMRA but more Tcm and Tem than PD-1-CD8+ T cells in two groups and both two treatments changed the ratio of memory T cells in PD-1+ and PD-1- cells. RT plus camrelizumab treatment reduced Naïve T cells and TEMRA subsets both in PD-1+ and PD-1- CD8+ T cells while elevated Tcm subset in PD-1+CD8+ T cells and Tem subset in PD-1-CD8+ T cells. CCRT elevated Tcm subset and reduced TEMRA subset in PD-1-CD8+ T cells while did not change any subset in PD-1+CD8+ T cells. Furthermore, patients undergoing radiotherapy plus immunotherapy were found to obtain better prognosis than those receiving CCRT. This study identified the dynamic changes of systematic immune status of patients undergoing treatment. The two treatments had similar activation effects on peripheral CD8+ T cells with different PD-1 properties but had different effects on their differentiation status. These results provided potential clues to the reasons underlying the difference in prognosis of the two treatments.

Tub-010, a Novel Antibody-Drug-Conjugate with Reduced Nonspecific Toxicity Profile Based on Tub-Tag Technology Widens the Therapeutic Window for the Treatment of CD30+ Malignancies

Introduction: Patients with R/R CD30+ malignancies such as PTCL, CTCL and cHL often lack effective and tolerable therapy options. Available treatments frequently provide only short-term disease control at the cost of significant or cumulative toxicities. Brentuximab-Vedotin (BV, Adcetris®), an MMAE-delivering anti-CD30-ADC, has a high response rate but dose-limiting AEs often result in premature dose reduction or even therapy discontinuation. The maleimide chemistry results in a heterogeneous product (DAR 0-8) with varying PK and potency properties and spontaneous deconjugation can cause premature transfer of linker payload to serum proteins and thereby reduce the tumor selectivity. In consequence, some of the BV-induced toxicity is likely design-based leading to CD30-independent cellular uptake and aggregation of the ADC. Objectives: We aimed to generate a novel CD30-ADC with improved biophysical properties to widen the therapeutic window using Tub-tag-conjugation technology. Material & methods: TUB-010 is a next-generation ADC, implementing a novel conjugation strategy, called Tub-tag® conjugation (Schumacher, Angewandte Chemie 2015). The recombinant Tub-tag sequence, a 14 amino acid peptide derived from α-tubulin and fused to the C-termini of the CD30 mAb light chains, has unique features in that it provides a significant degree of hydrophilicity to counterbalance the payload-derived hydrophobicity, thus enabling superior biophysical properties and product homogeneity. This new technology was used to generate a DAR2-ADC with stably linked MMAE via a Cathepsin B cleavable linker (vc-PAB) and chemoenzymatic conjugation of linker payload using Tubulin-tyrosine ligase (TTL). The resulting ADC was tested for physicochemical properties, homogeneity, stability, internalization, preclinical efficacy, nonspecific uptake and general cytotoxicity in primary cells, cell lines, rats & cynomolgus monkeys. Results: Tub-tag technology enabled us to create a DAR2 anti-CD30-ADC with a binding affinity, internalization and lysosomal release characteristics similar to BV. In contrast to BV, TUB-010 is a homogeneous DAR2 ADC. The linker-payload is chemically stably attached to the mAb with neglectable premature deconjugation while in circulation, whereas protease-mediated release of MMAE within the lysosome occurs normally as expected with the vc-PAB linker. The increased ADC stability prior to internalization via CD30 leads to reduced nonspecific transfer of linker-payload to serum proteins, and overall improved PK parameters in rats. The hydrophobicity of MMAE is counterbalanced by hydrophilicity provided by the Tub-tag peptides, which resulted in > 5-fold reduced ADC aggregation under stress conditions compared to BV. The increased hydrophilicity of the overall ADC reduced nonspecific cellular uptake and consecutive cellular toxicity vs BV within the clinically therapeutic range by factor 2- to >10-fold in various human primary cells (Fig. A). When normalized to the MMAE concentration, TUB-010 showed similar in vitro cytotoxic efficacy as well as similar bystander activity compared to BV on established cancer cell lines from cHL, PTCL and CTCL patients with various levels of CD30 expression (Fig. A). In vivo, TUB-010 exerted significantly improved tumor control compared to BV when both ADCs were dosed at equal MMAE concentration in the Karpas-299 cell-derived ALCL xenograft model (Fig. B). In cynomolgus monkey dose range finding and GLP toxicity studies, TUB-010 administered 2 or 4 times Q3W in a dose range of 6-15 mg/kg showed a 5-fold widened therapeutic window for the ADC as the MTD was not reached. Hematological toxicity was reduced compared to BV and no signs of anatomical of functional tissue damage were detectable. Conclusions: Tub-tag technology allows the reduction of nonspecific target-independent toxicity of ADCs in preclinical models by improving key design issues in current ADCs such as stability and overall biophysical features. TUB-010 is a promising, novel and potential best-in-class CD30-ADC which can deliver the cytotoxic payload to CD30+ malignancies with higher precision and with a wider therapeutic window than BV. These features suggest that TUB-010 may increase the clinical benefit with CD30-targeting ADC therapy for patients with CD30+ malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD19 Expression by IHC Alone Is Not a Predictor of Response to Loncastuximab Tesirine: Results from the LOTIS-2 Clinical Trial and Quantitative Systems Pharmacology Modeling

Introduction: CD19 is a clinically-validated target for the treatment of B-cell malignancies, and several CD19-targeted therapies have received approval, including chimeric antigen receptor T-cell (CAR-T) therapy, antibody-drug conjugates (ADCs), monoclonal antibodies, and bispecific agents. However, the optimal sequencing of these treatments has not yet been clarified. Loncastuximab tesirine (loncastuximab tesirine-lypl; Lonca) is an ADC comprising an anti-CD19 antibody conjugated to a pyrrolobenzodiazepine (PBD) dimer cytotoxin, indicated for relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) after ≥2 systemic treatments. Prior CAR-T therapy does not preclude a response to Lonca, and responses were also observed in patients who received CAR-T therapy post-Lonca. The present analysis was performed to determine the contribution of CD19 expression to responses in patients treated with Lonca. Methods: CD19 expression determined by immunohistochemistry (IHC) was evaluated in a cohort of patients enrolled in the LOTIS-2 clinical trial (NCT03589469) with available tissue samples obtained after their last anti-cancer systemic therapy and prior to Lonca. IHC was performed using the LE-CD19 antibody (DAKO), and expression was analyzed using the BenchMark ULTRA platform (Ventana). CD19 expression was assessed by semiquantitative scoring of both the percentage of positive tumor cells and the H-Score (semiquantitative assessment of the percentage of CD19 positive cells and staining intensity). Quantitative Systems Pharmacology (QSP) modeling was used to predict response to Lonca and to test hypotheses regarding patient-specific covariates. Results: The cohort included patients with any prior systemic therapies (n = 59), including patients who received CAR-T as the last therapy prior to biopsy (n = 9). Responses to Lonca were seen in patients across all levels of CD19 expression, including patients with extremely low or no detectable CD19 expression at baseline and extremely low H-Scores. On the basis of the QSP modeling, patients are anticipated to achieve disease response to Lonca with CD19 tumor cell-surface densities as low as 1,000 molecules/cell (Figure 2). Conclusions: Response to Lonca was observed in R/R DLBCL patients with very low CD19 tumor expression as measured by IHC. QSP modeling predicts that CD19 expression level by IHC is not predictive of response to Lonca, whereas the addition of CD19 surface density improves the response prediction. Patients responded to Lonca with estimated tumor cell surface densities as low as 1,000 molecules/cell, normally below the level of IHC detection. Our findings indicate that Lonca is an effective treatment option for patients with R/R DLBCL following ≥ 2 lines of treatment, even in patients expected to have a low level of CD19 expression. These results serve as a basis for future studies addressing sequencing of CD19-targeted agents. Funding: ADC Therapeutics SA; medical writing: CiTRUS Health Group. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of Epcoritamab and Rituximab Combination in Preclinical Models of B-Cell Non-Hodgkin's Lymphoma (NHL)

Background: Epcoritamab (Epco) is a CD3 bispecific antibody that induces selective, potent T-cell-mediated killing of CD20-positive malignant B-cells. In the phase 1/2 monotherapy study, subcutaneous administered Epco showed a favorable safety profile and promising efficacy, including complete responses in heavily pretreated patients with relapsed / refractory non-Hodgkin's lymphoma (Hutchings et al, Lancet 2021; Thieblemont et al, EHA 2022, LB2364; EPCORE NHL-1). Ongoing clinical studies combining Epco with standard-of-care therapies including combinations with Rituximab show promising preliminary anti-tumor activity in diffuse large B-cell lymphoma and follicular lymphoma (Falchi et al, ASCO 2022 abstracts 7523 and 7524; EPCORE NHL-2 arm 1 and 2; NCT04663347). Here we evaluated the competition for CD20 binding and functional interactions between these agents and modeled its impact on Epco, T-cell, and tumor cell trimer formation that drives Epco clinical activity. Aims: Investigate the preclinical binding and functional interaction between Epco and Rituximab to establish the potential for the combination of these agents in the treatment of B-cell NHL. Methods: Competition between Epco and Rituximab for binding to CD20 cells was assessed by Flow Cytometry after incubating the single labeled agent in the presence of increasing concentrations of the competitor and vice-versa (across 0.1 - 300 ug/ml evaluated for each agent), on a range of CD20 expressing lymphoma cell lines. Epco-induced-T cell activation and redirected T cell cytotoxicity was determined by co-culturing T cells with lymphoma cells with varying expression of CD20. Rituximab antibody-dependent cellular cytotoxicity (ADCC) activity was assessed by incubating fresh peripheral blood mononuclear cells with lymphoma cells in the presence of a CD3 control Epco antibody (DuoBody-ctrlxCD20). Data from binding and cytotoxicity was used to quantify the degree of binding curve shifts at different Rituximab:Epco concentrations as well as the impact on predicted trimer formation and cytotoxicity, based on modeling. Results: In binding assays, the addition of Rituximab resulted in a dose-dependent reduction of Epco binding to CD20 expressing lymphoma cell lines, with a significant reduction of Epco binding observed at clinically relevant Epco concentrations in cells with high CD20 expression levels (Figure 1). Similar binding interference interactions were observed for binding of Epco to cell lines with low and intermediate CD20 expression levels. Binding of Rituximab to cell lines expressing high CD20 levels was not significantly reduced by clinically relevant concentrations of Epco. In Epco functional studies, the addition of increasing concentrations of Rituximab did not affect Epco cytotoxic activity at clinically relevant range of Rituximab doses tested (Figure 2). Similarly, no effect of Rituximab on Epco-mediated T cell activation was observed at clinically relevant concentrations of Rituximab and Epco. ADCC activity at clinically relevant concentrations of Rituximab (10-250 ug/ml) was not impacted by incubation with the CD3 control Epco antibody (DuoBody-ctrlxCD20), even at concentrations exceeding the Epco clinical concentrations. Modeling and simulation of in vitro data showed that at clinically relevant concentration of Epco, the presence of clinically relevant concentration of Rituximab does not meaningfully impact Epco trimer formation, and the predicted trimer formation remains above EC90 relative to that needed for 50% cell kill. Conclusions: Our studies showed that while Rituximab exerts significant interference on Epco binding to CD20-expressing cells, tumor-cell cytotoxicity by Epco is minimally affected by clinically relevant concentrations of Rituximab. This conclusion is supported by preclinical modeling and simulations showing that Epco activity driven by trimer formation is not meaningfully impacted by clinical concentrations of Rituximab. Furthermore, we found a limited effect of Epco on Rituximab binding to CD20 and no impact on ADCC activity by Rituximab, thereby supporting potential synergy in the combination between these agents. Results from this study confirm the ability to combine Epco with CD20 antibodies, specifically with Rituximab, and the potential for synergistic activity of this combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PI3K and BTK Inhibition Induces the Upregulation of CD19 and Increases Sensitivity to CAR T Cells in a Model of Marginal Zone Lymphoma (MZL)

Background. Marginal zone lymphomas (MZLs) are characterized by an indolent course with median survival of over 10 years. Unfortunately, relapses/progressions are common and might heavily impact patients' survival. Chimeric antigen receptor (CAR) T cells revolutionized the treatment of several relapsing/refractory B cell malignancies. However, CAR T cells success in MZL is still limited (Jacobson et al, Lancet Oncol 2022) and none of the currently available products has been approved so far for this indication. Little is known about the reasons behind the limited efficacy of CAR T cells against MZL. Given the importance of CD19-antigen density in CAR T cell killing of B-cell malignancies, in this work we investigated the contribution of CD19 levels in sensitivity of MZL in vitro models to CAR T cells. We previously presented MZL-derived models with secondary resistance to BTK/PI3K inhibitors, also observing increased CD19 levels in some of the resistant cells (Arribas et al, ASH2019, ENA2019, Haematologica 2022). Based on these data, we hypothesized that the CD19 modulation induced by exposure to BTK/PI3K inhibitors might be a strategy to increase CAR T cell efficacy in MZL. Here, we tested this hypothesis using MZL models, parental or chronically exposed to BTK/PI3K inhibitors and with acquired resistance to the small molecules. Methods. CD19 expression was determined at protein level by flow cytometry. CD19-specific CAR T with CD28 co-stimulatory domain were produced from healthy donors using retroviral vectors. Transduction efficacy was evaluated by protein L staining. MZL cells were co-cultured with the CAR T cells at an E:T ratio of 1:1 after adjusting for transduction efficacy. Viability was measured by flow cytometry at 48h. CAR T cell-mediated cytotoxicity was calculated as percentage of cell death compared to MZL cells cultured alone. Longitudinal cell growth was measured by bioluminescence in mCherry/Luciferase transduced cells after normalization to day 0. Results. As we previously reported, CD19 expression levels are very heterogenous both across MZL clinical specimens (n.=41; Bonfiglio et al, Blood 2022; Figure 1A) and cell lines (n.=6; Hicks et al, Haematologica 2019; Figure 1B). We selected models expressing the highest (Karpas-1718) and the lowest (VL51) CD19 levels and tested their sensitivity to CAR T cell killing. CD19-specific CAR T cells displayed a robust cytotoxic activity against Karpas-1718 (median 74%, range 61-88%) while no CAR T cell-specific killing was observed against VL51 (Figure 1C). The results were in agreement with the CD19 levels, higher in Karpas-1718 and lower in VL51. PI3K or BTK inhibition led to CD19 upregulation on VL51 surface (Figure 1D). Compared to the non-treated (NT), co-incubation of parental VL51 with CAR T cells or control untransduced T (UT) cells did not inhibit tumor cell growth (Figure 1E, left panel). Conversely, CAR T cells, but not control UT cells, led to 43% growth inhibition at day 4 after co-incubation with idelalisib-treated VL51 (Figure 1E, middle panel) and completely inhibited ibrutinib-treated VL51 growth (Figure 1E, right panel). Conclusions. CD19 modulation by PI3K/BTK inhibition increased the sensitivity to CAR T cells in a CD19low MZL model. These data support exploring alternative modalities of CAR T cell treatment for MZL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genome-Wide CRISPR-Cas9 Screen Identifies KDM6A As a Modulator of Daratumumab Sensitivity in Multiple Myeloma

Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38, which triggers both direct and immune-mediated cytotoxicity of multiple myeloma (MM) cells in the bone marrow (BM) milieu. Importantly, Dara combination therapies have a high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse is commonly observed due to the development of Dara resistance. As an immune-targeting drug, the anti-tumor activity of Dara is closely associated with the expression of CD38, with nonresponders having a lower expression of CD38. To identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells, we performed a genome-wide CRISPR-Cas9 knockout (KO) screen in vitro. We identified 46 different sgRNAs that were positively correlated with MM cytotoxicity triggered by Dara and primary NK cells. Using the cells with the lowest expression of CD38 (bottom 5%), we performed a second round of screening and found that KDM6A was the top-ranked gene out of the set of overlapping genes from these two screenings. We confirmed that CD38 expression was significantly decreased (>2 times, p<0.01) by KDM6A KO and that re-introducing KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity. KDM6A encodes lysine-specific demethylase 6A, which demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. To confirm that CD38 expression was altered by the methylation status of H3K27, we compared the CD38 promoter area between KDM6A KO and control cells using ChIP-seq and ChIP qPCR and found that the H3K27me3 level was higher in KDM6A KO cells. H3K27 is methylated by EZH2, and we found that EZH2 inhibitor treatment significantly decreased the H3K27me3 level of the CD38 promoter area. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein expression in KDM6A KO cells. Importantly, EZH2 inhibitor also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity in vitro and in xenograft mouse model. We next re-introduced CD38 into KDM6A KO cells and found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment. This suggested that besides CD38 downregulation, there are other mechanisms underlying Dara resistance induced by KDM6A KO. We performed RNA-seq in KDM6A KO cells and found that CD48, an NK-activating ligand, was downregulated. Overexpression of CD48 in KDM6A KO cells significantly increased secretion of Granzyme B and Perforin, restoring the activity of NK cells and re-sensitizing KDM6A KO cells to Dara treatment. These data suggest that KDM6A not only regulates CD38 expression, but also NK cell activity by CD48 regulation, thereby modulating the efficacy of Dara. Taken together, our studies demonstrate that KDM6A regulates CD38 and CD48 expression in MM. Moreover, we validate that combination treatment with FDA-approved EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome.

Clonal Dynamics of HDR-Edited HSPCs Targeting the CD33 Locus in Rhesus Macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. Loss or damage of LT-HSCs with resultant oligoclonal hematopoiesis can increase the risk of malignant transformation or long-term hematopoietic and immune dysfunction. HDR has adverse impacts on LT-HSC function as revealed by ex vivo studies and murine xenograft models, likely due to p53 activation, intracellular innate immune activation, and/or other pathways. However, the impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not previously been studied. We tracked the clonal dynamics of CRISPR/HDR-edited barcoded HSPCs following autologous transplantation in rhesus macaques (RMs). HDR-favored gRNAs targeting the CD33 gene locus and an ssODN containing a 6 bp barcode ID and 14 bp random barcode sequences and flanked by CD33 homology arms were designed. Autologous CD34+ cells were electroporated with the Cas9 RNP and HDR cassette in the presence of i53 mRNA to inhibit p53-mediated cell death and reinfused into animal 1 following myeloablation. In animal 1, CD33 KO neutrophils were prevalent early following engraftment and then rapidly decreased over the next two months. Similar to CD33 expression level, we revealed that HDR knock-ins as well as total indel frequency drastically decreased following transplantation. This was in marked contrast to macaques transplanted with CD34+ HSPCs edited at the CD33 locus only for NHEJ, without inclusion of an HDR cassette (Kim et al, Cell, 2018, and Figure 1 below). We next performed a competitive transplantation comparing clonal dynamics of edited cells to barcoded lentiviral (LV)-transduced cells in two macaques. For each animal, CD34+ cells were fractionated into 2 aliquots, and half were transduced with a barcoded GFP-expressing LV vector and half electroporated with the Cas9 RNP and HDR cassette with higher concentrations of i53 mRNA to enhance protection of CRISPR edited LT-HSCs. The two cell populations were combined and reinfused into myeloablated animals. CD33 KO cells in both animals decreased rapidly, however the higher concentration of i53 mRNA facilitated a less steep reduction in CD33 total indel frequency in vivo over time. However, HDR in these two animals also markedly decreased, similar to the pattern in animal 1. In contrast, LV-transduced GFP+ cell engraftment stabilized at 20% after 2 months. We retrieved embedded barcodes from CRISPR/HDR cells and revealed that a diversity of HDR clones contributed to early hematopoietic reconstitution, with restriction to a much smaller set of long-term clones at steady state, proportional in the 3 animals to the level of HDR overall. The diversity of HDR-edited contributing clones was far less than LV-transduced contributing clones long-term post-transplantation. In conclusion, CRISPR/HDR edited cells were found to decrease rapidly after autologous transplantation in rhesus macaques despite substantial gene editing assessed in the infusion product prior to infusion, in contrast to much more stable LV-transduced cells. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which was somewhat improved by use of high dose i53 mRNA during editing. It will be important to find improved approaches to retain LT-HSC functional activity during the HDR editing process, or explore targeted gene correction approaches such as base or PRIME editing that may better preserve HSC activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The severity of COVID-19 is accompanied by a change in the expression of the surface marker CD38 in T lymphocytes and NK cells

BACKGROUND: COVID-19 mediates activation of immunocompetent cells. Little is known about the phenotypic marker CD38 on the surface of T and NK cells and its association with the severity of COVID-19.&#x0D; AIM: To analyze the distribution of the surface marker CD38 in subsets of T and NK cells at the different stages of their differentiation in the groups of patients with COVID-19 and convalescents, and to reveal the relationship between the level of CD38 expression in subpopulations of T and NK cells with the severity of COVID-19.&#x0D; MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) from patients and donors were isolated using ficoll density gradient followed by cytofluorimetric analysis of the surface markers: CD3, CD56, CD38.&#x0D; RESULTS: In PBMC samples the level of CD38 expression on NK cells (CD3CD56+), including CD56bright and CD56dim subsets, as well as on T lymphocytes, differentiating conventional CD56(CD3+CD56) and NKT-like (CD3+CD56+) cells, were analyzed. Among CD56 T cells and NKT-like cells the highest levels of CD38 expression were registered in the groups of patients of moderate severity and convalescents, and, in contrast, the decreased CD38 levels were detected in the group of patients from the intensive care unit.&#x0D; CONCLUSIONS: The findings suggest that the level of CD38+ T cell in COVID-19 may be of use as diagnostic value.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

CD20 Expression as a Possible Novel Prognostic Marker in CLL: Application of EuroFlow Standardization Technique and Normalization Procedures in Flow Cytometric Expression Analysis.

Simple SummaryUp to 50% of patients with chronic lymphocytic leukemia relapse within four years after anti-CD20-directed treatment with a need for a subsequent treatment, which can potentially include anti-CD20 agents again. To retrospectively study the influence of CD20 expression in CLL patients receiving anti-CD20 directed therapy on therapy response, we designed and evaluated a bead-based normalization approach for the analysis of CD20 expression levels to reduce variation of flow cytometric measurements due to technical issues. Normalization significantly reduced the variability of median fluorescence intensities of fluorochrome-conjugated beads without artificially rendering the instruments’ performances, and longitudinal biological variations of marker expression based on MFI values could be robustly assessed. In a cross-trial comparison, strong MRD response correlated with the CD20 expression level before therapy started in patients receiving Ofatumumab, but not in patients receiving Obinutuzumab.Background: CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL). Methods: Median fluorescence intensities (MFIs) of standard fluorescence beads from the daily calibration of flow cytometers according to EuroFlow protocols were used to establish a normalization approach to study CD20 expression on CLL cells. CD20 MFI was retrospectively assessed prior to and during treatment from flow cytometric measurements of peripheral blood in patients with different depths of molecular response in the four phase-II CLL2-BXX trials (BIG; BAG; BIO; BCG; N = 194) administering either Obinutuzumab or Ofatumumab in combination with targeted agents. Results: No significant difference was observed between the normalized and measured MFIs of CD19 and CD20 on CLL cells. During treatment, CD20 expression levels on CLL cells did not significantly differ between the four investigated different treatment schemes, but a strong molecular response to Ofatumumab seemed to correlate with higher CD20 expression prior to therapy. Conclusions: Standardized staining and instrument monitoring enable a robust assessment of longitudinal biological variations of marker expression based on MFI values. Obinutuzumab showed a higher proportion of patients with a strong MRD response independent from initial CD20 expression, whereas high pre-therapeutic CD20 expression levels seem to correlate with a profound response to Ofatumumab.

Molecular Determinants Underlying the Anti-Cancer Efficacy of CD38 Monoclonal Antibodies in Hematological Malignancies

CD38 was first discovered as a T-cell antigen and has since been found ubiquitously expressed in various hematopoietic cells, including plasma cells, NK cells, B cells, and granulocytes. More importantly, CD38 expression levels on malignant hematopoietic cells are significantly higher than counterpart healthy cells, thus presenting itself as a promising therapeutic target. In fact, for many aggressive hematological cancers, including CLL, DLBCL, T-ALL, and NKTL, CD38 expression is significantly associated with poorer prognosis and a hyperproliferative or metastatic phenotype. Studies have shown that, beyond being a biomarker, CD38 functionally mediates dysregulated survival, adhesion, and migration signaling pathways, as well as promotes an immunosuppressive microenvironment conducive for tumors to thrive. Thus, targeting CD38 is a rational approach to overcoming these malignancies. However, clinical trials have surprisingly shown that daratumumab monotherapy has not been very effective in these other blood malignancies. Furthermore, extensive use of daratumumab in MM is giving rise to a subset of patients now refractory to daratumumab treatment. Thus, it is important to consider factors modulating the determinants of response to CD38 targeting across different blood malignancies, encompassing both the transcriptional and post-transcriptional levels so that we can diversify the strategy to enhance daratumumab therapeutic efficacy, which can ultimately improve patient outcomes.

Response to Brentuximab Vedotin by CD30 Expression in Non-Hodgkin Lymphoma.

BackgroundThe safety and efficacy of brentuximab vedotin (BV), an antibody-drug conjugate directed to the CD30 antigen, has been assessed in several trials in patients with peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), or B-cell non-Hodgkin lymphoma (NHL). The objective of this research was to examine the relationship between CD30 expression level and clinical response to BV.Patients and MethodsWe analyzed response in patients treated with BV monotherapy in 5 prospective clinical studies in relapsed or refractory PTCL, CTCL, or B-cell NHL. CD30 expression was assessed by immunohistochemistry (IHC) using the Ber H2 antibody for 275 patients.ResultsAcross all 5 studies, 140 (50.9%) patients had tumors with CD30 expression <10%, including 60 (21.8%) with undetectable CD30 by IHC. No significant differences were observed for any study in overall response rates between patients with CD30 expression ≥10% or <10%. Median duration of response was also similar in the CD30 ≥10% and <10% groups for all studies.ConclusionsIn this analysis of studies across a range of CD30-expressing lymphomas, CD30 expression alone, as measured by standard IHC, does not predict clinical benefit from BV, making the determination of a threshold level of expression uncertain.

Brentuxinmab vedotin, alone or combine with bendamustine in the treatment of natural killer T cell lymphoma.

Natural killer (NK)/T cell lymphoma is a highly aggressive subtype of non-Hodgkin lymphoma. The prognosis of patients with natural killer T cell lymphoma (NKTCL) remains poor. More potent treatment strategies are urgently needed to improve the survival of these patients with R/R NKTCL. CD30 expression has been reported to occur in about 40% of NK/T cell lymphoma. Brentuximab vedotin (BV), a monomethyl auristatin E conjugated CD30 antibody, targets CD30 to kill cancer cells. Therapeutic combination of BV and bendamustine has been shown to be highly effective in Hodgkin lymphoma. We investigated efficacy of BV in treating NKTCL as a single therapy, and in combination with bendamustine in vitro and in vivo. We determined CD30 expression levels in 6 NKTCL cell lines. The efficiency of lymphoma cell inhibition by BV correlates with CD30 expression. We also determined the efficacy of BV in combination with bendamustine and found synergistic effects with bendamustine in NKTCL. Combined BV and bendamustine treatment exerted synergistic antiproliferation effect and enhanced cell apoptotic in vitro and in vivo. Brentuximab vedotin and bendamustine synergistically arrested cell cycle at the G2/M phase in NKTCL cell lines. The combination of BV and bendamustine was demonstrated to synergistically damage DNA in NKTCL. This study provides a reference for possible application on using BV for the treatment of NKTCL, either as a single agent or in combination with bendamustine.