Bispecific T-cell Engager Antibody Research Articles

Modified heated CGMD simulations for discovering stable docked conformations of BiTE antibody against CD3 and CD117/c-kit

ABSTRACT In cancer immunotherapy, the design and optimisation of bispecific antibodies hold great promise. Bispecific T-cell engager (BiTE) antibodies targeting CD3 and CD117/c-kit have shown significant potential in experimental settings. Nevertheless, knowledge on their stable docked conformations at molecular level is still limited. This study presents an approach of employing modified heated coarse-grained molecular dynamics (CGMD) simulations to elucidate the stable docked conformations of BiTE antibodies against CD3 and CD117/c-kit. We integrated molecular dynamics simulation with coarse-grained and temperature control to explore the conformational landscape of these complex interactions. The modified heated CGMD simulation aimed to re-assess the docked poses suggested by ClusPro webserver. Furthermore, the all-atomic trajectories unveiled the dynamic residues formed throughout the simulation process. The per-residue-energy-binding emphasised the crucial amino acids involved in binding within the complex especially between the complementarity-determining regions (CDR) of BiTEs and residues located at the N-terminal of CD117/c-kit and the C-terminal of CD3. The formation of three types of interactions, such as hydrogen bonds, salt-bridge contact and hydrophobic interactions plays a crucial role in the motion, configuration and the free energy landscape of the complexes. This method is a valuable tool for rational drug design especially in the field of cancer immunotherapy.

Educating and empowering health care teams who present to the hospital to recognize and intervene on cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) in patients on bispecific t-cell engager therapy (BiTEs): A quality improvement study.

e23212 Background: Treatment of oncologic disease with Bispecific T-Cell Engager antibodies (BiTEs) are an emerging novel immunotherapy with demonstrated efficacy in multiple hematologic malignancies. Notable side effects of BiTE therapy include cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), which can be life-threatening and must be rapidly identified. We aim to fill knowledge gaps surrounding CRS and ICANS for medical providers, particularly considering increased use of BiTE therapy. We hope that educating providers will allow faster recognition and treatment and will improve patient outcomes. Methods: Our study focused on the impact of educational presentations on awareness and identification of CRS/ICANS. Our intervention targeted the residents and attendings in the emergency department and the nurses, advanced practice providers, and hospitalists in the bone marrow transplant (BMT) unit at Stony Brook University Hospital. Anonymous surveys consisting of 9 multiple choice questions were conducted to assess baseline foundational knowledge of CRS/ICANS. QI consisted of a 15-minute educational presentation for both groups. The same surveys were repeated following the presentations to assess improvement in overall knowledge of CRS/ICANS. A two tailed independent T test was used to calculate significance. Results: The average pre-survey score for the emergency department (N = 37) was 39%. The average presurvey score for the BMT department (N = 13) was 45%. Significant improvements in scores after intervention were shown in both the emergency department (39% vs 66%, p = 0.0001) and the BMT department (45% vs 68%, p = 0.0006). Conclusions: Education among emergency department and hospitalist providers is essential in improving management of CRS and ICANS in patients treated with BiTEs. This quality improvement initiative confirmed a knowledge gap in CRS and ICANS amongst our healthcare teams. By providing educational resources to both departments, there was a significant improvement in this gap. Future directions for continued improvement in the detection and management of CRS/ICANS include patient medication identification cards, a central repository of treatment protocols, algorithms, and toxicities associated with BiTEs, and expanding our educational curriculum which are all currently under development at Stony Brook University Hospital.

Potent antitumor activity of a bispecific T-cell engager antibody targeting the intracellular antigen KRAS G12V.

Kirsten Rat Sarcoma viral oncogene homolog (KRAS) is one of the most frequent oncogenes. However, there are limited treatment options due to its intracellular expression. To address this, we developed a novel bispecific T-cell engager (BiTE) antibody targeting HLA-A2/KRAS G12V complex and CD3 (HLA-G12V/CD3 BiTE). We examined its specific binding to tumor cells and T cells, as well as its anti-tumor effects in vivo. HLA-G12V/CD3 BiTE was expressed in Escherichia coli and its binding affinities to CD3 and HLA-A2/KRAS G12V were measured by flow cytometry, along with T-cell activation. In a xenograft pancreatic tumor model, the HLA-G12V/CD3 BiTE's anti-tumor effects were assessed through tumor growth, survival time, and safety. Our results demonstrated specific binding of HLA-G12V/CD3 BiTE to tumor cells with an HLA-A2/KRAS G12V mutation and T cells. The HLA-G12V/CD3 BiTE also activated T-cells in the presence of tumor cells in vitro. HLA-G12V/CD3 BiTE in vivo testing showed delayed tumor growth without severe toxicity to major organs and prolonged mouse survival. This study highlights the potential of constructing BiTEs recognizing an HLA-peptide complex and providing a novel therapy for cancer treatment targeting the intracellular tumor antigen.

Allogeneic transplantation after immunotherapy for relapsed/refractory non-Hodgkin lymphoma: a comparison with a historical cohort

Background aimsNew immunotherapy drugs, such as bispecific T-cell engager antibodies, checkpoint inhibitors and antibody–drug conjugates, are commonly used as salvage therapy for patients with non-Hodgkin lymphoma relapsing after chimeric antigen receptor (CAR) T-cell therapy. Nevertheless, their potential long-term effects on the outcome of allogeneic stem cell transplantation (Allo-SCT) are not well known. MethodsWe retrospectively analyzed the outcomes of 27 relapsed/refractory non-Hodgkin lymphoma patients receiving Allo-SCT after immunotherapy in the pre-CAR T-cell therapy era and compared them with a historical cohort of 28 subjects undergoing Allo-SCT after conventional therapy. ResultsThe two cohorts had similar outcomes in terms of graft-versus-host disease/relapse-free survival (4 years, 59% versus 46%), overall survival (4 years, 77% versus 44%), non-relapse mortality (4 years, 19% versus 22%) and acute (6 months, 15% versus 21%) and chronic (4 years, 18% versus 24%) graft-versus-host disease. Of note, the cumulative incidence of relapse was lower after immunotherapy (4 years, 4% versus 14%), although significance was not reached. The cumulative incidence of cytomegalovirus and fungal infection did not differ among the two cohorts. ConclusionsConsolidation with Allo-SCT is a safe and curative option for patients achieving disease response after new immunotherapy drugs that could represent a desirable salvage strategy for patients relapsing after CAR T-cell therapy.

Patterns of CRS with teclistamab in relapsed/refractory multiple myeloma with or without prior T-cell redirection therapy

AbstractTeclistamab (Tec) is a first-in-class BCMA × CD3 bispecific T-cell engager antibody approved for treating multiple myeloma progressing after at least 4 lines of therapy. The objective of this study was to evaluate the rate of cytokine release syndrome (CRS) in patients who were treated with commercial Tec and had prior exposure to other T-cell redirection therapies. A retrospective chart review was performed to identify patients who completed the Tec step-up dosing phase between November 2022 and November 2023. Patients were divided into 2 cohorts based on prior exposure to T-cell redirection therapy (cohort 1: T-cell redirection therapy experienced; cohort 2: T-cell redirection therapy naïve). The primary objective was to compare the differences in the rates of CRS between the 2 cohorts. Univariate and multivariate logistic regression analyses were performed to assess the association between CRS rates with Tec and prior treatment with T-cell redirection therapy. A total of 72 patients were included in the analysis (27 in cohort 1 and 45 in cohort 2). The CRS rates were significantly lower in cohort 1 (37%, n = 10) compared with cohort 2 (80%, n = 36; P = .0004). Based on multivariate logistic regression analysis, patients without prior exposure to T-cell redirection therapy (cohort 2) had about a fourfold increase in the incidence of CRS (95% confidence interval, 1.40-14.90; P = .0002) with Tec. In our study, prior exposure to T-cell redirection therapy reduced the risk of CRS with Tec during the step-up dosing phase. This observation will allow for the optimization of CRS prophylactic strategies for Tec.

Perils and Problems in Bispecific T-Cell Engager Antibodies.

Bispecific antibodies (BsAbs) are promising immunotherapies for cancer treatment designed to engage both tumour and immune cells. However, their use is associated with potential toxicities, including cytokine release syndrome (CRS), neurotoxicity, and on-target, off-tumour toxicity. CRS, characterized by cytokine release, is the most common, potentially life-threatening toxicity. Neurotoxicity presents as neurological symptoms and on-target, off-tumour toxicity damages healthy cells. Incidence and severity vary based on BsAb type, dose, patient factors, and tumor characteristics. For this study, articles pertaining to BsAb toxicity were searched on PubMed. Moreover, the management involves early recognition, dose modification, supportive care, and, in severe cases, immunosuppressive therapy or treatment discontinuation. Clinicians must carefully assess risks and benefits, considering individual patient profiles. Close monitoring and multidisciplinary collaboration are crucial for effective BsAb therapy. All in all, while toxicity is a concern, with vigilant management, BsAbs remain a valuable cancer treatment option.

Geographic and Racial Disparities in Chimeric Antigen Receptor–T Cells and Bi-Specific T-Cell Engager Antibodies Trials Access for Diffuse Large B-Cell Lymphoma

Geographic and Racial Disparities in Chimeric Antigen Receptor–T Cells and Bi-Specific T-Cell Engager Antibodies Trials Access for Diffuse Large B-Cell Lymphoma

BR109, a Novel Fully Humanized T-Cell-Engaging Bispecific Antibody with GPRC5D Binding, Has Potent Antitumor Activities in Multiple Myeloma.

At present, multiple myeloma (MM) is still an essentially incurable hematologic malignancy. Although BCMA-targeted therapies have achieved remarkable results, BCMA levels were found to be downregulated in patients with MM who relapsed after these treatments. Therefore, the search for other antigens specific to MM has become a priority. Independently of BCMA expression, G-protein-coupled receptor family C group 5 member D (GPRC5D) is mainly expressed in the plasma cells of MM patients, while it is expressed in a limited number of normal tissues. Combining MM-specific antigen GPRC5D and T-cell-mediated therapies would be a promising therapeutic strategy for MM. Recently, we constructed a new anti-GPRC5D × anti-CD3 T-cell-engaging bispecific antibody (TCB), BR109, which was capable of binding to human GPRC5D and human CD3ε. Moreover, BR109 was proven to have relatively good stability and antitumor activity. BR109 could specifically trigger T-cell-mediated cytotoxicity against many GPRC5D-positive MM cells in vitro. Meanwhile, antitumor activity was demonstrated in MM cell line xenograft mouse models with human immune cell reconstitution. These preclinical studies have formed a solid foundation for the evaluation of MM treatment efficacy in clinical trials.

EX103, a Novel CD20xCD3 Bispecific Antibody, Showed a Favorable Safety and Potent Single-Agent Antitumor Activity in Heavily Pretreated Relapsed/Refractory Patients with B-Cell Non-Hodgkin Lymphoma

Introduction Non-Hodgkin lymphoma (NHL) is one of the most common cancers in the United States, and relapsed or refractory (R/R) B cell NHL had a poor prognosis. In recent years, T cell engaging bispecific antibody (TCB) emerged as a promising therapy. Nevertheless, severe cytokine release syndrome (CRS) remains to be a significant challenge in TCB treatment. To overcome this issue, we designed a new CD20xCD3 bispecific antibody (EX103) with a lower affinity to CD3. In the non-human primate tox study, EX103 induced much lower IL-6 release. Preliminary data from dose escalation part of Phase I clinical study demonstrates that EX103 has an encouraging safety and robust single-agent antitumor activity trend, even at low doses, in heavily pretreated R/R B-NHL patients. Method The dose-escalation part (8 dose cohorts, ranging from 1.2 to 36 mg) of this Phase I, multicenter, single-arm, open-label clinical trial has enrolled R/R CD20+ B-cell non-Hodgkin lymphoma subjects at 4 study sites in China. Objectives include safety, dose finding (RP2D), and antitumor activity. Eligible patients in each cohort received intravenous(iv) step-up doses of EX103 (with 1 priming dose and 2 intermediate doses followed by 1 target dose of EX103) administered in a 28-day dose limiting toxicity (DLT) observation period, and then received a target dose every 2 weeks for up to 12 cycles until disease progression or unacceptable toxicity. Response rates reported are based on the Lugano criteria (2014). Results As of June 2023, 5 cohorts (target dose from 1.2 to 18 mg) of the dose-escalation were completed. Among 13 evaluable patients , 9 of them are aggressive B-NHL (5 Diffuse large B-cell lymphoma (DLBCL), 3 Follicular lymphoma (FL) grade 3B, and 1 Mantle cell lymphoma (MCL)), 2 of them are indolent B-NHL (1 FL grade 1-3A, 1 Marginal zone lymphoma (MZL)), and 2 of them are Chronic lymphocytic leukemia (CLL). No DLT was observed, and the maximum tolerated dose (MTD) was not reached. The most common adverse events (AEs) were CRS, pyrexia, neutropenia, thrombocytopenia. All CRS were grade (Gr) 1-2, no Gr 3 or higher CRS. Most CRS events occurred in first or second treatment cycle, and all CRS related clinical symptoms were resolved within 48 hours. No Immune effector cell-associated neurotoxicity syndrome (ICANS) or other clinically significant neurologic AEs were reported. Among those patients, the median age was 51 years (range 43-66) and median number of prior lines of treatment was 5 (range 2-8). The preliminary Disease Control Rate (DCR) is 84.6% (11/13). Among 9 aggressive NHL patients, the overall response rate (ORR) was 67.7 % (6/9), including 3 DLBCL patients (1 each in 6 or 12 or 18 mg cohorts) and 1 MCL patient (in 3 mg cohort) achieving CR, and 2 grade 3B FL patients achieving PR (1 each in 6 or 18 mg cohorts). Both indolent NHL patients (1 FL (Grade 1-3A) and 1 MZL in 12 mg cohort) achieved PR and the preliminary ORR for indolent NHL was 100.0 % (2/2). Of 2 CLL patients, 1 achieved SD (3mg cohort). It is worth mentioning that two of the patients who failed prior CAR-T treatment had achieved PR after received 6-14 week treatment of EX103. Both patients are still under EX103 treatment. Conclusions Although this study has a limited number of subjects so far and the findings are preliminary, the available dose-escalation data demonstrate that single-agent EX103, even at low doses (3 or 6 mg), exhibits remarkable antitumor activity and favorable safety. Furthermore, EX103 demonstrates promising efficacy in heavily pretreated patients with R/R B-NHL, including some who have previously failed CAR-T treatment. The safety and efficacy of EX103 will be further evaluated in an ongoing clinical study.

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

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

Trial in Progress: A Phase I/II Study of Tafasitamab Plus Lenalidomide in Patients with Relapsed CNS Lymphoma

Background and Significance The development of novel, more effective biological strategies to prevent secondary CNS lymphoma (CNSL) and to treat primary and secondary CNSL would have significant impact. It is well established that the CD19 transmembrane glycoprotein is expressed by B-cells throughout differentiation, by the majority of B cell malignancies, and by follicular dendritic cells. Given the high incidence of neurotoxicity associated with blood-brain barrier disruption with CD19-directed CAR-T cells and bi-specific T-cell engager antibodies, Parker et al. recently investigated the possibility of CD19 expression by previously unrecognized cell population(s) in the brain. Single-cell RNA sequencing data demonstrated CD19 expression in brain mural cells, an integral component of the neurovascular unit that regulates integrity of the blood-brain barrier.(Cell, 2020; 183:126-42). Given these observations and the compelling problem of relapsed primary and secondary CNS lymphoma, we are evaluating tafasitamab, an anti-CD19 humanized monoclonal antibody, as a novel therapeutic for refractory CNS lymphomas. We hypothesize that tafasitamab may modulate blood-brain barrier integrity by perturbation of CD19 expressed on mural cells; but without recruitment of cytotoxic T cells and resultant cytokine release syndrome. Our rationale is supported by the Phase II L-MIND study, in which tafasitamab plus lenalidomide exhibited synergistic efficacy against relapsed/refractory aggressive non-Hodgkin lymphoma, with overall response rate of 60%, complete response rate of 43%, and a favorable toxicity profile compared to CAR-T without neurotoxicity. Notably, the L-MIND trial excluded patients previously treated with immunomodulatory drugs as well as CNS lymphoma. (Lancet Oncology 2020: 21:978-88). We also recently reported that tafasitamab plus lenalidomide induced brain parenchymal regressions in two consecutive CNS lymphoma patients with disease refractory to lenalidomide, pomalidomide and rituximab. (British Journal of Haematology 2023 201(1):154-157). Study Design and Methods This single-arm, open-label, multicenter Phase I/II study (NCT05351593) assesses safety, CNS pharmacokinetics and preliminary efficacy of tafasitamab plus lenalidomide in patients with relapsed primary and secondary CNSL. The Phase I portion of the study will determine tolerated dose (MTD) and recommended Phase II dose (RP2D) of lenalidomide in combination with tafasitamab. We are applying 3+3 dose escalation rules to examine lenalidomide 10mg, 15mg and 20 mg plus tafasitamab at 12 mg/kg. After the RP2D is identified, the Phase II portion of the study will begin. The primary objective of the Phase II expansion is to evaluate the clinical benefit rate (stable disease or better) via Simon's minimax two-stage design. Exploratory endpoints include assessment of effect of tafasitamab on blood-brain barrier permeability, correlation of the relationships between tumor response or resistance with tumor mutational profile, and changes in CSF and blood immune cell phenotypes. The study plans to enroll up to 35 patients. Eligibility mandates age ≥ 18 years, anticipated survival >2 months, ECOG PS 0-1, and receipt of at least one prior systemic therapy. Patients with prior exposure to lenalidomide or pomalidomide or concomitant systemic lymphoma are allowed. Patients with HIV infection, CNS PTLD, and those who previously received a CD19-targeting CAR-T product are ineligible. The study is currently enrolling at the University of California San Francisco, with plans to expand enrollment to other institutions within the United States.

Pediatric Patients with Acute Lymphoblastic Leukemia Treated with Blinatumomab in a Real-World Study

Introduction Pediatric B-cell acute lymphoblastic leukemia (B-ALL) involves chromosomal abnormalities and genetic alterations at an early stage of differentiation of lymphoid precursor cells. Most children (~around 80%) with B-ALL can achieve long-term survival with conventional chemotherapy but a sizable number of patients relapse or do not attain complete remission. Blinatumomab is a bispecific T-cell engager antibody designed to redirect CD3+ T cells to bind to CD19+ target cells. Although blinatumomab has been approved for the treatment of relapsed or refractory B-ALL, it has shown efficacy in newly diagnosed B-ALL also. Here, we studied the effect of blinatumomab in newly diagnosed B-ALL (including infantile B-ALL) and post-induction therapy measurable residual disease (MRD) positive B-ALL in a real-world study. Methods Pediatric patients with B-ALL including newly diagnosed infants (infantile type) or with MRD positivity during the induction remission phase, were enrolled. The patients received a 7-day pre-treatment VCP regimen consisting of intravenous infusions of cyclophosphamide 800 mg/m 2 on day 1, vindesine 3 mg/m 2 on day 1, prednisone 1-2 mg/kg on days 1 to 7 (VCP regimen), followed by 28 days of treatment with blinatumomab at 5µg/m 2/day on the first 1 to 7 days and 15 µg/m 2/day on the next 8 to 28 days. Four patients with economic burden received blinatumomab for 14 days: 5µg/m 2/day for days 1 to 4 and 15 µg/m 2/day from day 5 to 14, after pre-treatment with the VCP regimen. Bone marrow assessment of MRD was done on days 14 and 28. Results Between July 2022 and July 2023, 24 pediatric patients received VCP-blinatumomab. Among them, 11 (45.8%) patients had high-risk disease, and 5 (20.8%) had standard risk. The median age of the cohort was 8.5 years. Among the cohort, there was 1 patient with infantile B-ALL and 1 patient with Philadelphia chromosome-positive (Ph+)-ALL; besides 3 patients were TEL-AML positive, 3 patients were CRLF2 positive, 1 patient was E2A-PBX1 positive, 1 patient was SSBP2-CSF1R positive, and 1 patient was EVI1 positive. Blinatumomab was administered to 5 patients in the induction remission period, and for 2 patients, blinatumomab was administered in consolidation after inotuzumab ozogamicin (INO). While 18 patients were treated with blinatumomab for 28 days, 6 patients were treated with blinatumomab for 14 days. Four patients opted for a 14-day regimen due to financial reasons and 2 patients were transplanted with hematopoietic stem cell transplantation after 14 days of treatment with blinatumomab. All the treated patients achieved MRD negativity (100%). There were no ≥ grade 3 treatment-related toxicity and no deaths reported. Conclusion Our study results showed that a 100% MRD negativity rate was achieved in children with B-ALL after treatment with the blinatumomab consolidation following the chemotherapy and no significant safety concerns were observed. Blinatumomab consolidation therapy was also effective in patients pretreated with INO. Blinatumomab consolidation after induction therapy could be a new and effective strategy for the treatment of patients with newly diagnosed B-ALL.

Novel CD123xCD3 Bispecific Igm Antibody, Igm-2537, Potently Induces T-Cell Mediated Cytotoxicity of Acute Myeloid Leukemia Cells In Vivo and in Vitro with Minimal Cytokine Release

Introduction: New therapeutics are needed for the effective treatment of acute myeloid leukemia (AML), as standard chemotherapeutics are poorly tolerated and ~50% of patients relapse primarily due to the incomplete elimination of leukemia stem cells (LSCs). CD123, the IL-3Rα chain, is highly expressed on leukemic blasts and LSCs, and is further increased in patients with poor prognostic factors. Bispecific T-cell engager (TCE) antibodies and CAR-T cells targeting CD123 have shown promising clinical efficacy in AML patients, but cytokine release syndrome limits their therapeutic window and remains a major safety concern. IGM-2537 is a novel pentameric IgM bispecific TCE antibody engineered with ten anti-CD123 binding sites, and an anti-CD3ε single chain Fv domain fused to the joining chain to engage T-cells. Here, we report that IGM-2537 not only potently induces T-cell mediated cytolytic killing of AML cells in vitro and in vivo but also demonstrates minimal cytokine release, and little to no effect on normal hematopoietic progenitor cells and human vascular endothelial cells (HUVECs) with in vitro and ex vivo studies. Additionally, an improved tolerability, excellent pharmacodynamic (PD) response and lower cytokine release was observed with a cynomolgus cross-reactive IgM TCE in monkeys. Materials and Methods:Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity and epitope mapping, respectively. T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 target cytotoxicity, T cell activation and cytokine release. Ex vivo AML colony formation assays were used to assess the activity on AML blasts. Human MV-4-11 and MOLM-13 xenograft tumor models in humanized NSG dKO mice were utilized to determine in vivo anti-tumor activity of IGM-2537. Healthy human PBMCs and bone marrow (BM) samples as well as HUVECs were used in ex vivo assays to address potential safety concerns of IGM-2537 mediated effects on hematopoietic progenitor cells and HUVECs, respectively. Lastly, to confirm that a CD123 directed IgM-based TCE molecule can potentially provide better safety profile in vivo, a cynomolgus-cross reactive IgM TCE comparator was assessed in monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound with high affinity and avidity to CD123. In vitro, IGM-2537 engaged both CD123 and CD3 to induce potent T-cell activation and T-cell mediated cytotoxicity of CD123 + AML cells, and autologous CD123 + basophils and plasmacytoid dendritic cells (pDCs). Though IGM-2537 demonstrated comparable maximal killing activity to a comparator IgG TCE, IGM-2537 demonstrated minimal cytokine release. In ex vivo patient-derived AML or normal BM colony formation assays, IGM-2537 eliminated AML colony forming cells at physiologically relevant effector/target (E/T) ratios but had little or no effect on normal hematopoietic progenitors. In addition, IGM-2537 demonstrated no cytotoxicity against HUVECs regardless of their CD123 expression at basal levels or at maximal levels upregulated by TNF-a and IFN-g, in contrast to a comparator IgG TCE. In vivo, IGM-2537 completely inhibited tumor growth in two AML xenograft tumor models, a subcutaneous MV-4-11 model, and a systemic MOLM-13 model in humanized NSG dKO mice. To further evaluate the potency and safety of the CD123xCD3 IgM bispecific TCE format in vivo, a cynomolgus cross-reactive CD123xCD3 IgM bispecific TCE was evaluated for tolerability and PD responses in cynomolgus monkeys using single doses. All animals tolerated the cross-reactive bispecific IgM TCE well at doses up to 10 mg/kg (the maximal dose level evaluated), a dose 100-fold greater than published doses of a comparator IgG TCE. Complete depletion of CD123 + basophils and substantial reductions of pDCs were seen in blood and BM with minimal to no cytokine induction. In summary, IGM-2537 demonstrated potent in vitro and in vivo T-cell mediated cytotoxicity of AML cell lines with minimal cytokine induction. The encouraging preclinical safety profiles were further supported by little/no effect on normal hematopoietic progenitor cells and HUVECs. These preclinical data support the clinical development of IGM-2537 for the treatment of AML and other hematological malignancies.

Treatment with Blinatumomab in Combination with Venetoclax for Mixed Phenotype Acute Leukemia

Background: Mixed phenotype acute leukemia (MPAL) is a rare and heterogeneous type of acute leukemia that displays an ambiguous pattern of antigen expression to a degree that assigning a single lineage of origin is not possible due to which, understanding of the pathophysiology of the disease, diagnosis and treatment are limited. The lack of well-established optimal treatment for MPAL has paved path for requirement of a more effective and less toxic approach. Blinatumomab, a bispecific CD19-directed CD3 T-cell engager (BiTE) antibody and venetoclax, a potent, highly selective B-cell leukemia/lymphoma-2 (BCL2) inhibitor was approved for the treatment relapsed/refractory (r/r) B-acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), respectively. Here, we present 6 patients with CD19-positive MPAL that are successfully treated with blinatumomab in combination with venetoclax and/or azacytidine in front line setting. Methods: All 6 patients met the criteria for MPAL as defined by European Group for Immunological Characterization of Acute Leukemias (EGIL) or World Health Organization (WHO 2022). Flow cytometry and polymerase chain reaction (PCR) was used to assess MRD and BCR::ABL1 or KMT2A gene, respectively. As pre-phase treatment, patients received glucocorticoid for 7 days. Blinatumomab at a dose of 9 mg/day for 1 week followed by 28 mg/day for 3 weeks combined with venetoclax at a dose of 100 mg/day for 14 to 21 days and/or azacytidine at a dose of 75 mg/m 2/day was given as induction therapy subcutaneously for 7 days. The patient with BCR::ABL1 received blinatumomab in combination with olverembatinib. Intrathecal (IT) chemotherapy with cytarabine and methotrexate was administered to central nervous system (CNS) as prophylaxis treatment. Results: Of the six patients included five were male and only one was female; five were diagnosed with B-cell/myeloid MPAL, while one with B-cell/T-cell MPAL. Abnormalities in KMT2A gene was observed in three patients while one patient had abnormality in BCR::ABL1 gene, two patients were negative for both gene abnormalities. After one cycle of blinatumomab combination treatment, all the six patients achieved complete response (CR) with full hematologic recovery. In 5 patients, MRD was <0.1% of these 3 had MRD-negative remission (<0.01%) and two patients had molecular remission. Patients were switched to chemotherapy post induction therapy, after one cycle of post-reinduction, the patients who still had the CD19-positive flow MRD or persistent molecular MRD received sequencing chemotherapy and blinatumomab. The treatment and MRD was presented in table 1. The median follow-up time was 10 months. Except 2 patients, all other patients achieved continuous MRD and molecular remission, of which two patients underwent an allogeneic hematopoietic stem cell transplant (allo-HSCT). The most frequent grade 3 or 4 adverse events (AEs) observed were anemia (6 patients), neutropenia (6 patients) and thrombocytopenia (4 patients). No AEs led to treatment interruptions and no serious AEs were reported. Conclusion: The results provide preliminary evidence of effective and safe treatment with blinatumomab and venetoclax combination as first-line therapy in MPAL resulting in the achievement of normal bone marrow function with less toxicity than using conventional chemotherapy.

Patterns of Cytokine Release Syndrome with Teclistamab in Relapsed/Refractory Multiple Myeloma with or without Prior T-Cell Redirection Therapy

Introduction: Numerous therapeutic options with novel mechanisms of actions are currently available for treating patients with relapsed/refractory multiple myeloma (RRMM). Teclistamab (Tec) is a first-in-class BCMA X CD3 bispecific T-cell engager antibody that was recently approved for patients with RRMM who have received at least 4 lines of therapy (LOT). In the landmark MajesTEC-1 study, cytokine release syndrome (CRS) with Tec was reported at a rate of 72% (Usmani S, et al. Lancet 2021, Moreau P, et al. N Engl J Med 2022); however, patients who were previously treated with T-cell redirection therapies (TCRT) were excluded from the study [TCRT: chimeric antigen receptor (CAR) T-cells and/or other bispecific antibodies]. Given this knowledge gap, we investigated whether prior TCRT exposure impacts CRS rates with Tec in a real-world setting. Methods: Our institutional plasma cell disorders database was queried to identify RRMM patients who received commercial Tec from November 2022 to July 2023. Patients who completed the Tec step-up dosing phase (first step-up, second step-up and first full doses) were divided into two cohorts based on prior TCRT exposure before treatment with Tec (prior TCRT exposure: cohort 1 and no prior TCRT exposure: cohort 2). Data collection included patient demographics, disease characteristics, prior LOT, and rates/grades of CRS and immune effector cell-associated neurotoxicity syndrome (ICANS). The Chi-square/Fisher exact test was used to compare differences in CRS rates between the two cohorts. Univariate and multivariate logistic regression analyses were performed to assess impact of prior TCRT exposure on CRS rates with Tec. The results were presented as odds ratios (ORs) with 95% confidence intervals (CIs). For cohort 1, the Mann-Whitney U-test was used to compare the median time elapsed from the last prior TCRT dose until the first Tec step-up dose between patients who developed CRS with Tec compared to those who did not. All statistical analyses were performed using SPSS software (version 29). Results: 50 patients (cohort 1: 25 and cohort 2: 25) were included in the final analysis. Baseline characteristics were comparable between the two cohorts except for prior LOT, where patients in cohort 1 represented a more heavily pretreated population (Table 1). The CRS rates were significantly lower in cohort 1 (40%, n=10) compared to cohort 2 (76%, n=19, p=0.02; Figure 1). CRS grades 1 and 2 occurred in 32% (n=8) and 8% (n=2) of patients in cohort 1 and 48% (n=12) and 28% (n=7) of patients in cohort 2, respectively (p=0.40). Among the CRS events, 60% (6/10) and 68% (13/19) occurred following the first Tec step-up dose in cohorts 1 and 2, respectively. In univariate logistic regression analysis, no prior exposure to a TCRT was associated with about a 5-fold increase in the incidence of CRS with Tec (OR= 4.8, 95% CI: 1.5-17.1.2, p=0.02). After adjusting for age, extramedullary disease (EMD) and elevated LDH levels, the impact of prior exposure to TCRT remained significant with an OR of 4.5 (95% CI: 1.3-18.6, p=0.01). In cohort 1 (prior TCRT exposure), the median time elapsed between the last TCRT dose and the first Tec step-up dose was 321.5 days (range: 50-808 days) in patients who developed CRS with Tec compared to 265 days (range: 41-1617 days) in those who did not (p=0.90). Conclusion: In our study, prior exposure to a TCRT was associated with a significantly lower incidence of CRS during the Tec step-up dosing phase, which may be suggestive of T-cell exhaustion. This observation will allow for optimization of CRS prophylactic strategies for RRMM patients receiving Tec treatment.

The Effectiveness of Blinatumomab in Clearing Next-Generation Sequencing Measurable Residual Disease in Pediatric Patients with B-Cell Acute Lymphoblastic Leukemia

Introduction Blinatumomab is a bispecific T-cell engager antibody designed to redirect CD3+ T cells to bind to CD19+ target cells in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). Measurable residual disease (MRD) is a significant prognostic factor for relapse and overall survival in B-ALL. Blinatumomab consolidation therapy has been shown to improve MRD remission (<10 -4), as detected by multiparameter flow cytometry (MFC) or reverse transcriptase-polymerase chain reaction (PCR), and survival outcomes in children with high-risk first relapsed B-ALL. The next-generation sequencing (NGS)-MRD is a highly sensitive technique that can detect very low levels of MRD (<10 -6) in patients who were considered “MRD negative” by less sensitive MFC or PCR technologies. Early achievement of NGS-MRD negativity (<10 -6) can help in identifying patients who have a very low risk of relapse. It has been suggested that blinatumomab treatment could help to attain deeper responses than that measured with a MRD sensitivity level of 10 -4. However, data on blinatumomab clearing MRD with high sensitivity of 10 -6 remain scarce. Hence, we evaluated the effectiveness of blinatumomab in eradicating extremely low level of MRD (<10 -6), as detected by NGS, in children with B-ALL. Methods Pediatric B-ALL patients who were MFC-MRD negative (<10 -4) but NGS-MRD positive (sensitivity of 10 -6) after chemotherapy followed by blinatumomab consolidation therapy between October 2021 and February 2023, were studied retrospectively. At diagnosis, NGS analysis was performed by high-throughput deep level sequencing using the Seq-MRD ® (ImmuQuad Biotech, Hangzhou, China) to detect clonal rearrangements. Genomic DNA was extracted from bone marrow samples using QIAamp DNA Mini kit (Qiagen, Hilden, Germany), amplified by quantitative PCR and sequenced using the Illumina® Novaseq PE150 (Illumina ® Novaseq, San Diego, USA) platform. The sequences of immunoglobulin heavy chain (IGH) variable (V), diversity (D), and joining (J) (IGH -VDJ), IGH-DJ, immunoglobulin kappa light chain (IGK), immunoglobulin kappa deleting element (IGKDE) and immunoglobulin light chain (IGL) were assessed for clonality with dominant index sequence(s). Results Overall, 19 patients were enrolled. The baseline patient characteristics are shown in Table 1. Median age at diagnosis was 4.4 years (range 0.3-10.6), with males accounting for 84% of patients. Eight patients (42%) had at least 1 unfavorable genetic abnormality (i.e., KMT2A rearrangement, E2A-PBX1). Seventeen (89%) patients had ≥2 Ig clonal rearrangement at diagnosis. In total, NGS detected 71 clonal rearrangements, including 27 IGH clonal rearrangements in 15 (79%) patients, 9 IGH-DJ in 6 (32%), 14 IGK in 8 (42%), 6 IGKDE in 9 (32%), and 12 IGL in 8 (42%) patients. All patients achieved hematological complete response (CR) with MFC-MRD <10 -4 after the induction or consolidation chemotherapy. However, all these patients were identified to be MRD positive by NGS, including NGS-MRD >10 -6 and positive but not quantifiable (NGS-MRD <10 -6). Though the patients were MFC-MRD negative, 19 IGH clones, 4 IGH-DJ clones, 8 IGK clones, 7 IGKDE clones and 3 IGL clones were still persisted. After blinatumomab, MRD negativity rate by MFC was 95% (18/19) and the NGS-MRD negativity rate was 68% at 10 -6 (13/19). The rate of MRD clearance after blinatumomab per NGS was 69% and 67% in CR1 and CR2 patients, respectively. The clearance rates of IGH, IGK, IGL, IGH-DJ, and IGKDE were 75%, 67%, 33%, 100%, and 83%, respectively (Table 2). Three NGS-MRD negative patients with available NGS-MRD at pre-, mid-, and post-treatment achieved MRD clearance rapidly after blinatumomab treatment that was maintained durably through day 28. Three of 6 NGS-MRD positive patients relapsed after blinatumomab with 3 being IGH positive and 2 being IGL positive. Conclusion To the best of our knowledge, for the first time it has been demonstrated that blinatumomab could further eradicate MRD in patients achieving MFC-MRD <10 -4. Advanced MRD detection using NGS and blinatumomab made deeper molecular remission in B-ALL patients achievable. Further large-scale studies are warranted to verify the prognostic value of NGS in detecting lower levels of MRD in ALL patients treated in the current immunotherapy era.

Binding domain on CD22 molecules contributing to the biological activity of T cell-engaging bispecific antibodies

CD22, as the B-cell malignancies antigen, has been targeted for immunotherapies through CAR-T cells, antibody-drug conjugates (ADCs) and immunotoxins via interaction of antibodies with binding domains on the receptor. We hypothesized that avidity and binding domain of antibody to target cells may have significant impact on the biological function in tumor immunotherapy, and T cell-engaging bispecific antibody (TCB) targeting CD22 could be used in the therapy of hematologic malignancies. So, to address the question, we utilized the information of six previously reported CD22 mAbs to generate CD22-TCBs with different avidity to different domains on CD22 protein. We found that the avidity of CD22-TCBs to protein was not consistent with the avidity to target cells, indicating that TCBs had different binding mode to the protein and cells. In vitro results indicated that CD22-TCBs mediated cytotoxicity depended on the avidity of antibodies to target cells rather than to protein. Moreover, distal binding domain of the antigen contributed to the avidity and biological activity of IgG-[L]-scfv-like CD22-TCBs. The T cells' proliferation, activation, cytotoxicity as well as cytokine release were compared, and G5/44 BsAb was selected for further in vivo assessment in anti-tumor activity. In vivo results demonstrated that CD22-TCB (G5/44 BsAb) significantly inhibited the tumors growth in mice. All these data suggested that CD22-TCBs could be developed as a promising candidate for B-cell malignancies therapy through optimizing the design with avidity and binding domain to CD22 target in consideration.

A simplified function-first method for the discovery and optimization of bispecific immune engaging antibodies.

Bi-specific T-cell engager antibodies (BiTEs) are synthetic fusion molecules that combine multiple antibody-binding domains to induce active contact between T-cells and antigen expressing cells in the body. Blinatumomab, a CD19-CD3 BiTE is now a widely used therapy for relapsed B-cell malignancies, and similar BiTE therapeutics have shown promise for treating various other forms of cancer. The current process for new BiTE development is time consuming and costly, requiring characterization of the individual antigen binding domains, followed by bi-specific design, protein production, purification, and eventually functional screening. Here, we sought to establish a more cost-efficient approach for generating novel BiTE sequences and assessing bioactivity through a function first approach without purification. We generate a plasmid with a bi-modular structure to allow high-throughput exchange of either binding arm, enabling rapid screening of novel tumour-targeting single chain variable (scFv) domains in combination with the well-characterized OKT3 scFv CD3-targeting domain. We also demonstrate two systems for high throughput functional screening of BiTE proteins based on Jurkat T cells (referred to as BiTE-J). Using BiTE-J we evaluate four EGFRvIII-scFv sequenced in BiTE format, identifying two constructs with superior activity for redirecting T-cells against the EGFRvIII-tumour specific antigen. We also confirm activity in primary T cells, where novel EGFRvIII-BiTEs induced T cell activation and antigen selective tumor killing. We finally demonstrate similar exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BiTEs, we were able to identify highly active BiTE molecules with desirable functional activity for downstream development. In summary, BiTE-J presents a low cost, high-throughput method for the rapid assessment of novel BiTE molecules without the need for purification and quantification.

An engineered Trop2/CD3 bispecific antibody for treatment of gastric, pancreatic, and colorectal cancers.

e14521 Background: Given its over-expression and poor prognosis in a variety of solid tumors, trophoblast cell surface antigen 2 (Trop2) is regarded as a potential therapeutic target. Trop2-directed antibody-drug conjugates have been developed for treatment of solid tumors; however, their clinical benefits can be limited by the associated undesirable effects which is likely attributed to the on-target off-tumor effect arising from the low basal Trop2 expression on the surface of multiple normal epithelial tissues. The present study aims to develop a humanized Trop2/CD3 bispecific T-cell engager antibody that can promote immunological synapse formation with tumor cells overexpressing Trop2, redirecting T cell-mediated killing specifically to cancers with reduced risk of on-target off-tumor effects. Methods: A Trop2/CD3 bispecific antibody was generated by linking a humanized anti-Trop2 monoclonal antibody with a CD3-binding scFv. To tune the affinity of the bispecific antibody for tumor target, in silico paratope prediction was firstly done using ProABC-2 (Ambrosetti F et al., 2020), following by alanine substitution of the identified paratope residues individually. A total of 6 CDR variants were generated with their binding to Trop2 examined using biolayer interferometry. Selected candidates were further characterized for their binding to Trop2-positive cancer cells and activation of T cells upon target engagement. Redirection of T cell-mediated killing on cancer cells expressing high Trop2 level was also studied in vitro. Results: Substitution of a complementarity-determining residue (CDR) on the heavy chain of anti-Trop2 antibody with an alanine significantly reduced binding affinity to Trop2. This CDR variant was selected for further functional characterization. Apart from weaker binding to Trop2-positive tumor cells, the variant also demonstrated a slower cytokine induction, a less acute T-cell activation, and a cytotoxic effect which was more restricted to Trop2-high cancer cells. Conclusions: Our new Trop2/CD3 bispecific antibody demonstrates potent anti-tumor activity, while at the same time shows a reduced risk of on-target off-tumor toxicity. The drug candidate warrants further investigations including toxicity study in monkeys. [Table: see text]

A Novel bispecific T-cell engager (BiTE) targeting CD22 and CD3 has both in vitro and in vivo activity and synergizes with blinatumomab in an acute lymphoblastic leukemia (ALL) tumor model

Immunotherapy has revolutionized cancer therapy. Two recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or chimeric antigen receptor T (CAR-T) cells. Blinatumomab, an FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating effector-target cell contact and T-cell activation that results in effective elimination of target B cells. Although CD19 is expressed by essentially all B-cell malignancies at clinical presentation, relapses with loss or reduction in CD19 surface expression are increasingly recognized as a cause of treatment failure. Therefore, there is a clear need to develop therapeutics for alternate targets. We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments. Target binding of the anti-CD22 and anti-CD3 moieties was confirmed by flow cytometry. CD22-BiTE promoted in vitro cell-mediated cytotoxicity in a dose and effector: target (E:T)-dependent fashion. Additionally, in an established acute lymphoblastic leukemia (ALL) xenograft mouse model, CD22-BiTE demonstrated tumor growth inhibition, comparable to blinatumomab. Further, the combination of blinatumomab and CD22-BiTE yielded increased efficacy in vivo when compared to the single agents. In conclusion, we report here the development of a new BiTE with cytotoxic activity against CD22+ cells which could represent an alternate or complementary therapeutic option for B-cell malignancies.