Antigen Loss Research Articles

Pro-Active 4SCAR2.0 Therapy with NGS-MRD Disease Monitoring in High-Risk B Cell Leukemia Management

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HCT) is a standard treatment for high-risk relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, about 30-40% of patients still relapse after HCT. Chimeric antigen receptor-modified T cell (CAR-T) therapy holds great promise as a salvage approach. Although CD19-targeted CAR-Ts have demonstrated a rapid and significant anti-tumor effect in patients with B-cell malignancies, many still suffer disease relapse due to the decrease or loss of CD19 antigen. Therefore, novel curative approaches are needed. PURPOSE: The aim of this study is to determine the best timing for applying CAR-T therapy in high-risk B-ALL patients based on high-sensitivity analysis of tumor cells by next-generation sequencing (NGS) detecting minimal residual disease (MRD). An innovative CAR-T therapy concept, CAR2.0, has been proposed, which uses multi-antigen-targeted CAR-Ts to prevent antigen escape and prolong remission. Based on NGS-MRD detection, patients with potential tumor relapse can be identified early and managed accordingly. On the other hand, if extended negative NGS-MRD is found, the patient is deemed at low risk and no further treatment is needed. The study proposes to treat high-risk B-ALL patients in the first remission period with CAR2.0 followed by continued disease monitoring by NGS-MRD to avoid HCT. METHODS: B-ALL patients who have high tumor burden and high-risk genetic indication is enrolled with the understanding that CAR-T therapy will be applied in the first remission and disease will be continuously monitored by NGS-MRD. The tumor cells from initial diagnosis will be analyzed for target antigens by immunostaining and to detect clonally rearranged immunoglobulin gene sequences by NGS-MRD with sensitivity ≤10-6. Patients will receive 4SCAR19 (CD19scFv-CD28/CD27/CD3ζ-iCasp9) therapy in the first remission and will be followed up for CAR-Ts, B cells and IgG/IgA/IgM in the blood, and bi-monthly NGS-MRD for residual tumor cells in the bone marrow (BM). A booster CAR-T treatment targeting additional tumor antigen such as CD22 will be applied 2 months later. A consolidation CAR-T treatment based on known target antigens will be applied 6 months after the first CAR-T infusion. The cyclophosphamide/fludarabine chemo-conditioning will be included prior to the first CAR-T treatment only. The CAR-T infusion dose is 1-5x106 CAR-Ts/kg. The quality of apheresis cells, efficiency of gene transfer, T cell proliferation, CAR-T infusion dose and blood CAR copies are quantitatively documented. The end point for this study is the establishment of NGS-MRD negativity for 1 year, with continued follow-up for 5 years. RESULTS: A 20 years old female was diagnosed with BCR/ABL1 P190 positive B-ALL, with 82.52% tumor blasts in the BM. NGS identified two high-frequency IgH clones at 66.29% and 30.41%. The patient received induction chemotherapy (VTCP, CAM, VP+MTX) combined with TKI therapy (Nilotinib) and achieved complete remission (CR) after the first round of treatment based on both flow cytometry and NGS MRD analyses. During the fourth round of chemotherapy (Methotrexate-Cytarabine-Dexamethasone), the patient suffered severe oral and intestinal mucositis, along with kidney and liver toxicity and required week-long dialysis. A decision was made to stop chemotherapy and start the first 4SCAR19 therapy of 3.53x106 CAR-Ts/kg, while maintaining the TKI therapy. The CAR-T cells in blood, the B cell count and Ig levels were monitored, and a booster CD22/CD19 dual CAR-Ts (4SCAR2219) was given at a dose of 3.35 x106 CAR-Ts/kg on day 96 (see figure). Consistent with 4SCART safety profile, the patient experienced no greater than grade 1 CRS. NGS-MRD detection of tumor cell clones remained negative on day 96 after the first CAR-T infusion and at 6 months into the remission. In summary, the 4SCAR2.0 therapy regimen appeared safe and effective in the innovative NGS-MRD/4SCAR2.0 B-ALL study regimen with in vivo persistency for more than three months. The simultaneous expansion of CD19 mono-specific and CD22/CD19 bi-specific CAR-Ts supports a stable maintenance treatment with good life quality as compared with conventional chemotherapy. CONCLUSIONS: The combination of 4SCAR2.0 with NGS-MRD detection of tumor clones in the management of high-risk B-ALL appears to be safe and may be a logical option for patients not elected for HCT therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Impaired Death Receptor Signaling Mediates Cross-Resistance to Immunotherapy in MM

The treatment landscape in various cancers is currently shifting from genotoxic drugs to immunotherapy, such as monoclonal antibodies, immunoconjugates, T-cell engaging antibodies (TCE) or CAR T-cells (CARTs). This is in particular true for multiple myeloma (MM), in which unprecedented treatment efficiencies are induced by these novel therapies; however, patients continue to relapse and a subset even faces primary resistance, with the underlying mechanisms poorly understood. The role of the death receptor signaling proteins FADD and BID in MM resistance towards immunotherapy has not yet been explored. Therefore, we first generated FADD and BID CRISPR-Cas9 knock out (KO) models in two independent MM cell lines, AMO1 and L363. We confirmed the functional impact of the KO genes on the apoptotic activity using apoptosis inducing, CD95 activating antibody (anti-FAS Ab). As hypothesized, no apoptosis was observed in our FADD KO clones following 24 hours treatment with 15µg/ml Fas antibody, compared to a 50% increase of apoptosis in the WT cells via Annexin-V PI staining. Activity of Caspase 3/7 and 8 increased 3-4 folds in the WT cells and no increase was seen in our KO models by using commercially available fluorometric assays. Detection of cleaved PARP in WT and its absence in KO clones via Western blotting further validated the inactivation of the apoptotic pathways in our KO models. Next, we treated our KO cells with 0,1µg/ml Daratumumab (Dara) along with 10:1 ratio of PBMCs from healthy donors and 5:1 and 10:1 effector to target ratio of BCMA-CARTs: Of note, killing efficacy by Daratumumab was significantly lower in our KO models compared to the WT cells (22% vs 47% cell death) after 24 hours of treatment. Similarly, BCMA-CARTs efficiency was dramatically reduced with 58% survival in our KO models compared to around 10% in WT cells. Strikingly, we did not observe such a difference in IC50 between the KO and the WT cells when we treated for 3-5 days with Bortezomib, Lenalidomide, Doxorubicin, Melphalan, or the approved ADC Belantamab mafodotin. To determine the functional profile of the CART cells we performed a commercially available cytokine quantification assay (Human IL-2, Human IFN- γ) in response to 20 hours of antigen stimulation with WT and KO cells and no difference in IL-2 and IFN-γ production was observed. In addition, we excluded CD38 or BCMA antigen loss in our KO model by Direct Stochastic Optical Reconstruction Microscopy (dSTORM) and confirmed no significant differences in receptor density /µm2 or antigen surface distribution between the WT and the KO cells. To determine the clinical relevance of our findings we screened for decreased FADD and BID expression levels in a pilot cohort of 24 newly diagnosed (NDMM) and 54 relapsed refractory MM (RRMM) patients and normalized expression levels with 26 CD138+ bone marrow plasma cell samples from healthy donors. We did not find significant expression differences between the cohorts (that did not include patients post TCE or CARTs). However, when we ranked the patients according to their gene expression of FADD and BID the ten patients with the lowest values were predominantly Daratumumab resistant (7/10 and 9/10, respectively), whereas in the patients with highest FADD BID expression Daratumumab resistance was less pronounced (3/10 and 4/10 respectively). In conclusion, our study highlights induction of apoptosis as the main mode of action of T-cell based immunotherapies in MM. Furthermore, we provide first evidence, that gene expression of FADD and BID is essential for the action of Daratumumab and BCMA CARTs but may be dispensable for the anti-tumor effects in MM of ADC, IMiD, PI or chemotherapeutic agents. In our in vivo screen, we identified patients with low expression of FADD and BID being resistant to Daratumumab, suggesting a potential resistance mechanism.

Be Cautious to Adopt a Second CAR T-Cell Infusion after Failure of CD19/CD22 Cocktail CAR T-Cell Therapy in Relapsed/Refractory B-NHL

Objective: Chimeric antigen receptor (CAR) T-cell infusion (CTI) has emerged as a breakthrough therapy for relapsed/refractory B-cell non-Hodgkin's lymphoma (R/R B-NHL) patients. Nevertheless, there are still a significant number of patients suffering treatment failure (failed to respond or eventually relapsed after a partial or complete response, PR/CR) post the first time CTI (CTI1). The issue how to salvage this group of patients should be urgently addressed. Our study aimed to provide clues for exploring the potentially effective salvage strategies and finally improving the prognosis of them by analyzing the efficacy and safety especially between the second time CTI (CTI2) and other different therapies (non-CTI2). Methods: Herein,a retrospective analysis was performed on total 61 patients after failure of CD19/22 cocktail CTI1 (ChiCTR-OPN-16008526) between January 2017 and May 2022 in our hospital. Among them, 27 of 61 patients with a prior determination of no antigen loss received CTI2 for compassionate use. The other 34 patients were administrated as non-CTI2 therapies. The clinical characteristics, efficacy, safety, survival analysis and outcome-related risk factors were all evaluated. Results: The CTI2 group achieved a higher overall response rate (ORR) at 30 days assessment (8/27 vs. 2/34, P=0.017). Nevertheless, the non-CTI2 group showed a better event-free survival (EFS) (P=0.007) (Figure1, left panel) and overall survival (OS) (P=0.048) (Figure1, middle panel) than the CTI2 group with a median follow-up of 6.7 months (range, 3-45.5) and 4.7 months (range, 1.1-53). Further analysis on copies of CAR transgene in peripheral blood at 90 days detection revealed that the better CAR T cells expansion was related to a better response in CTI2 group (P=0.012)(data not shown). Additionally, neither of the CTI2 and non-CTI2 was the independent risk factor of survival (HR: 1.665, 95%CI: 0.883-3.141, P=0.115). Either of Ann Arbor stage (HR: 1.970,9 5%CI: 1.944-3.997, P=0.029) or bulky disease (HR: 2.879, 95%CI: 1.321-6.276, P=0.008) at disease onset was the related independent risk factor instead. As shown in Figure1 right panel, the best responses to different salvage therapies suggested that the regimen (Polatuzumab vedotin,-pola, Bendamustine, Rituximab, pola+BR) was likely to be a potentially optimal non-CTI2 salvage therapy, even without statistically significant differences due to the limitation of the sample size. The adverse effects of CTI2 were generally similar to those of non-CTI2. Remarkably the infection-related mortality rate was significantly higher (59% vs. 21%, P=0.025) in CTI2 group. Conclusion: Our study provides novel insights and clues that clinicians should be cautious to adopt a CTI2 after failure of the CD19/22 cocktail CTI1 in R/R B-NHL. It is noteworthy that the choice of pola+BR regimen probably has an advantage in salvage strategies. Large-scale prospective studies are warranted in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antibody-Mediated Antigen Loss Can Switch an Augmented Immune Response to Antibody-Mediated Immunosuppression

Antibody-Mediated Antigen Loss Can Switch an Augmented Immune Response to Antibody-Mediated Immunosuppression

A Phase 1 Study of ADI-001: Anti-CD20 CAR-Engineered Allogeneic Gamma Delta1 (γδ) T Cells in Adults with B-Cell Malignancies

Introduction: ADI-001 is a first-in-class allogeneic gamma delta (γδ) CAR T cell therapy targeting the B-cell antigen CD20. ADI-001 has both adaptive and innate cytotoxic effector functions to complement CAR targeting, potentially enhancing efficacy and reducing the possibility of tumor escape due to antigen loss. ADI-001 expresses MHC-independent γδ T cell receptors, thus lowering the risk of graft-versus-host disease (GvHD) without the need for gene editing. Methods: This multicenter phase 1 clinical trial is evaluating ADI-001 in adults with relapsed / refractory B-cell lymphoma. Eligibility criteria included the presence of measurable lesions, expression of CD20 on tumor cells and ≥ 2 prior systemic therapies. All patients received conditioning therapy with fludarabine and cyclophosphamide. ADI-001 can be administered at four dose levels (DL) (DL1:3E7, DL2:1E8, DL3:3E8 and DL4:1E9 CAR+ cells) in a 3+3 dose-escalation scheme. Patients who completed the 28-day DLT period were considered evaluable. In DL3, patients could receive a second course of conditioning therapy and be re-dosed with ADI-001 if there was no DLT during the first 28 days, no progressive disease on PET/CT assessment on Day 28, and have recovered from cytopenias. Treatment-emergent adverse events were graded by CTCAE v5.0, and Immune Effector Cell Associated Neurologic Syndrome (ICANS) and Cytokine Release Syndrome (CRS) assessments were performed per ASTCT criteria. Objective response rates (ORR) were evaluated by independent radiographic review per Lugano 2014 criteria. Results: As of 15 July 2022, 11 patients were enrolled and nine were evaluable. Of these nine patients, six (67%) were male and the median age was 62 years (range 45-75). Eight patients had large B-cell lymphoma (LBCL) and one had mantle cell lymphoma (MCL). Of the eight patients with LBCL, five had diffuse-large B-cell lymphoma (DLBCL), two had high-grade B-cell lymphoma (HGBCL) with double/triple hit, and one had HGBCL not otherwise specified. At baseline, the median IPI score was four (range 2-4); the median tumor burden was 2,974 (150-7,919) mm2, and 89% (8/9) had stage III/IV disease. The median number of prior therapies was four (range 2-5). Four patients had prior anti-CD19 CAR T cell therapy (two Liso-cel and two Axi-cel). Among nine evaluable patients, three patients were treated at each of DL1, DL2, and DL3. Two patients at DL3 were re-dosed with a second course of ADI-001. Two patients developed CRS: one Grade 1 and one Grade 2. One patient developed a Grade 1 ICANS which resolved within 24 hours. There were no ≥ Grade 3 CRS or ICANS. The only related SAEs were Grade 2 CRS, Grade 1 ICANS and Grade 3 adenoviraemia. There was no reported GvHD or protocol-defined DLT events. The best ORR was 78% (7/9), and the complete response (CR) rate was 78% (7/9). For the four patients who had prior CD19 CAR T therapies, the ORR was 100% (4/4) and CR rate was also 100%. As of the data cut-off date, of the seven patients who had achieved CR, two patients progressed, one died while in complete remission and four were still in CR and in active follow-up, with a range of follow-up time between 1.2 and 8.8 months. CAR+ γδ T cell kinetics improved in a dose-dependent manner with peak cell expansion occurring between Days 7 and 10 at DL3 based on flow cytometry. Conclusions: ADI-001 γδ CAR T cells maintained a favorable safety profile. Preliminary efficacy showed encouraging CR rate and sustained durability in patients, including those previously exposed to CAR T therapy. Additional data will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rapid Tumor Debulking of Relapsed/Refractory CLL Patients By PI3Kδ Inhibition and Anti-CD20 Monoclonal Antibody Treatment

Introduction Effective multi-targeted drug combinations could rapidly achieve deep remissions in CLL allowing for defined duration treatment. The combination of a novel highly-specific phosphoinositide 3-kinase delta (PI3Kδ) inhibitor and casein kinase 1 epsilon (CSK1ε) inhibitor, umbralisib (UMB), and a glycoengineered chimeric monoclonal antibody (mAb) targeting a unique epitope on CD20, ublituximab (UBL), with the BCL2 inhibitor venetoclax (VEN) may decrease drug resistance, reduce risk of tumor lysis syndrome (TLS) and achieve higher levels of undetectable minimal residual disease (MRD). This triple agent therapy is being evaluated in relapsed or refractory CLL patients (U2-VEN, ClinicalTrials.gov NCT03379051). Patients received oral 800 mg UMB once-daily. For Cycle 1, UBL was given via IV on Day 1 (150 mg), Day 2 (750 mg), Day 8 (900 mg), Day 15 (900 mg), and then on Day 1 in Cycles 2 and 3 (900 mg); for 3 cycles of 28 days, followed by consolidation with UMB and VEN for cycles 4 to 24. Patient (n = 25) blood samples were collected Pre- and Post-treatment on days of UBL administration during cycles 1-3. Our studies focused on UMB and UBL treatment effects on: 1) The decrease in the circulating CLL tumor burden and thus the subsequent risk of TLS from the addition of VEN to the regimen; and 2) CD20 antigen loss on CLL cells, a major mechanism of anti-CD20 mAb resistance. Methods Blood was analyzed by clinical laboratory automated cell counter and then stained with antibodies against CD3, CD5, CD19, and CD20 for flow cytometry analysis. CLL cell counts were calculated from flow cytometry and clinical laboratory results. To quantitate CD20 surface molecules, peripheral blood mononuclear cells were isolated and stained for flow cytometry analysis with the addition of a Molecules of Equivalent Soluble Fluorochrome standard curve. All human specimen collection and usage was conducted with written informed consent after approval of our Institutional Research Subjects Review Board according to the ethical guidelines of the Declaration of Helsinki. Results Average CLL cell counts (47 x 109/L) decreased rapidly after administration of the first dose of UMB and UBL (C1D1 Post, 26 x 109/L), which rebounded the next day (C1D2 Pre, 43 x 109/L) and then decreased after the second treatment (C1D2 Post, 24 x 109/L). By Day 15, the average CLL cell count had dropped to 9 x 109/L, which decreased further to 5 x 109/L by the start of cycle 3 (Fig. 1). Notably, the variation in CLL cell counts (error bars) decreases over treatment time, suggesting that most CLL patients' tumor burdens are decreasing to a similar low level independent of starting CLL cell count. The change in average CLL cell count between Pre and Post measurements is initially very high (45% decrease C1D1) with decreasing effectiveness over treatment time (8% decrease C3D1). This change could be due to the loss of CLL cell membrane CD20 levels leading to mAb resistance. Pre-treatment (C1D1 Pre) average CLL cell membrane CD20 levels (n=18) were 29,000 CD20 molecules/cell. This decreased to 9,000 molecules/cell after the first treatment (C1D1 Post), stabilized at 9,100 molecules/cell on the next day (C1D2 Pre) and then decreased further after the second treatment (C1D2 Post) to 1600 molecules/cell. After day 8 treatment, CD20 levels remained at 1,000 molecules/cell or less until the end of this study (C3D1). Discussion UMB and UBL rapidly decrease circulating CLL counts over three cycles of treatment, reducing most CLL patient tumor burden to similar low levels. This clinical response could facilitate addition of VEN in the 4thcycle of therapy by decreasing the risk of TLS. The rapid loss of surface CD20 from CLL cells to 1,000 CD20 molecules/cell or less by C1D8 Post has the potential to cause acquired resistance to anti-CD20 mAb therapy. This rapid and sustained loss of CD20 levels is consistent with that reported at standard doses for other anti-CD20 mAbs. However, an effect of concomitant UMB on CD20 levels cannot be ruled out. Previous work suggests that lower anti-CD20 mAb doses can prevent these large decreases in CD20 levels and increase UBL efficacy. In conclusion, our initial data show that treatment with a selective PI3Kδ and CSK1ε inhibitor (UMB) combined with an anti-CD20 mAb (UBL) is effective in decreasing CLL cell counts in all patients assessed in the U2-VEN clinical trial. However, treatment efficacy could be limited by large decreases in the CLL surface CD20 levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HDAC Inhibition Involves CD26 Induction on Multiple Myeloma Cells Via the c-Myc/Sp1-Mediated Prompter Activation and Overcomes Therapeutic Resistance By Humanized Antibody

Background: CD26, a 110-kDa transmembrane glycoprotein, which is expressed on several tumor cells including malignant lymphoma has been implicated in tumorigenesis, whereas, little is known about its roles in plasma cell malignancies. Recently, we have identified that CD26 is uniformly and intensely expressed in osteoclasts, while its expression in plasma cells of patients with multiple myeloma (MM) reveals heterogenous due to its cellular diversity or immune escape, leading to a marked heterogeneity of response to humanized anti-CD26 monoclonal antibody (mAb) therapy in MM. Decreased expression levels of CD26 in MM cells are one of the mechanisms underlying innate and acquired resistance to CD26mAb therapy in MM. Aim: Here, we clarify the potential impact of epigenetic modification by HDAC inhibition (HDACi) with isoform selective- or broad- inhibitors on the regulation of CD26 expression in MM cells and elucidate its mechanisms, thereby affecting the performance of CD26mAb in refractory MM. Methods and Results: Immunostaining of bone marrow tissues of MM showed that CD26pos/CD138pos plasma cells were detected in several patients but not in others. First, we investigated the impact of HDACi on CD26 expression of MM cells. Although the cell surface expression of CD26 was relatively low or not detected on 5 MM cell lines (KMS26, KMS27, KMS28, KMS11, RPMI8226), cultured alone, the increased expression in CD26 levels of MM cells was detectable within 24 hr of the treatment with HDAC1i; FK228, HDAC3i; BG45, MS-275, RG2833 or HDAC6i; nexturastat A, tubastatin A, ACY-1215 as well as broad HDACi; LBH-589, SAHA by flow cytometry. Its expression increased further during the exposure to each HDACi and its maximum increase was observed at 72 hr of the treatment. Then, subsequent removal of HDACi for 48 hrs resulted in a decline of CD26 expression on MM cells to reduced or baseline levels. In addition, the levels of CD26mRNA were concomitantly augmented, which was paralleled with an increase in the induction of CD26 protein and its DPPIV enzymatic activity in MM cells. We also examined the effect of each HDACi on the viability of CD26neg MM cells in the absence or presence of CD26mAb.CD26mAb alone did not induce lysis of CD26neg MM cell lines at any doses, whereas, combining each HDACi plus CD26mAb at 10 μg/ml synergistically facilitated lysis of CD26neg MM cells via direct effects as well as NK cell- mediated ADCC by CD26mAb. Of note, HDACi plus CD26mAb in combination readily augmented lysis of CD26neg cell populations, refractory to HDACi or CD26mAb alone. Next, gene expression profiles in KMS11, KMS27 and RPMI8226, left untreated or treated with LBH589 or RG2833 revealed that Myc is one of the overlapped genes, altered in its expression. The 5'-flanking region of human CD26 gene, representing the promoter activity contains potential binding sites of several transcriptional factors including Sp1 and c-Myc. So, further to elucidate the mechanisms by which CD26 expression is induced in MM cells by HDACi, we examined the epigenetic status in histone and non-histone at the CD26 promoter of MM cells with the treatment of HDACi. First, ChIP-qPCR demonstrated that the acetylation in histone 3 lysine 27 is increased at the CD26 promoter of 5 MM cell lines after exposure to each HDACi, suggesting parts of mechanisms in the CD26 promoter activation. Moreover, the expression level of c-Myc was time-dependently decreased in KMS11, KMS27 and RPMI8226 with the treatment of LBH-589 or RG2833 both at mRNA and protein levels and was significantly down-regulated after 12 to 24 hr of exposure. On the other hand, the acetylation of c-Myc on K323 was significantly increased after 12 to 24 hr of exposure to each HDACi. ChIP-qPCR revealed that the recovery % IP/INPUT showing the binding of c-Myc to the CD26 promoter was exposure time-dependently reduced in KMS11, KMS27 and RPMI8226 by the exposure to LBH589 or RG2833. Namely, in the absence of HDACi, c-Myc was present on the CD26 promoter via binding to Sp1 on the proximal G-C box and repressed CD26 promoter in MM cells, whereas, in the presence of HDACi, c-Myc was highly detached from Sp1 on the CD26 promoter after 12 to 24 hr with its increased acetylation, leading to activate CD26 promoter and initiate CD26 transcription in MM cells. Conclusion: HDACi not only shows anti-MM activity itself but sensitizes MM cells with CD26 antigen loss to CD26mAb via c-Myc/Sp1-mediated CD26 induction and augments its cytotoxicity.

CD22-Targeted CAR-Modified T-Cells Safely Induce Remissions in Children and Young Adults with Relapsed, CD19-Negative B-ALL after Treatment with CD19-Targeted CAR T-Cells

Introduction: Children and young adults with B-lymphoblastic leukemia (B-ALL) who relapse with CD19 antigen loss after CD19-targeted CAR T-cell therapy have a dismal prognosis, with a median overall survival of 9.7 months. Novel immunotherapies are urgently needed for this population. CD22 can serve as a potential alternative target. In our initial pilot trial of CD22-targeted/4-1BB CAR T cells, clinical activity was unexpectedly poor. Based on additional preclinical work demonstrating that shorter scFv linkers improve T-cell effector function, a novel CD22/4-1BB CAR construct with a short scFv linker (CART22-65s) was developed and investigated in a phase 1 study (NCT02650414). Methods: After fludarabine/cyclophosphamide lymphodepletion, patients (pts) were infused with CART22-65s using a 3-day fractionated dosing scheme with the 2nd/3rd doses held for early signs of cytokine release syndrome (CRS). The primary endpoint was safety. Key secondary endpoints were feasibility, efficacy, and CAR T cell expansion and persistence. Results: Nineteen pts were enrolled and had a product successfully manufactured. Of 17 pts infused (median age, 16 years; range 3-28 years), all had CD19-negative, relapsed disease after prior CD19-directed therapy (CART19, n=16; blinatumomab, n=1); 8 had prior inotuzumab; 9 had prior stem cell transplant (SCT); and 3 had multiple prior SCTs. Median bone marrow blasts immediately pre-infusion was 31% (range, 0-99%); 8 patients had >75% blasts. The median cell dose infused was 4x106 CART22-65s cells/kg (range, 0.8 to 10 x 106). In vivo cellular kinetics by qPCR demonstrated robust initial CAR T cell proliferation peaking at a median of 20 days post-infusion, and persistence that correlated with clinical response (Figure A). At day 28, 13/17 (77%) pts achieved a complete remission (CR) of which 10 (77%) had undetectable minimal residual disease (uMRD) by flow cytometry. One additional pt cleared MRD at month 2 without intervention. Notably, 4/5 (80%) pts refractory to prior inotuzumab achieved a CR with CART22-65s. Of 11 pts with a best response of uMRD CR, 5 proceeded to consolidative SCT, including 3 with prior SCT: 2 remain in CR post-SCT, 2 are in CR after a brief course of alternative therapy for emergence of MRD, and 1 had a CD22+ relapse post-SCT. Of 6 pts with uMRD CR who did not proceed to SCT (5 had a prior SCT), 5 experienced relapsed disease with a range of CD22 expression and 1 remains in CR 30 months after infusion with ongoing CAR T persistence. Of 2 pts with MRD+ CR (1 CD22+, 1 CD22-), both progressed to morphologic relapse; one received alternative therapy prior to progression. Of 4 pts with non-response, all died of disease within 9 months. Thus, with a median follow-up of 29 months, median relapse-free, event-free, and overall survival were 5.3 (95% CI, 1.9-NR), 5.8 (95% CI, 2.1-NR), and 16.5 (95% CI, 9.3-NR) months, respectively (Figure B). In addition, 2 patients received CART22-65s retreatment for CD22+ relapsed disease after initial infusion. Both engrafted, but only 1 achieved uMRD CR. CRS was observed in 15 pts (grade 1, n=10; grade 2, n=5), and mild neurotoxicity in 6 pts (grade 1, n=5; grade 2, n=1). In the retreatment setting, 1 pt developed grade 3 CRS, which resolved, and grade 3 encephalopathy/hypotonia, but died of progressive disease prior to resolution. Autopsy findings were consistent with leukomyelopathy of the spinal cord, which was inconclusive in its relation to CART22-65s. Other SAEs included: (1) platelet refractoriness and recurrent, severe inflammatory reactions to platelet transfusions in 1 pt that improved with corticosteroids and anakinra; and (2) delayed hemophagocytic lymphohistiocytosis in 1 pt after CART22-65s retreatment that resolved without intervention. Conclusion: CART22-65s induced remissions in 77% of children and young adults with highly refractory, CD19-negative B-ALL, including in 4/5 pts refractory to prior CD22-directed therapy. Manufacturing was successful, and the overall toxicity profile was favorable even in pts with high disease burden. Several uncommon toxicities were observed, but the only grade 3 CRS and neurotoxicity events occurred in the retreatment setting. Our observation of high initial response rates, but later recurrences, highlights the need to use CART22-65s as part of combination therapy. These promising preliminary data provide support for a combined CD19/22 approach using the CART22-65s construct. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-23. BICISTRONIC CAR-T CELLS TARGETING EGFR AND IL13RΑ2 MITIGATE ANTIGENIC HETEROGENEITY IN GLIOMA

Abstract Chimeric antigen receptor (CAR)-T cells have had a significant effect in hematological diseases, with a recent study confirming that the cells can last for ten years in vivo. Unfortunately, to date, CAR-T cells have had little effect in solid tumors. One cause of the treatment difficulty is antigenic heterogeneity. Our experience with CAR-T cells in glioblastoma suggests that addressing antigenic heterogeneity may improve the clinical efficacy. Targeting the epidermal growth factor receptor (EGFR) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) individually could lead to tumor control in the short term, but it will be marked by tumor recurrence through antigen loss or downregulation in the long term. This resistance mechanism can be inhibited by combinatorial targeting. To this end, we have designed a bicistronic construct that targets both EGFR and IL13Rα2 simultaneously, to decrease the potential of antigen escape and tumor recurrence. The bicistronic CAR-T cells employ an “OR” logic gate to modify the T cells and kill the tumor cells in vitro and in vivo, in order to retain effectiveness when one target antigen is lost but the second is still present. Our experimental results demonstrate that and validate the safety and possibility of clinical translation. In addition, the efficiency of transfecting T cells with the single bicistronic vector was higher than two single vectors on monovalent CARs. In summary, the bicistronic CAR-T structure is a promising strategy to address the clinical problem of targeting antigenic heterogeneity in solid tumors and potentially level the barriers for targeted T-cell therapy. Educational Objectives: After completion, participants will be able to explain the importance of antigenic heterogeneity in solid tumors in the context of tumor escape. Participants will also be able to identify a strategy to reducing the problem of antigen escape and tumor recurrence through the implementation of bicistronic CARs.

State-of-the-art and emerging antivirals for chronic hepatitis B infection

ABSTRACT Introduction Current treatment options for chronic hepatitis B virus (HBV) infection cannot achieve functional cure [hepatitis B surface antigen (HBsAg) loss]; therefore, new approaches are under investigation. This review summarizes the most promising approaches in emerging antivirals against HBV, after search in Medline (2016–2022) and European and American liver meetings (2019–2022). Areas covered Classes of antivirals include entry inhibitors (bulevirtide), capsid assembly modulators (CAMs), translation inhibitors [small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs)], and HBsAg secretion inhibitors [nucleic acid polymers (NAPs)]. Bulevirtide has good efficacy in hepatitis B and D coinfection, but there is limited data in HBV monoinfection. CAMs profoundly reduce serum HBV DNA/RNA levels, but have minimal effects on antigen levels. siRNAs and ASOs mostly reduce HBsAg levels, but small proportions of patients reach HBsAg seroclearance. NAPs reduce serum HBV DNA and especially HBsAg levels offering substantial HBsAg seroconversion rates, but having limited data over a long period. Combinations of agents of different classes are starting to be evaluated. Expert opinion Continued efforts are required in order to address many unanswered questions about the optimal combined regimens of finite duration which will be safe and well tolerated achieving functional cure in a substantial proportion of chronic HBV patients.

Abstract 14503: Cardiac Arrest Secondary to Inferior ST-Segment Elevation Myocardial Infarction in Patient With Paroxysmal Nocturnal Hemoglobinuria and COVID 19 Infection

Background: Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) have attacks of complement-mediated hemolysis that can be spontaneous or secondary to precipitating factors such as infections. During these episodes, these patients have an increased risk of thrombosis, typically venous and rarely arterial. We present a rare case of cardiac arrest due to ventricular fibrillation (VF) and inferior STEMI in a patient with PNH and a recent COVID infection. Case: 63 years old male patient with a medical history of PNH presented with typical chest pain, fever, cough, jaundice, and dark-colored urine. On examination, he was hemodynamically stable with jaundiced eyes. A few minutes later, he had cardiac arrest due to VF. He achieved return of spontaneous circulation (ROSC) after two defibrillation shocks. EKG showed inferior STEMI. Labs showed hemoglobin of 6.4 g/dl, elevated cardiac markers, serum LDH, and indirect bilirubin. Serum haptoglobin was < 1 mg/dl. His COVID 19 PCR test was positive. Decision making: He received 2 units of packed RBCs and underwent a coronary angiogram (CA) which revealed total proximal occlusion of the right coronary artery (Figure A). He underwent successful PCI by two drug-eluting stents (figure B). His peripheral blood immunophenotyping and flow cytometry was positive for loss of glycosylphosphatidylinositol-linked antigens and decreased expression of CD 59/14/24. He was started on Ravulizumab, a humanized monoclonal antibody complement five inhibitor. Conclusion: PNH and COVID increase the risk of thrombosis. Thrombosis mechanism in PNH includes platelets and coagulation system activation via complement cascade and impairment of the fibrinolytic system. COVID infection increases the risk of thrombosis by endothelial cell dysfunction and release of cytokines. Due to the patient’s acute presentation, he needed to undergo immediate CA and lifesaving PCI regardless of his hematological disorder or active COVID-19 infection.

Two new Scianna variants causing loss of high prevalence antigens: ERMAPmodel and 3D analysis of the antigens.

Scianna (Sc) antigens, seven high and two of low prevalence, are expressed on erythrocyte membrane-associated protein (ERMAP). We investigated SC (ERMAP) in individuals who made antibodies to high prevalence Scianna antigens, and propose a 3D model for ERMAP to precisely localize the residues associated with the known antigens. Serological testing and DNA sequencing was performed by standard methods. A 3D structural model was built using a multi-template homology approach. Protein structures representing missense variants associated with the loss or gain of an antigen were generated. Residue accessibility and intraprotein interactions were compared with the wild-type protein. Two new SC alleles, one with c.349C > T (p.Arg117Cys) in a woman from South India with anti-Sc3 in her plasma, and a c.217_219delinsTGT (p.Arg73Cys) in an African-American woman with an antibody to a new high prevalence antigen, termed SCAC, were identified. Six structural templates were used to model ERMAP. 3D analysis showed that residues key for Scianna antigen expression were all exposed at the surface of the extracellular domain. The p.Arg117Cys change was predicted to abolish interactions between residues 93 and 117, with no compensating interactions. We confirm the extracellular location of Scianna residues responsible for antigen expression which predicts direct accessibility to antibodies. Loss of intraprotein interactions appear to be responsible for a Sc null and production of anti-Sc3 with p.117Cys, SC*01 N.03, and for loss of a high prevalence antigen with p.73Cys, termed SCAC for Sc Arg to Cys. Comparative modeling aids our understanding of new alleles and Scianna antigen expression.

The Efficacy of Antiviral Treatment for Chronic Hepatitis B Patients with Normal ALT Levels: A Systematic Review and Meta-analysis

Context: When nucleos(t)ide analogues (NAs) were applied clinically to manage chronic hepatitis B virus infection, the prognosis of chronic hepatitis B (CHB) patients greatly improved. However, certain CHB patients with normal alanine aminotransferase (ALT) levels were not used to be considered as the population with the need for antiviral treatment. Objectives: This systematic review and meta-analysis collected and analyzed data from clinical trials to assess and compare the efficacy of antiviral treatment among patients with elevated and normal ALT levels. Methods: A systematic search was performed to gather studies published from 1990.01 to 2022.08 in PubMed and Web of Science databases. The quality of the literature was assessed, and 16 studies were included for further analysis. Basic information on included studies and study populations was collected. A meta-analysis was carried out to evaluate three major outcomes of viral response, hepatitis B envelope antigen (HBeAg) loss, and HBeAg seroconversion after NAs treatment based on data extracted from these studies. Odds ratios (ORs) with 95% confidence intervals (CIs) for all outcomes were calculated using fixed-effects models. Results: In the 16 relevant studies, 5,345 patients met the inclusion criteria, including 3,687 patients receiving NAs treatment. All patients were grouped into one with elevated ALT and another with normal ALT based on whether their pretreatment ALT levels > 1*upper limit of normal (ULN). For patients receiving lamivudine, the viral response showed no significant difference between the groups with elevated and normal ALT levels (pooled log OR: 0.51 [-0.23 - 1.26], P = 0.79); the pooled log OR for HBeAg loss was 1.19 (0.63 - 1.76, P = 0.03) and pooled log OR for HBeAg seroconversion was 2.19 (0.91 - 3.47, P = 0.40). For patients receiving first-line therapy with tenofovir disoproxil fumarate (TDF) and entecavir (ETV), the viral response showed no significant difference between the two groups: Pooled log OR (0.38 [-0.22 - 0.97], P = 0.10). The pooled log OR for HBeAg loss and HBeAg seroconversion was (-0.07 [-0.81 - 0.67], P = 0.68) and (0.40 [-0.84 - 1.63], P = 0.88), respectively. Conclusions: The efficacies of first-line therapy with TDF and ETV treatments were similar in groups with elevated and normal ALT levels for the outcomes of viral response and HBeAg loss. These findings may support further treatment of CHB patients with normal ALT levels.

Dynamic changes of estimated glomerular filtration rate are conversely related to triglyceride in non-overweight patients.

Correlation between Triglyceride (TG) and estimated glomerular filtration rate (eGFR) remains largely unknown in overweight and non-overweight patients. To investigated the dynamic changes of eGFR and lipid profiles during 3-year tenofovir disoproxil fumarate (TDF) treatment in patients with chronic hepatitis B (CHB) and overweight. A total of 202 CHB patients who received TDF treatment at the Third People's Hospital of Changzhou (Changzhou, China) and Nanjing Drum Tower Hospital (Nanjing, China) between January 2016 and May 2018 were retrospectively enrolled. According to the body mass index (BMI) at the initiation of TDF treatment, CHB patients were divided into overweight (BMI ≥ 25 kg/m2) and non-overweight (BMI < 25 kg/m2) groups. Logistic regression was applied for the analysis of risk factors for eGFR < 90 mL/(min·1.73 m2). There is no significant difference in hepatitis B virus DNA (HBV DNA) negativity and hepatitis Be antigen (HBeAg) loss between patients with overweight and non-overweight (both P > 0.05). More patients in non-overweight group achieved alanine aminotransferase normalization compared with those in overweight group (χ 2 = 11.036, P < 0.01). In non-overweight patients, the eGFR significantly declined in the 1st year (P < 0.01), then remained at a relatively lower level. TG significantly declined in the 2nd year (P = 0.02) and increased in the 3rd year. Moreover, TG was negatively correlated with GFR at the four-time points (P = 0.002, 0.030, 0.007, 0.008, respectively). In overweight patients, eGFR and TG remained relatively stable during the 3-year treatment, and eGFR showed no significant relationship with TG. Moreover, multivariate analysis showed that age [P < 0.01, 95%CI (0.97-1.005)] and baseline eGFR [P < 0.01, 95%CI (5.056-33.668)] were independent risk factors for eGFR < 90 mL/(min·1.73 m2) at the 3rd year. Dynamic changes in renal function were conversely related to TG during TDF treatment in patients with CHB and normal BMI, but not with overweight.

Multispecific CAR T Cells Deprive Lymphomas of Escape via Antigen Loss.

Chimeric antigen receptor (CAR) modified T cell therapy has transformed the management of relapsed/refractory B cell malignancies. Despite high overall response rates, relapse post CAR T treatment remains a clinical challenge. Loss of target antigen, specifically CD19, is one well-defined mechanism of disease relapse. The mechanism of CD19 loss and which patients are at higher risk of CD19 loss remain poorly understood. To overcome CD19 loss, CARs targeting multiple antigens are being tested in clinical trials. CD19/20 and CD19/22 bispecific CARs demonstrate cytotoxicity against CD19-negative cells in preclinical studies. These CARs have also shown efficacy, safety, and a relatively low rate of CD19-negative relapse in phase I trials. These small studies suggest that multispecific CAR T cells can deprive lymphomas of escape via antigen loss. However, the selection of an ideal target, the right CAR construct, and whether these multispecific CARs can induce long-term remissions are still under investigation.

Tumor Microenvironment-Activated Hydrogel Platform with Programmed Release Property Evokes a Cascade-Amplified Immune Response against Tumor Growth, Metastasis and Recurrence.

In situ tumor vaccines (ITV) have been recognized as a promising antitumor strategy since they contain the entire tumor-specific antigens, avoiding tumor cells from evading immune surveillance due to antigen loss. However, the therapeutic benefits of ITV are limited by obstacles such as insufficient antigen loading, inadequate immune system activation, and immunosuppressive tumor microenvironments (TME). Herein, a tumor microenvironment-activated hydrogel platform (TED-Gel) with programmed drug release property is constructed for cascaded amplification of the anti-tumor immune response elicited by ITV. Both doxorubicin (Dox) and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG) are released first, in which Dox induces immunogenic tumor cell death causing additional tumor antigen release and leading the dying primary tumor cells into autologous tumor vaccine, and the released CpG promotes antigen presenting cell activation. Subsequently, the decomposed scaffold materials in conjunction with CpG, turn the anti-inflammatory M2-like macrophages into the M1 type, reversing the immunosuppressive TME. With decomposition of the TED-Gel, large amounts of macromolecule anti-PD-L1 antibodies are liberated, reinvigorating the exhausted effector T cells. In vivo studies demonstrate that TED-Gel significantly inhibits the primary, distant and rechallenged tumor growth. Overall, the simple and powerful TED-Gel provides an alternative strategy for the future development of tumor vaccines with broad application.

Interleukin-2 promotes pegylated interferon alpha for hepatitis B surface antigen loss: A retrospective pragmatic clinical study at the Fourth Affiliated Hospital of Zhejiang University Medical College.

Interleukin-2 (IL-2) can be used as an adjuvant therapy when pegylated interferon alpha (Peg-IFN-α) does not effectively promote hepatitis B surface antigen (HBsAg) loss, but the relevant timing, kinetic patterns, and prognostic associations of this intervention are unclear. A total of 115 patients with chronic hepatitis B (CHB) treated at our institution between October 2018 and March 2021 were included in this retrospective analysis. They were divided into two kinetic patterns by using K-medoids cluster analysis. Profile and prognostic associations were statistically analyzed between the two patterns. After baseline standardization, before the intervention, the relative HBsAg level showed a continuously increasing trend, but after the intervention, it showed a continuously decreasing trend. Based on the relative change in the HBsAg level, two kinetic patterns, namely, a fluctuation platform pattern and a stepwise growth pattern, were identified by using K-medoids cluster analysis for all 115 patients before IL-2 intervention. Profile analysis showed that there were statistically significant differences between the two patterns before IL-2 intervention (p < 0.05), but their profiles showed the same trend after 2 weeks of IL-2 intervention. Prognostic association analysis showed that CD8+ T cells, alanine transaminase (ALT), age, natural killer (NK) cells, neutrophils, and course of treatment before IL-2 intervention were the six main indicators affecting the relative decrease in the HBsAg level. For CHB patients who have received continuous Peg-IFN-α treatment, IL-2 intervention should be given as early as possible when the HBsAg level has not decreased for four consecutive weeks or a fluctuation platform pattern is observed. After the intervention, a downward relative change in the HBsAg level can be maintained over 4weeks. CD8+ T cells, ALT, NK cells, and neutrophils are baseline indicators closely related to the prognosis of this intervention.

Some HBeAg-negative chronic hepatitis B patients treated with nucleos(t)ide analogue can achieve HBsAg loss after drug withdrawal: stop-to-cure may be coming

Nucleoside/Nucleotide analogues (NAs) are widely used for the antiviral treatment of chronic hepatitis B (CHB), however, it is difficult to achieve serum hepatitis B surface antigen (HBsAg) loss with NAs therapy. In recent years, several prospective trails have reported that HBsAg loss (functional cure or clinical cure) also occurs in a small number of hepatitis B e antigen (HBeAg) negative CHB patients who discontinued long-term treatment with NAs. Accordingly, the "stop-to-cure" strategy is proposed. Although the mechanism has not been fully elucidated, the known factors related to serum HBsAg loss with NAs withdrawal include HBV genotype, duration of NAs treatment, serum HBsAg and HBV RNA levels at end-of-treatment, and ethnic differences. In the review, we discuss the best time to stop NAs therapy, the potential markers for predicting relapse after cessation of NAs and the possible mechanism of "stop-to-cure" in HBeAg-negative CHB patients, and propose some suggestions on the time of retreatment.

Towards a Functional Cure for Hepatitis B Virus: A 2022 Update on New Antiviral Strategies.

Chronic infection with hepatitis B virus (HBV) represents one of the main causes of the development of cirrhosis and its complications. Treatment with potent third-generation nucleos(t)ide analogues (NUCs) results in >99% HBV DNA undetectability, and prevents fibrosis progression and liver-related complications. However, NUCs are not able to induce the so-called functional cure, which is hepatitis B surface antigen (HBsAg) loss and anti-HBs seroconversion. Consequently, NUC treatment is currently intended as being long-term or lifelong, resulting in the need for clinical monitoring and potentially suffering from compliance issues. Consequently, drug development in HBV has the goal of developing new agents in order to achieve a functional cure for HBV. Currently, the three main strategies include the following: inhibition of viral replication, inhibition of viral antigens, and immune modulation. This review summarizes the most recent updates concerning HBV compounds among these three main classes.

Type D lymphomatoid papulosis with pityriasis lichenoides et varioliformis acuta-like features in a child with parvovirus infection: a controversial diagnosis in the spectrum of lymphoid proliferations: case report and literature review

BackgroundLymphomatoid papulosis (LyP) is a rare condition in pediatrics; LyP histological type D has been reported in only 7 children. The differential diagnosis of LyP in the spectrum of lymphoid proliferation remains controversial.Case presentationA 6-year-old boy presented to Emergency Department with a 3-week history of an erythematous papulo-vesicular itchy eruption over the submandibular regions, trunk and extremities. History, symptoms and laboratory tests were unremarkable. SARS-CoV-2 antigen was negative. The clinical suspicion of pityriasis lichenoides et varioliformis acuta (PLEVA) was posed, and topical steroids were introduced. One week after, he returned with an extensive painful scaly papulo-erythematous rash, with some ulcerated and necrotic lesions, and fever; therefore the child was hospitalized. Biochemical results were within reference limits, except for high level of C-reactive protein, aspartate aminotransferase, alanine transaminase and bilirubin. Due to a persistently high fever, systemic corticosteroid treatment was administered, with a good clinical response and an improvement of the skin lesions. Anti-PVB-19 Immunoglobulin M was detected. Elevated levels of IL-6, IL-10 and IFN-γ were also recorded. Five days post-admission, most of the lesions had cleared, and the child was discharged. Methotrexate was started, with a positive response. At skin biopsy a “PLEVA-like” pattern was apparent, with a dense, wedge shaped lymphoid infiltrate featuring epidermotropism and morphologically comprising pleomorphic and blastic cells. The pattern of infiltration was highlighted by immunohistochemical stains, which prove the process to feature a CD8+/CD30 + phenotype, the latter being intense on larger cells, with antigenic loss. Polymerase chain reaction for T-cell receptor gamma (TCRG) chain clonality assessment documented a monoclonal peak. A diagnosis of LyP type D was favored.ConclusionThe reported case encompasses most of the critical features of two separated entities—PLEVA and LyP—thus providing further support to the concept of them representing declinations within a sole spectrum of disease. Studying the role of infectious agents as trigger potential in lymphoproliferative cutaneous disorders and detecting novel markers of disease, such as cytokines, could have a crucial impact on pathogenic disease mechanisms and perspective therapies.