Quantitative Assessment of the Evolution of Therapeutic Target Antigen Expression Level in Diffuse Large B-Cell Lymphoma in Response to Treatment

Introduction: Approximately one-third of patients (pts.) with diffuse large B-cell lymphoma (DLBCL) develop relapsed or refractory (R/R) disease after frontline rituximab-(R) based chemo immunotherapy. Variability in expression of the B-cell surface antigen CD20, and possibly CD19, is thought to be an important mechanism of treatment failure, but there is vast heterogeneity in the reported incidence, with most reports utilizing Immunohistochemical (IHC) staining which is non-quantitative in nature. New therapeutics including CAR T-cells, antibodies (Ab) (tafasitimab) and Ab-drug conjugates (loncastuximab teserine) target CD19, while several bispecific engager Abs. in development target CD20. Understanding the evolution of target antigen expression in R/R DLBCL can provide information regarding selection and sequencing of therapies. We sought to quantitate the change in surface expression of CD19 and CD20 in R/R DLBCL after R-based chemo immunotherapy relative to pretreatment levels, to provide insight into the relative stability of these targets. Methods: Pts. diagnosed with DLBCL after January 2014, who were R/R to frontline R-based chemo immunotherapy (R-CHOP or R-EPOCH) were retrospectively identified. Pt. and disease related clinical variables at diagnosis (Dx) and first R/R were recorded. Data from IHC were obtained, as were archival flow cytometry assays performed on tumor biopsies at Dx and in the R/R setting. Using FCS Express® software, neoplastic cells were gated, and fluorescence intensity (FI) of CD19 and CD20 expression were reported (using fluorochromes APC-A for CD19 and PECy7 for CD20, respectively). Multivariate linear mixed model was used to compare median and geometric mean FI of CD19 and CD20 between Dx and R/R, while adjusting for other clinical variables. Results: A total of 51 flow cytometry assays (26 Dx and 25 R/R) were analyzed for 33 pts. Median age at Dx was 64 (range, 41-76) yrs., 24 pts. (73%) were male and 29 (88%) had IPI ≥ 2. 11 (33%) pts. had a prior indolent lymphoma. Cell of origin at Dx was GCB in 16 (49%) and non-GCB in 12 (36%) pts., while two had a double-hit rearrangement at diagnosis. Treatment was R-CHOP in 27 cases (82%) and R-EPOCH in 6 (18%). Median time to R/R was 10.4 months. There was a significant reduction in median FI of CD20 from Dx [median: 40,610 (range: 167 - 259,962)] to R/R [median: 11,596 (range: 63 - 79,592)], representing a mean reduction of 63% at R/R relative to Dx (P= 0.01; 95% CI: 20-73%). Similar change was observed in geometric mean FI of CD20, which was reduced 65% at R/R relative to Dx (P< 0.01; 95%CI: 31-82%). In comparison, on IHC, CD20 was reported to be negative at R/R relative to Dx in only one of 16 pts. for whom IHC results were available. Median and geometric mean FI of CD19 at R/R were 38% and 20% lower compared to Dx, respectively, but these differences were not statistically significant (P= 0.08 and 0.39, respectively) (Table). When examining the relative change in FI at R/R in individual cases, compared to the mean FI of all Dx cases, we observed that 21 out 25 R/R cases (84%) had reduction of CD20 whereas only 14/25 (56%) had reduction of CD19. Interestingly, 7/25 (28%) R/R cases had an increase in CD19 expression by >80% (Figure).When adjusting for clinical variables such as age, sex, presence of B symptoms, bone marrow (BM) involvement, PS and prior indolent lymphoma, the change in CD20 median FI from Dx to R/R maintained statistical significance (p=0.01). Reduction in CD20 geometric mean FI from Dx to R/R was significantly associated with age >60 years (p=0.04), BM involvement (p <0.01) and >1 site of extra nodal involvement (p= 0.03) at Dx. Conclusions: Quantitative assessment by flow cytometry revealed a significant decline in expression of CD20 at R/R compared to Dx in the majority of patients with DLBCL treated with R-based chemo immunotherapy. CD19 expression was unchanged in most R/R cases but was found to be dramatically upregulated in a subset of R/R cases. Given the role of CD19 mediated pathways in B-cell NHL and its association with PI3K pro-survival signaling, these data merit further exploration as a potential mechanism of treatment resistance. These findings also highlight the importance for repeat tissue biopsy at the time of suspected R/R DLBCL, with focus on therapeutic target expression, as it may influence treatment decisions or enrollment in clinical trials exploring efficacy of newer agents. Figure 1 Figure 1. Disclosures Caimi: TG Therapeutics: Honoraria; Seattle Genetics: Consultancy; Amgen Therapeutics.: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); Verastem: Consultancy; Genentech: Research Funding; Kite Pharmaceuticals: Consultancy; ADC Theraputics: Consultancy, Research Funding. Hill: Celgene (BMS): Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel Support, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Gentenech: Consultancy, Honoraria, Research Funding; Incyte/Morphysis: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding.

Ikaros Mediates Antigen Escape Following CD19 CAR T Cell Therapy in r/r B-ALL

Despite impressive response to front-line treatment, relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) remains one of the most common causes of death in children with cancer. In pediatric and adult r/r B-ALL, CD19 CAR T cell (CART19) therapy has shown impressive results with over 80% of patients achieving complete remission. However, as an unintended consequence of long-term persistence of CAR T cells and the resulting sustained pressure against CD19 expressing cells, 30 - 70% of initial responders will eventually relapse with CD19 loss. Early identification of patients that will respond successfully to CART19 therapy vs. those at risk for CD19 Neg relapse would support clinical decision making and improve patient care. To address this problem, we profiled by mass cytometry (CyTOF) 25 pre-CART19 samples including patients that achieved a durable complete remission (CR, n = 6), patients that underwent CD19 Neg (n = 11) or CD19 Pos relapse (n = 4) and patients that were refractory to CART19 (n = 4). For 14 of those patients, we also analyzed paired post-CART19 samples: 8 from CD19 Neg relapse, 2 from CD19 Pos relapse, and 4 from refractory tumors. We observed a significant increase in the proportion of leukemic early progenitor non-B cells (called early-non-BI) after CD19 Neg relapse, suggesting that CD19 loss is associated with the loss of other B cell features. In addition, in the pre-CART19 samples we identified a subpopulation of CD19 Pos pro-B cells that resemble early-non-BI cells. We hypothesized that these pro-B "discordant" cells are able to lose CD19 expression and escape the immune pressure exerted by the CART19, leading to CD19 Neg relapse. Closer analysis revealed lower levels of IKAROS in pro-B discordant cells from patients that underwent CD19 Neg relapse, than in those that achieved a durable CR. To further explore the difference between pro-B discordant cells from patients that achieved a durable CR or underwent CD19 Neg relapse, we performed single cell REAP-seq (RNA expression and protein sequencing) from 3 pre-CART19 CR and 3 pre-CART19 CD19 Neg relapse r/r B-ALL samples. From this analysis, we confirmed a subpopulation of leukemic pro-B like cells with decreased expression of IKZF1 in those patients that underwent CD19 Neg relapse. IKAROS is a transcription factor that plays a crucial role in B cell lineage specification and commitment and is a known B-ALL tumor suppressor. Genetic alterations affecting the IKAROS gene, IKZF1, have been associated with poor response to front-line treatment and they are a factor in relapse risk stratification. However, a role for IKAROS in CART19 failure has not been described. To assess if there is a correlation between the levels of IKAROS and CD19 surface expression, we manipulated IKAROS levels in B-ALL cell lines through RNA silencing and targeted protein degradation approaches. Consistently, we observed that CD19 surface expression diminishes upon IKAROS downregulation. To determine how IKAROS regulates CD19 expression, we performed CyTOF, RNA-seq and ATAC-seq analysis of isogenic B-ALL cell lines with wild-type or reduced (IKAROS KD) IKAROS levels. While the chromatin accessibility to CD19 regulatory regions did not change, CD19 mRNA levels were lower in IKAROS KD cells. Interestingly, we found that IKAROS regulates several genes associated with mRNA splicing. In particular, CD19 mRNA preferentially retains intron 10 in IKAROS KD cells. The inclusion of intron 10 introduces several premature stop codons. Moreover, IKAROS KD B-ALL cells more closely resemble myeloid cells based on their gene expression profiles, suggesting a loss in their B cell identity. In summary, this is the first description of a leukemia-specific subpopulation of CD19 Pos pro-B discordant cells in patients prior CART19 administration. Furthermore, lower levels of IKAROS in this subpopulation correlates with poor response to CART19 therapy. Preliminary molecular analysis suggests that low IKAROS result in retention of CD19 intron 10 and loss of B-cell identity (Figure 1). Future research will focus on in-depth characterization of the myeloid-like phenotype induced by low levels of IKAROS expression, and the impact that intron 10 retention has on CD19 mRNA stability and surface expression. Figure 1 Figure 1. Disclosures Mullighan: Pfizer: Research Funding; Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Barrett: Tmunity Therapeutics: Current Employment. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Characterization of Aleta-001, a CAR T Engager Designed to Optimize Responses and Prevent Relapses from CAR-19 Therapy

Adoptive T cell therapies directed to the B cell malignancy antigen CD19 (CAR19 T cells) have transformed the care of otherwise refractory, last-line leukemia and lymphoma patients. The overall response rates achieved are very high, routinely above 60%, and a substantial number of responding patients have durable responses that can last many years. However, 50% of responders relapse within 6 months, and outcomes for these relapsed patients are poor. Relapses occur most often in patients whose initial CAR19 T cell expansion is suboptimal, and whose tumor cells reduce or lose expression of the target antigen, CD19. We have created a CAR T Engager protein that is designed to improve responses to anti-CD19 CAR T treatment and is further designed to prevent relapses. This modular protein contains an anti-CD20 llama VHH linked to an optimized CD19 protein and further linked to an anti-albumin llama VHH. Thus, this CAR T Engager, called Aleta-001, binds to CD20 on B cell tumor cells, displays CD19 on the tumor cell surface thereby activating anti-CD19 CAR T cells, and binds to albumin, providing for a long half-life upon injection. Aleta-001 increases CD19 antigen density and/or replaces lost CD19 expression by coating cell surface CD20 with CD19. Here we present studies that establish the efficacious dose to support the upcoming Phase 1/2 clinical trial to be run in collaboration with Cancer Research UK. Extensive in vitro modeling established that the Aleta-001 CAR T Engager specifically bound to CD20-positive/CD19 negative lymphoma cells, JeKo-1-CD19KO, that represent the phenotype of a lymphoma relapsing after losing CD19 expression. In the presence of CAR19 T cells, the Engager protein mediated cytotoxicity against the JeKo-1-CD19KO cells at sub-nM concentrations. The JeKo-1-CD19KO cells induced a rapid and lethal lymphoma when implanted into NSG mice. Administration of the Aleta-001 CAR T Engager and CAR19 T cells eliminated the JeKo-1-CD19KO lymphoma at administered protein concentrations as low as 0.5mg/kg. These results support the clinical development of the Aleta-001 Engager protein. The protein is designed to be administered to patients who have received CAR19 T cell therapy and who fail to achieve a complete response at the time of their first clinical evaluation, or who relapse from a complete response thereafter. Patients who are enrolled will be dosed with the Aleta-001 Engager protein every two weeks. Aleta-001 has entered GMP production and IND enabling studies and will then enter Phase 1 dose escalation clinical studies in the UK. Figure 1 Figure 1. Disclosures Rennert: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Su: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Wu: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Lobb: Aleta Biotherapeutics Inc.: Consultancy, Current holder of stock options in a privately-held company. Ambrose: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company.

The Impact of CD19 Expression Combined with Measurable Residual Disease on Post-Remission Treatment and Outcome in Adult t(8;21) Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) with t(8;21) has considerable clinical heterogeneity. Only 50% of these patients receiving multiple cycles of high-dose cytarabine as post-remission treatments could achieve long term survival, and relapse occures in up to 40% of them. Therefore, further risk stratification is needed to guide appropriate post-remission treatment for t(8;21) AML patitents in first complete remission(CR1). Measurable residual disease (MRD) monitored by RUNX1-RUNX1T1 transcript levels after treatment is established as a powerful marker to predict relapse and guide treatment. Recent studies revealed cell-surface antigen CD19 negativity (CD19-) was a risk factor for relapse in t(8;21) AML. However, reports about the impact of CD19 expression combined with RUNX1-RUNX1T1 MRD on post-remission treatment remains absent to date. Methods: A total of 155 consecutive patients diagnosed with t(8;21) AML were enrolled in this study, including 69 patients who received chemotherapy (CMT) and 86 who received allogeneic stem cell transplantation (allo-SCT) as post remission treatment in CR1. MRD+ was defined as a < 3 log reduction of RUNX1/RUNX1T1 level after the second consolidation therapy. Results: With a median follow-up of 40 months (range, 4-133 months), the 3-year OS, LFS and CIR rates were 75.57% ,70.46% and 27.50% for the entire population, respectively. The 3-year CIR was dramatically decreased in allo-SCT than in CMT group (13.47% vs 49.19%, p<0.001). Allo-SCT demonstrated a significantly superior 3-year OS (85.78% vs 58.89%, p< 0.001) and LFS (85.64% vs 45.84%, p< 0.001) over CMT, respectively. The 3-year cumulative incidence of TRM was 9.27% and 7.04% in the CMT and allo-HSCT groups, respectively (p=0.890). KIT exon 17 mutation was a risk factor for OS in multivariate analysis, CD19 negativity was a risk factor for RFS in univariate analysis. Allo-SCT was a beneficial factor for LFS and OS in multivariate analysis when taking CMT as a reference. For CD19- patients (n=59), allo-HSCT demonstrated significantly lower CIR (17.20% vs 51.03%, p=0.006) , better OS (95.24% vs 51.55%, p < 0.001) and LFS (82.8% vs 39.71%, p = 0.001) than CMT. On the other hand, CMT had a comparable CIR (26.70% vs 11.40, p=0.064 ), OS (73.19% vs 86.01%, p=0.153) and LFS (71.58% vs 87.76%, p=0.067) with allo-SCT for CD19+ patients (n=80). Notably, when combined with RUNX1-RUNX1T1 transcripts after the second consolidation, allo-SCT preserved a significantly lower CIR (6.67% vs 66.67%, p=0.002), improved OS (100% vs 33.33%, p< 0.001) and LFS (93.33% vs 33.33%, p=0.001) than CMT in CD19-MRD+ group (n=22), respectively. In contranst, CMT revealed similar OS (80.0% vs 87.5%, p=0.677 ), LFS (60% vs 62.5%, p=0.663) and CIR (40.00% vs 37.50%, p=0.795 ) with allo-SCT in CD19-MRD- patients (n=13), respectively. For CD19+ patients, addition of MRD status had no impact on the outcome of allo-SCT and CMT. CMT preserved comparable OS (75% vs 84.66%, p =0.516), LFS (85.71% vs 96.30%, p =0.357 ) and CIR (12.50% vs 3.70%, p =0.367) with allo-SCT for CD19+MRD+ patients (n=36), and comparable OS (100% vs 92.31%, p =0.459), LFS (83.33% vs 85.29%, p =0.940 ) and CIR (16.67% vs 13.54%, p =0.998) with allo-SCT in CD19+MRD- patients (n=33). Conclusion: Combination of CD19 expression and RUNX1-RUNX1T1 MRD improved risk stratification and treatment guidance for t(8;21) AML in CR1. Allo-SCT might be recommended for the CD19-MRD+ patients, while CMT might be a reasnonable choice for CD19-MRD+ and CD19+ patients. Multicenter prospective studies are warranted to confirm the current results. Key words: t(8;21) AML, CD19, measurable residual disease, post-remission treatment, allo-SCT, chematherapy Disclosures No relevant conflicts of interest to declare.

SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism

The SARS-CoV-2 infection causes severe immune disruption. However, it is unclear if disrupted immune regulation still exists and pertains in recovered COVID-19 patients. In our study, we have characterized the immune phenotype of B cells from 15 recovered COVID-19 patients, and found that healthy controls and recovered patients had similar B-cell populations before and after BCR stimulation, but the frequencies of PBC in patients were significantly increased when compared to healthy controls before stimulation. However, the percentage of unswitched memory B cells was decreased in recovered patients but not changed in healthy controls upon BCR stimulation. Interestingly, we found that CD19 expression was significantly reduced in almost all the B-cell subsets in recovered patients. Moreover, the BCR signaling and early B-cell response were disrupted upon BCR stimulation. Mechanistically, we found that the reduced CD19 expression was caused by the dysregulation of cell metabolism. In conclusion, we found that SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism, which may provide a new intervention target to cure COVID-19.

Hypermethylation of CD19 promoter enables antigen-negative escape to CART-19 in vivo and in vitro

BackgroundAnti-CD19 chimeric antigen receptor T cells (CART-19) frequently induce remissions in hemato-oncological patients with recurred and/or refractory B-cell tumors. However, malignant cells sometimes escape the immunotherapeutic targeting by CD19 gene mutations, alternative splicing or lineage switch, commonly causing lack of CD19 expression on the surface of neoplastic cells. We assumed that, in addition to the known mechanisms, other means could act on CD19 to drive antigen-negative relapse.MethodsHerein, we studied the mechanism of antigen loss in an in vivo CD19-negative recurrence model of chronic lymphocytic leukemia (CLL) to CART-19, established using NOD-scid IL2Rgnull mice and HG3 cell line. We validated our findings in vitro in immortalized B-cell lines and primary CLL cells.ResultsIn our in vivo CLL recurrence model, up to 70% of CART-19-treated mice eventually recurred with CD19-negative disease weeks after initial positive response. We found that the lack of CD19 expression was caused by promoter DNA hypermethylation. Importantly, the expression loss was partially reversible by treatment with a demethylating agent. Moreover, this escape mechanism was common for 3 B-cell immortalized lines as well as primary CLL cells, as assessed by in vitro coculture experiments.ConclusionsEpigenetically driven antigen escape could represent a novel, yet at least partially reversible, means of CD19 loss to CART-19 in B-cell tumors.

A T-cell independent universal cellular therapy strategy through antigen depletion

CD19-targeting chimeric antigen receptor (CAR) T-cell therapeutics is a revolutionary, novel and successful treatment for B-cell malignancies. However, while CD19-CAR-T therapy can obtain high rates of complete responses in these patients, a significant fraction of patients may experience CD19-negative relapse. Moreover, the dependency on T-cell mediated cytotoxicity restricts CAR-T therapy as a patient-specific individualized therapy with severe side effects such as cytokine-release syndrome (CRS). Whether CAR-T therapy can be substituted by a non-T-cell based universal cellular therapy is largely unknown. Surprisingly, we have demonstrated here that T-lymphocytic cells, as well as non-lymphocytic cells, can cause CD19 internalization and subsequent depletion when they are armed with a CD19-recognizing moiety. This CD19 antigen depletion can efficiently induce T-cell independent apoptosis in target cancer cells whose survival is dependent upon CD19 expression, suggesting that CD19 antigen depletion constitutes a crucial tumor destroying mechanism for CD19-CAR-T, especially for its long-term efficacy. We therefore proposed a universal strategy for CRS-free cellular therapeutics, utilizing artificial antigen-recognizing cells (AARC), which can be manufactured universally and standardly as off-the-shelf mesenchymal stromal cells (MSCs) or other types of non-autologous cell expressing anergic CARs. Our results not only uncovered an unrecognized mechanism for CAR-T cytotoxicity and antigen loss, but also shed new insight into a shift in cellular therapeutics from unique patient-specific autologous therapeutics, to universal and standardized allogeneic treatment.

Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.