CD3 Bispecific T-cell Engager Research Articles

A novel B7-H3 x CD3 bispecific T-cell engager for the treatment of B7-H3+ tumors.

e14509 Background: B7-H3 (CD276) is a transmembrane protein highly expressed in solid tumors but with limited expression in normal tissues. Its presence is strongly associated with the inhibition of T-cell activity, cancer progression, and invasion. Furthermore, B7-H3 is strongly associated with poor prognosis and reduced patient survival. Given its crucial role in tumor biology, B7-H3 has emerged as an attractive therapeutic target for drug development. We utilized ExMab heterodimer platform and common light chain technology to develop EX105, a next-generation B7-H3×CD3 bispecific antibody. This innovative T-cell engager exhibits excellent anti-tumor activity in mouse models and good safety profile in NHP tox study. Methods: EX105 was generated using ExMab heterodimer platform and common light chain technology to form a heterodimeric IgG1 structure. Preclinical pharmacology studies, efficacy, and safety were evaluated. Anti-tumor activity study of EX105 includes in vitro T-cell cytotoxicity assays and in vivo PBMC reconstituted xenograft mice models. Preclinical safety evaluation of EX105 was performed through GLP single-dose and repeat-dose tox studies in non-human primates (NHPs). Results: Based on ELISA and FACS analyses, EX105 can bind to both human and cynomolgus monkey B7-H3 and CD3 antigens. Furthermore, SPR data suggest that EX105 exhibits a high affinity to B7-H3 and a much lower affinity to CD3. In consistent with that, EX105 only induced relatively low cytokine release. Nevertheless, EX105 showed a potent in vitro T cell cytotoxicity against B7-H3+ tumor cell lines. The in vivo anti-tumor activity of EX105 was further determined in several tumor xenograft models. Treatment with EX105 resulted in remarkable tumor regression in both “hot” tumor model and “cold” tumor model, indicating its effectiveness across a range of tumor types. Tox study in NHPs showed minimal adverse effects and no liver impairment after EX105 administration, even at high doses. The cytokine release data observed in this study suggests a good safety profile of EX105, with much lower cytokine release compared to other T cell engagers. Conclusions: These findings collectively indicate that EX105 has the potential to become a promising therapeutic agent for the treatment of various B7-H3 positive cancers. The first-in-human clinical trial for patients with advanced B7-H3 positive cancers is due to start.

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.

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.

Abstract LB126: Generation of a novel ROR1 x CD3 bispecific T-cell engager for better tumor killing and minimal cytokine release

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) expression is a highly specific tumor associated antigen expressed in multiple hematological malignancies and solid tumors. EMB-07 is a novel monovalent asymmetrical tandem Fab (MAT-Fab) antibody composed of two different Fab fragments targeting ROR1 and CD3ε respectively, and a “knob-in-hole” Fc fragment maintaining FcRn binding and Ig-like pharmacokinetics in vivo. To avoid light chain miss-pairing, the anti-ROR1 light chain was directly connected to the N-terminus of the anti-CD3 heavy chain. The ROR1 binding arm has a high affinity of 0.52 nM and recognizes a membrane distal epitope, which is different from a reference BiTE molecule currently in clinical trial. In comparison with this BiTE molecule, EMB-07 showed similar tumor killing efficacy to ROR1 expressing tumor cell lines while it induced much lower levels of cytokines both in vitro and in vivo. Moreover, comparing with a clinical stage ROR1-vc-MMAE ADC, EMB-07 demonstrated ~500-fold increased tumor lysis potency in vitro. In PD-1 resistant tumor xenograft mice models, EMB-07 showed potent tumor growth inhibition and synergistic activity in combination with PD-1 blockade. In a preclinical GLP toxicological study, EMB-07 was well tolerated in cynomolgus monkeys with no evidence of lethality, life-threatening toxicity, or obvious EMB-07-related macroscopic and histopathological adverse findings up to 50 mg/kg. By using a highly specific IHC clone, we validated membrane-associated ROR1 expression in multiple tumor types, including triple negative breast cancer, ovarian cancer, endometrial cancer, lung adenocarcinoma, and pancreatic cancer, while the observation of its expression in normal tissues is very limited. This study supports the clinical development of EMB-07 for treating multiple solid tumors with ROR1 expression. A FIH study of EMB-07 in locally advanced/metastatic solid tumors or relapse/refractory lymphoma is currently ongoing (NCT05607498). Citation Format: Danqing Wu, Shiyong Gong, Xuan Wu, Gaowa Naren, Liqin Dong, Stephan Lensky, Chengbin Wu. Generation of a novel ROR1 x CD3 bispecific T-cell engager for better tumor killing and minimal cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB126.

Spatially Resolved Transcriptomics Unveils Unique T-Cell Dysfunctional States and Prognostic Gene Expression Signatures in TP53-Mutated Acute Myeloid Leukemia

Introduction Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 8% to 10% of AML and correlate with primary induction failure, high risk of relapse, and dismal prognosis. We have previously shown that TP53 abnormalities are associated with a pro-inflammatory and immunosuppressive tumor microenvironment (TME), including high CD274 and FoxP3 expression, with dysfunctional natural killer (NK)/CD8 + T-cell states and with response to flotetuzumab, a CD123 × CD3 bispecific T-cell engager. In the current study, we explore the cellular neighborhoods of TP53-mutated ( TP53-m) AML using spatially resolved transcriptomics and we employ machine learning to identify prognostic RNA signatures. Methods We analyzed the expression of >1,800 immune and TME genes in bone marrow (BM) formalin-fixed paraffin-embedded (FFPE) sections (5-µm) from a discovery cohort of 11 patients with chemotherapy-naïve AML (N=5 TP53-m and N=6 TP53-wt cases) using the GeoMx™ Digital Spatial Profiling (DSP) platform (Cancer Transcriptome Atlas with next generation sequencing read-out; NanoString Technologies, Seattle, WA). Imaging was performed at 20× magnification and a total of 151 circular regions of interest (ROIs) with a 300-600 µm diameter were selected (Fig. 1A). We then generated a ROI-by-gene count matrix for deconvolution and downstream pathway analysis. For each ROI, enrichment scores reflecting immune senescence/dysfunction and leukemia stemness were calculated with gene set variation analysis (GSVA), using a manually curated compendium of published gene signatures as input. We also employed public bulk and single-cell RNA-sequencing (sc-RNA-seq) AML cohorts for in silico modeling and validation. Results We initially captured transcripts from ROIs of TP53-m and TP53-wt AML with varying degrees of BM infiltration with T cells (CD3 hotspots) and AML blasts, and we found that TP53-m is enriched in T cell-specific, type I/II interferon (IFN) and pro-inflammatory genes ( IL1B), as well as leukemia stem cell (LSC)-related gene programs (LSC17). After dichotomizing ROIs based on a median split of CD3D RNA expression, we observed that T-cell low ROIs express higher levels of monocyte-macrophage and myeloid derived suppressor cell (MDSC)-related genes, including S100A9 and S100A8. These findings point to previously unappreciated links between BM infiltration with immune-modulatory myeloid cell types and leukemia stemness / T-cell abundance. We then tested for differential cell density in neighborhoods (regions of transcriptionally similar cells) across TP53-m and TP53-wt AML using MiloR in a sc-RNA-seq AML dataset annotated by cell type (N=42 cases, of which N=3 with TP53-m; Lasry A, et al. Nat. Cancer 2023). This orthogonal approach highlighted a significantly higher frequency (FDR <0.05) of multipotent and granulocyte-macrophage progenitor neighborhoods in TP53-m AML, alongside suppression of adaptive immune system pathways (Fig. 1B). We next inferred active receptor-ligand pairs mediating the crosstalk between T cells and leukemic blasts in the TME of TP53-m AML, and we uncovered decreased MHC class I/II-mediated signaling, curbed CD8 +/CD4 + T cell-to-AML interactions and enhanced CD6/ CDCP1( CD318) signaling (Fig. 1C). We also found that CD8 + and CD4 + T cells from TP53-m AML are enriched in effector memory (T EM) / terminally differentiated effector memory (T EMRA) and regulatory T-cell gene programs, respectively, compared with their cellular counterparts from TP53-wt patients (Fig. 1D). In particular, T EM cells from TP53-m AML expressed genes known to regulate T-cell dysfunctionality, foremost PRDM1, and transcription factors ( ZEB2) expected to drive the differentiation of killer cell lectin-like receptor (KLR) + exhausted T-cell states with residual cytolytic potential. Finally, we applied the least absolute shrinkage and selection operator (LASSO) statistical method for feature selection to our discovery AML dataset, and we constructed a TP53-m-related ‘spatial metagene’ that predicted poor overall survival in TCGA-LAML (N=170) and Beat-AML2 cases (N=444; Fig. 1E). Conclusions Spatial gene programs of leukemia stemness, T-cell dysfunction and immune suppression are enriched in TP53-m AML. It remains to be determined whether TP53-m AML with a pre-existing but ineffective T-cell response may be amenable to T cell-targeting immunotherapies.

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.

Bivalent BCMA Binding and Low Affinity CD3 T-Cell Engagement By Abbv-383 Drives Sustained Activation with Reduced T-Cell Exhaustion in Preclinical Models of Multiple Myeloma

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by >100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

Microenvironment-responsive anti-PD-L1 × CD3 bispecific T-cell engager for solid tumor immunotherapy

Bispecific T-cell Engager (BiTE) antibodies can redirect T-cells to tumor cells, and turn on the targeted lysis of tumor cells. However, BiTE has been challenging in solid tumors due to short plasma half-life, "off-target" effect, and immunosuppression via PD-1/PD-L1 axis. This study designed a safe, long-acting, and highly effective Protease-Activated PSTAGylated BiTE, named PAPB, which includes a shielding polypeptide domain (PSTAG), a protease-activated linker, and a BiTE core. The BiTE core consists of two scFvs targeting PD-L1 and CD3. BiTE core bound PD-L1 and CD3 in a dose-dependent manner, and PAPB can release BiTE core in response to MMP2 in the tumor microenvironment to exert antitumor activity. The plasma half-life of PAPB in mice was significantly prolonged from 2.46h to 6.34h of the BiTE core. In mice bearing melanoma (A375) xenografts, PAPB significantly increased infiltration of T lymphocytes in tumor tissue, and inhibited tumor proliferation without activating T-cells in the peripheral blood. Overall, the engineering protein PAPB could be a promising drug candidate for solid tumor immunotherapy.

Transcriptomic Features of Immune Exhaustion and Senescence Predict Outcomes and Define Checkpoint Blockade-Unresponsive Microenvironments in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Reinstating immunological control of AML is highly desirable to eradicate chemotherapy-resistant clones and provide long-term disease control. We recently identified bone marrow (BM) microenvironmental transcriptomic profiles that stratify patients with newly diagnosed AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction beyond that afforded by current prognosticators (Vadakekolathu J et al., 2020). We have also shown that CD8 + T cells from patients with AML exhibit features of immune exhaustion and senescence (IES), including heightened expression of killer cell lectin-like receptor subfamily G member 1 (KLRG1) and B3GAT1 (encoding CD57) (Knaus H et al., 2018). Whether deranged T-cell functions affect the likelihood of responding to antitumor therapy, including immune checkpoint blockade (ICB), is an outstanding question in AML.In the current study, we analyzed 183 BM samples collected longitudinally at time of AML onset, response assessment and disease relapse from multiple cohorts of patients with AML treated with standard-of-care induction chemotherapy, and from 33 elderly AML patients with newly diagnosed or chemotherapy-refractory/relapsed AML treated with azacitidine, and the PD-1 checkpoint inhibitor pembrolizumab (NCT02845297). Primary patient specimens and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki on research protocols approved by the Institutional Review Boards of the participating Institutions. RNA (150-200 ng) was extracted from BM aspirates and was processed on the nCounter FLEX analysis system (NanoString Technologies, Seattle, WA) using the PanCancer Immune profiling panel, as previously published (Vadakekolathu J et al., 2020). The correlation between transcriptomic features of IES, clinical characteristics, therapeutic response and patient outcome was validated using publicly available RNA-sequencing and NanoString data from 1,698 patients with AML, including samples from the TCGA-AML (n=147 cases), Beat-AML Master Trial (n=264 cases, of which 240 with survival data and 195 with chemotherapy response data) and Children's Oncology Group (COG)-TARGET AML series (n=145 cases).We initially showed that, compared with their non-senescent CD8 +CD57 -KLRG1 - counterpart, senescent CD8 +CD57 +KLRG1 + T cells are functionally impaired in terms of their ability to effect AML-blast killing mediated by an anti-CD33/CD3 bi-specific T-cell engager antibody construct (kindly provided by Amgen, USA; effector/target [E/T] ratio = 1:5). We then used gene set enrichment analysis (GSEA) to derive a transcriptomic signature of IES encompassing natural killer (NK)-cell and stem-like CD8 + T-cell markers, and showed that IES states correlate with lymphoid infiltration, adverse-risk molecular lesions (TP53 and RUNX1 mutations), experimental gene signatures of leukemia stemness (LSC17 score; Ng et al., 2016) and poor outcome in response to standard induction chemotherapy (Fig. 1A). In independent validation cohorts of children and adults with AML, the IES score was higher at baseline in patients with primary induction failure (following a standard 2 cycles of chemotherapy) compared with complete remission, increased in post-chemotherapy BM specimens, and predicted survival with greater accuracy than the ELN cytogenetic risk classifier (Fig. 1B). In the immunotherapy setting, high IES scores at baseline defined a checkpoint blockade-unresponsive AML tumor microenvironment and correlated with significantly shorter overall survival (9.1 versus 15.56 months in patients with high and low IES scores, respectively; HR = 3.32 (95% CI = 1.19-9.25); log-rank P = 0.021; Fig. 1C). Finally, the IES-related gene set also predicted for long-term outcomes and objective responses, based on RECIST criteria, to single-agent nivolumab or pembrolizumab, or combination anti-PD-1 + anti-CTLA-4, in 106 patients with melanoma (PRJEB23709 and GSE93157 series), a tumor type known to derive durable clinical benefit from ICB (Fig. 1D).Our findings encourage the pursuit of immune senescence reversal as a strategy to functionally reinvigorate T cells and could inform the delivery of ICB and other T cell-targeting immunotherapies to patients who are likely to benefit. [Display omitted] DisclosuresRadojcic: Syndax Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allakos: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding.

Recent updates on bispecific T-cell engager (BiTE) antibodies in relapsed and refractory multiple myeloma.

e20034 Background: Bispecific T-cell engager (BiTE) antibodies are currently being studied for various hematological malignancies including relapsed and refractory multiple myeloma (RRMM). BiTE antibodies that target CD3 on T cells and antigens GPRC5D, BCMA or FcRH5 on myeloma cells are being studied in RRMM patients (pts). Methods: A comprehensive literature search was done on Pubmed, Embase, and Cochrane. The data presented in various conferences (ASH, ASCO) were also included. Results: Six clinical trials are currently evaluating anti-BCMA, anti CD3 BiTE in RRMM. Madduri et al. reported ORR of 39% in 49 pts treated with REGN5458 an anti BCMA and CD3 BiTE antibody. Grafall et. al reported an ORR of 73% with Teclistamab at 1500µg/kg SC. Harrison et al. reported ORR of 83% in 9 pts treated with 9mg of AMG 701. Lesokhin et al. reported an ORR of 33% among 18 RRMM pts at all dose levels and ORR of 75%. at dose level of 215μg/kg weekly (n=4), and 360 μg/kg weekly (n=4). Rodriguez et al. reported an ORR of 80% among 15 RRMM pts treated with TNB-383B at the dose of 40-60mg IV every 3 weeks. Topp et al reported an ORR of 70% among 10 pts treated with AMG420/BI-836909 at MTD of 400µg/d. Chari et al reported results of Talquetamab which targets GPRC5D which showed an ORR of 66% in 55 evaluable pts. ORR at RP2D (405 µg/kg SC) was 69% in 19 pts treated at this dose. Cohen et al reported results on BFCR4350A, an anti FcRH5 BiTE which showed an ORR of 53% at dose ≥3.6/20mg Q3W. Cytokine release syndrome (CRS) is a serious side effect of BiTE antibodies and was reported in 39%, 64%, 65%, 61%, 45%, 38%, 54% and 76% in studies by Madduri et al, Grafall et al., Harrison et al., Lesokhin et al., Rodriguez et al., Topp et al., Chari et al., and Cohen et al., respectively. Most of the CRS cases were grade 1-2 and resolved either by themselves or required treatment with tocilizumab and steroids. Other most common side effects are reported in table. Conclusions: The most recent data on BiTE antibodies in RRMM has shown promising results with good tolerability. However, the trials are ongoing and a longer follow up is needed.[Table: see text]

Efficacy and preclinical safety of ARB202, a potential first-in-class anti-CDH17/CD3 bispecific T-cell engager, for treatment of pancreatic and colorectal cancers.

405 Background: Globally, gastrointestinal (GI) cancers, which account for around 28% of all cancers, has increased constantly at a CAGR of 2% (2014-2019), while the treatment options remain limited and often ineffective. This is largely due to late diagnosis and lack of new tumor associated antigens for most of the GI malignancies. Cadherin-17, CDH17 (aka CA17), is being evaluated as a potential therapeutic target and molecular marker for GI cancers, of which aberrant expression of CDH17 was reported in 50-90% in tumor samples (including stomach, pancreas, liver/bile duct and colon) by multiple groups. The present study aims to develop a humanized CDH17-CD3 bispecific T-cell engager antibody, ARB202, to treat advanced pancreatic and metastatic colon cancers. Methods: The ARB202 bispecific was generated from anti-CDH17 monoclonal ARB102 as previously described (Lum Y et al, Mol. Pharm. 2020) by linking a CD3-binding scFv in the format of IgG4-scFv. Biophysical properties, efficacy and safety were evaluated. Efficacy was addressed in cytotoxicity assays, IL2-release assays and subcutaneous xenograft models using multiple GI tumor target lines. Preclinical safety profiles include Tissue cross-reactivity assays and GLP repeat-dose toxicity studies in NHP. Results: The ARB202 bispecific antibody was efficiently produced (yield ~1 g/L) and remained stable for at least 3 months under accelerated stress tests. Binding affinity (Kd [M]) of ARB202 to CDH17 and CD3 was at 10-10 and 10-9 respectively, with cellular affinities of 10-9 and 10-8. In vitro cytotoxicity assays demonstrate that ARB202-mediated tumor cell killing is CDH17-dependent and requires immune synapse formation with effector T cells. By contrast, normal cells and CDH17-negative cancer cells are resistant. Pancreatic AsPC1 xenograft model revealed an ARB202 (co-inoculated with human PBMC) dose-dependent inhibition of tumor growth. Human IL-2 release in mice blood samples was consistent with functional CDH17 and CD3 engagement. ARB202 PK profile in rodents and NHP was found to be excellent relative to other bispecifics and GLP toxicity studies showed no significant findings nor adverse effects. Conclusions: ARB202 bispecific antibody promises to be a first-in-class biologic, which may tackle approximately 50% of GI cancers that which have been reported CDH17 positive. First-in-human clinical study with advanced GI cancers is scheduled to start in Q1 2021.

Fn14-targeted BiTE and CAR-T cells demonstrate potent preclinical activity against glioblastoma

ABSTRACT T cell-engaging therapies involving bispecific T cell engager (BiTE) and chimeric antigen receptor T (CAR-T) cells have achieved great success in the treatment of hematological tumors. However, the paucity of ideal cell surface molecules that can be targeted on glioblastoma (GBM) partially reduces the immunotherapeutic efficacy. Recently, high expression of Fn14 has been reported in several solid tumors, so the strategy of exploiting this specific antigen for GBM immunotherapy is worth studying. Consequently, we constructed Fn14× CD3 BiTE and Fn14-specific CAR-T cells and investigated their cytotoxic activity against GBM in vitro and in vivo. First, expression of Fn14 was confirmed in glioma tissues and GBM cells. Then, we designed Fn14-specific BiTE and CAR-T cells and tested their cytotoxicity in GBM cell cultures and mouse models of GBM. Fn14 was highly expressed in GBM tissues and cell lines, while it was undetectable in normal brain samples. Fn14× CD3 BiTE, Fn14 CAR-T cells and Fn14 CAR-T/IL-15 cells were antigen-specific and highly cytotoxic, showing good antitumor activity in vitro and causing significant regression of established solid tumors in xenograft models. However, the xenografts treated with Fn14 CAR-T cells regrew, whereas xenografts treated with Fn14 CAR-T/IL-15 cells did not. IL-15 engineering augmented the antitumor activity of Fn14 CAR-T cells and resulted in significant antitumor effects similar to those of Fn14× CD3 BiTE. Our results suggest that Fn14 is an appropriate target for GBM. Anti-Fn14 BiTE and Fn14-specific CAR-T/IL-15 cells may be exciting immunotherapeutic options for malignant brain cancer.

Safety and Efficacy of Blinatumomab As Monotherapy Vs Combination Therapy in the Treatment of Relapsed/Refractory Adult ALL: A Systematic Review

Background: Acute lymphoblastic leukemia (ALL) is rare but aggressive. For refractory and relapsed ALL (R/R ALL), various treatment strategies are adopted, of which the use of blinatumomab has shown promising results in recent decades. Blinatumomab is a CD-19 directed CD3 bispecific T-cell engager antibody that has been approved by the FDA for the treatment of both adults and pediatric ALL. We report the efficacy and toxicity of blinatumomab in relapsed and refractory ALL adult patients. Materials/Methods: Following the PRISMA guideline, we performed a comprehensive literature search PubMed, Embase, Cochrane Library, Web of Science, and Clinicaltrials.gov. We used the following keywords, "acute lymphocytic leukemia" and "Blinatumomab" from the inception of data till 06/17/2020. We screened 1199 articles and included 7 trials and 6 retrospective studies. We excluded all case reports, case series, preclinical trials, review articles, and meta-analysis. We extracted the data for efficacy (complete response, relapse, overall survival, etc.) and safety (≥Grade 3 adverse events). Results: A total of 1393 patients with relapsed/refractory ALL were included. Monotherapy: In phase 3 TOWER trial, a total of 405 patients with heavily pretreated B-cell precursor ALL were randomized in a 2:1 ratio to either receive blinatumomab (n=271) or standard of care (SOC) (N=134). The mean age of both groups was 40.8 years and 41.1, respectively. The dose of blinatumomab (9-28 ug/d) was instituted to the intervention group, whereas the control group received investigator-chosen chemotherapy regimens. The CR/CRi rate for both groups was 95/267 (35.5 %) and 27/134 (31.34%), respectively (p<0.01). The median overall survival was 7.7 (5.6 to 9.6 months) vs. 4.0 (2.9 to 5.3 months); the hazard rate for death: 0.71 (95% CI 0.55-0.93) p=0.01 and MRD negativity was 76 % and 48 %, respectively. The most common grade 3 and 4 adverse events (AEs) were neutropenia 45.7%) vs. 61.94% and cytopenia 67.80% vs. 81.34% respectively. In 6 phase 1b/2 studies (N=469), the starting dose of blinatumomab was 5 µg /m2/d and subsequently increased to 30 µg/m2/d. The pooled CR/CRi for these studies was 180/469 (38.37%). The most common hematological grade ≥3 adverse events were neutropenia 89/448 (19.87%), anemia 45/422 (10.70%), and thrombocytopenia 41/422 (9.7%). A total of 509 patients were included in 5 observational studies. The overall CR/CRi reported was 129/509 (25.34%). Grade ≥3 neutropenia and thrombocytopenia were reported as 14/52(26.92%) in one study.CRS was reported in another study as 5/14 (35 %). Combination regimens: In a phase II trial of Blinatumomab when given with a TKI (Dastanib), CR/CRi achieved was 27/47 (56.3%). Blinatumomab related neurotoxicity was observed in 2/12 (16.67%) patients. A total of 38 patients were included in 3 observational studies involving Blinatumomab and TKIs. The overall CR/CRi reported was 25/30(83.33%). In a multicenter phase I/II trial blinatumomab when given with PD-1 inhibitor pembrolizumab (pembro) achieved a CR/CRi of 2/4 (50%). In a phase II study, Blinatumomab and PD-1 and CTLA-4 inhibitors were used in combination in 8 patients. The CR/CRi achieved was 4/5 (80%). Grade ≥3 non-hematological adverse events (AEs) were observed in 1/5 (20%) patients. Blinatumomab has been used in combination with Hyper-CVAD in a phase II study. The ORR and CR achieved have been 14/14 (100%) and 13/14 (93%) respectively. In a recent study, blinatumomab has been used along with miniHCVD + INO in 17 patients. The CR/CRi achieved was 13/17 (76.47%). Conclusion: Blinatumomab alone and in combination with TKIs, PD-1 inhibitors, CTLA-4 inhibitors,hyper-CVAD and miniHCVD + INO were well tolerated by adult patients with acute lymphocytic leukemia. Blinatumomab in combination therapy has shown superior efficacy in adult ALL patients as compared to blinatumomab monotherapy. Additional multicenter randomized, double-blind clinical trials are needed to confirm these results. Disclosures Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.:Honoraria, Research Funding, Speakers Bureau.

Evaluating Blinatumomab for the Treatment of Relapsed/Refractory ALL: Design, Development, and Place in Therapy.

Although adults with B-cell acute lymphoblastic leukemia (B-ALL) achieve high complete remission (CR) rates following treatment with intensive multi-agent chemotherapy regimens, up to two-thirds of these patients eventually relapse. Unfortunately, adults with relapsed or refractory (R/R) B-ALL have a poor prognosis, with variable responses to salvage chemotherapy regimens and allogeneic stem cell transplant. As such, the need to develop effective and well-tolerated treatments for this patient population has been of paramount importance over the past decade. In this regard, treatment options for R/R B-ALL patients have expanded considerably over a relatively short period of time, with the approvals of blinatumomab, inotuzumab ozogamicin and tisagenlecleucel occurring within only the past six years. Blinatumomab, a CD19 x CD3 bispecific T-cell engager (BiTE) was the first of these immune therapies to receive approval, and for many patients, is used as first-line salvage therapy. A number of large clinical trials have demonstrated improved progression-free survival and overall survival for R/R B-ALL patients receiving blinatumomab as compared to those receiving conventional salvage chemotherapy. In addition to being approved for both Philadelphia chromosome-negative and Philadelphia chromosome-positive R/R B-ALL, blinatumomab is also the only ALL therapy that carries approval for the treatment of measurable residual disease (MRD). Although blinatumomab has changed the therapeutic landscape for adults with R/R B-ALL, a number of important clinical considerations and questions remain, including the potential role of blinatumomab in the frontline setting, mechanisms of resistance, optimal goal MRD level, the role of transplant following MRD clearance, the optimal place for blinatumomab in the context of other recently approved immune-mediated therapies, and real world outcomes for patients treated outside the context of clinical trials. These issues are the focus of ongoing studies, which will hopefully inform future clinical practice regarding the utility of blinatumomab in the treatment of B-ALL patients.

Phase I study of pasotuxizumab (AMG 212/BAY 2010112), a PSMA-targeting BiTE (Bispecific T-cell Engager) immune therapy for metastatic castration-resistant prostate cancer (mCRPC).

124 Background: mCRPC has a poor prognosis and immunotherapies are largely ineffective. PSMA is a promising therapeutic target in mCRPC. Pasotuxizumab is a PSMA x CD3 BiTE immune therapy that mediates killing of tumor cells by T cells. Methods: NCT01723475 was a first-in-human, multicenter, dose-escalation study in patients (pts) with mCRPC refractory to standard therapy. Pts received continuous IV infusion of pasotuxizumab in cohorts of 3–4 pts. Dose-escalation followed a continuous reassessment methodology. The primary objective was to determine safety and maximum tolerated dose (MTD); secondary objectives included pharmacokinetics, biomarkers, and tumor response. Results: 16 pts were enrolled into 5 dosing cohorts (5 µg/d, n=3; 10 µg/d, n=4; 20 µg/d, n=3; 40 µg/d, n=4; 80 µg/d, n=2). All pts had ≥1 AE of any grade; most common were fever (94%), chills (69%), and fatigue (50%). 13 pts (81%) had ≥1 AE of grade ≥3; most common were decreased lymphocytes and infections (both 44%). No grade 5 AE occurred. A serious drug-related AE was reported for 1 pt (fatigue, 20 µg/d). No antidrug antibodies were observed. Recruitment was stopped before MTD was reached to allow initiation of a new study sponsored by Amgen. Antitumor activity as indicated by PSA serum level decline was dose dependent, with a mean best PSA change per dosing cohort vs baseline of +0.74% (5 µg/d), −17.9% (10 µg/d), −37.4% (20 µg/d), −42.5% (40 µg/d) and −54.9% (80 µg/d). PSA decreases ≥50% occurred in 3 pts (n=1 each in 20 µg/d, 40 µg/d, 80 µg/d cohorts). One long-term PSA responder was treated for 14 months (40 µg/d) and one for 19.4 months (80 µg/d). The latter pt showed a complete regression of soft-tissue metastases and marked regression of bone metastases by PSMA-PET/CT, >90% reduction in PSA and alkaline phosphatase, and a significant and durable improvement in disease related symptoms. Conclusions: Pasotuxizumab had an acceptable safety profile and dose-dependent clinical activity in mCRPC pts. There were two long term responders in the dose escalation. This is the first clinical study showing that a BiTE immune therapy can be efficacious in solid tumors. Clinical trial information: NCT01723475.

Phase 1 study of pasotuxizumab (BAY 2010112), a PSMA-targeting Bispecific T cell Engager (BiTE) immunotherapy for metastatic castration-resistant prostate cancer (mCRPC).

5034 Background: mCRPC has a poor prognosis and immunotherapies are largely ineffective. PSMA is a promising therapeutic target in mCRPC, and pasotuxizumab is a PSMA x CD3 BiTE that mediates tumor cell killing. Methods: NCT01723475 was a first-in-human, multicenter, dose-escalation study in patients (pts) with mCRPC refractory to standard therapy. Pts received pasotuxizumab as a continuous intravenous infusion in cohorts of 3–4 pts. Dose-escalation followed a continuous reassessment methodology design. The primary objective was to determine safety and maximum tolerated dose (MTD); secondary objectives included pharmacokinetics, biomarkers, and tumor response. Results: 16 pts were enrolled into 5 dosing cohorts (5 µg/d, n = 3; 10 µg/d, n = 4; 20 µg/d, n = 3; 40 µg/d, n = 4; 80 µg/d, n = 2). All pts had ≥1 AE of any grade; most common were fever (94%), chills (69%), and fatigue (50%). 13 pts (81%) had ≥1 AE of grade ≥3; most common were decreased lymphocytes and infections (both 44%). No grade 5 AE occurred. A serious AE related to study drug was reported for 1 pt (fatigue, 20 µg/d). No anti-drug antibodies were observed. Recruitment was stopped before MTD was reached to facilitate initiation of a new study sponsored by Amgen. Antitumor activity as indicated by PSA serum level decline was dose dependent, with a mean best PSA change per dosing cohort versus baseline of +0.74% (5 µg/d), –17.9% (10 µg/d), –37.4% (20 µg/d), –42.5% (40 µg/d) and –54.9% (80 µg/d). PSA decreases of ≥50% occurred in 3 pts (n = 1 each in 20 µg/d, 40 µg/d, and 80 µg/d cohorts). One long-term PSA responder was treated for 14 months (40 µg/d) and one for 19.4 months (80 µg/d). The latter pt showed a complete regression of soft-tissue metastases and marked regression of bone metastases as assessed by PSMA-PET/CT, > 90% reduction in PSA and alkaline phosphatase, and a significant and durable improvement in disease related symptoms. Conclusions: Pasotuxizumab had an acceptable safety profile and dose-dependent clinical activity in mCRPC pts. There were two long term responders in the dose escalation. This is the first clinical study showing that a BiTE immunotherapy can be efficacious in solid tumors. Clinical trial information: NCT01723475.

Benefit-Risk Assessment of Blinatumomab in the Treatment of Relapsed/Refractory B-Cell Precursor Acute Lymphoblastic Leukemia.

Blinatumomab is the first-and-only Food and Drug Administration (FDA)-approved cluster of differentiation (CD) 19-directed CD3 bispecific T-cell engager (BiTE®) immunotherapy. It is currently FDA approved for the treatment of adults and children with Philadelphia chromosome-positive (Ph+) and Philadelphia chromosome-negative (Ph−) relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukemia (ALL) and B-cell precursor ALL with minimal residual disease. Similarly, initial marketing authorization for blinatumomab in the European Union was granted for the treatment of adults with Ph− R/R B-cell precursor ALL. The benefits of treating R/R B-cell precursor ALL patients with blinatumomab include increased overall survival, more favorable hematologic remission and molecular response rates, and a lower incidence rate of selected adverse events when compared with standard-of-care chemotherapy. The key risks associated with blinatumomab treatment include cytokine release syndrome, neurotoxicity, and medication errors. Here, we review the benefits and risks of blinatumomab treatment and describe how these risks can be mitigated.

TIM-3 Expression on CD4+ Bone Marrow T Cells Predicts Relapse of Pediatric B-Precursor Acute Lymphoblastic Leukemia

IntroductionPediatric acute lymphoblastic leukemia (ALL) is a cancer entity of minimal mutational load and low immunogenicity. The interaction of ALL cells with bone marrow (BM) T cells has not been investigated as a pathogenic driver or prognostic marker for pediatric ALL. We defined BM T cells of pediatric ALL patients as tumor-infiltrating lymphocytes (TILs) and investigated the prognostic relevance of co-stimulatory and co-inhibitory signals between ALL and BM T cells.MethodsBM samples of 100 pediatric ALL patients were analyzed at time of initial diagnosis. T-cell subpopulations and expression of co-stimulatory and co-inhibitory molecules were defined by flow cytometry and correlated with clinical outcome of the patients. To investigate the role of TIM-3 for the interaction between T cells and leukemic cells, CRISPR/Cas9-mediated TIM-3 knockout (KO) was performed in primary T cells by ribonucleoprotein electroporation. T-cell activation and proliferation after contact with leukemic target cells were analyzed in TIM-3 KO cells and compared to wildtype T cells and T cells with retroviral TIM-3 overexpression. Interaction of T cells with leukemic target cells was induced by addition of anti-CD19/-CD3 bispecific T-cell engager (BiTE). Fold change (FC) of T-cell activation and proliferation was analyzed before and after co-culture. BM expression levels of known TIM-3 inducers were identified by RNA next generation sequencing of the bone marrow samples.ResultsMultivariate analyses identified high TIM-3 expression on CD4+ BM T cells at initial diagnosis as strong predictor for relapse of pediatric acute lymphoblastic leukemia (relapse free survival (RFS) 94.6% vs. 70.3%). The risk to develop ALL relapse was 7.1-fold higher in the group of TIM-3 high expressing patients (n=37) compared to TIM-3 low expressing patients (n=37). Expression levels of known TIM-3 ligands and inducers in the bone marrow of the patients were analyzed by RNA next generation sequencing and compared between patients with high TIM-3 expression (n=12) and low TIM-3 expression (n=15) on BM T cells. Presence of known TIM-3 ligands HMGB1 (High-Mobility-Group-Protein B1) and Galectin-9 was confirmed, but expression levels did not show significant differences. Known TIM-3 inducers IL-2, -7, -15 and -21 were not expressed on RNA level indicating that another mechanism must be responsible for TIM-3 overexpression. In vitro experiments showed that the interaction with leukemic cells induces TIM-3 expression on the surface of T cells (mean TIM-3 expression 51.1% vs. 29.7% on T cells with vs. without addition of leukemic cells, n=3). To investigate the functional relevance of TIM-3 expression in pediatric leukemia, TIM-3 KO and overexpression was performed on primary T cells. TIM-3 KO T cells showed higher activation levels after co-culture with leukemic cell lines plus CD3-/CD19-specific BiTE compared to wildtype (WT) T cells (FC of CD69 surface expression 5.0 vs. 3.2, n=3). FC of anti-leukemic proliferation was impaired in TIM-3 overexpressing T cells compared to WT T cells (FC 1.6 vs. 2.3, n=3) whereas TIM-3 KO T cells showed a higher proliferation FC compared to controls (FC 6.5 vs. 2.4, n=3).ConclusionsOur study identifies TIM-3 expression on CD4+ bone marrow T cells at initial diagnosis as a strong predictor for pediatric ALL relapse. TIM-3 expression is induced by interaction of T cells with leukemic cells and results in impaired anti-leukemic T-cell activation and proliferation. TIM-3-mediated T-cell inhibition represents a new mechanism of impaired immune surveillance in pediatric ALL and blockade of this axis may be of importance for future immunotherapy in ALL. DisclosuresNo relevant conflicts of interest to declare.

Resistance to anti-CD19/CD3 BiTE in acute lymphoblastic leukemia may be mediated by disrupted CD19 membrane trafficking

AbstractThe CD19 antigen is a promising target for immunotherapy of acute lymphoblastic leukemia (ALL), but CD19− relapses remain a major challenge in about 10% to 20% of patients. Here, we analyzed 4 CD19− ALL relapses after treatment with the CD19/CD3 bispecific T-cell engager (BiTE) blinatumomab. Three were on-drug relapses, with the CD19− escape variant first detected after only 2 treatment courses. In 1 patient, the CD19− clone appeared as a late relapse 19 months after completion of blinatumomab treatment. All 4 cases showed a cellular phenotype identical to the primary diagnosis except for CD19 negativity. This argued strongly in favor of an isolated molecular event and against a common lymphoid CD19− progenitor cell or myeloid lineage shift driving resistance. A thorough molecular workup of 1 of the cases with early relapse confirmed this hypothesis by revealing a disrupted CD19 membrane export in the post–endoplasmic reticulum compartment as molecular basis for blinatumomab resistance.

T-cell redirected killing and cure of pancreatic and gastric cancer xenografts by targeting Trop-2.

262 Background: Trop-2 [also called tumor-associated calcium signal transducer 2 (TACSTD2), EGP-1 (epithelial glycoprotein-1), GA733-1, or M1S1]is a 35 kDa transmembrane glycoprotein that is overexpressed relative to normal tissues in a variety of human cancers, including pancreatic and gastric carcinomas, where increased expression correlates with poor prognosis. Trop-2 appears to be more tumor-specific than the related molecule, EpCAM (Trop-1). MT110, the EpCAM antibody x CD3 bispecific T-cell engager (BiTE), is currently undergoing a Phase I study in various solid tumors, including lung, gastric, colorectal, breast, prostate, and ovarian cancers. We produced a similar T-cell redirecting bispecific tandem scFv, E1-3, using the variable domains of hRS7 (humanized anti-Trop-2 mAb) and Okt-3 (anti-CD3 mAb). Methods: T-cell activation, cytokine induction and cytotoxicity were evaluated ex vivo using PBMCs or purified T cells with human pancreatic (Capan-1 and BxPC3) and gastric (NCI-N87) cancer cell lines as target cells. In vivo activity was assayed with NCI-N87 xenografts that were inoculated s.c. in a mixture with twice the number of human PBMCs and matrigel. Results: In the presence of target cells and PBMCs, E1-3 potently induced T-cell activation, proliferation, and dose-dependent cytokine production of IL-2 (>2 ng/mL), IL-6 (>1 ng/mL), IL-10 (>7 ng/mL), TNF-α (>1 ng/mL) and IFN-γ (>50 ng/mL). In vitro, E1-3 mediated a highly potent T-cell lysis of BxPC3 [IC50=0.09(±0.04) pM], Capan-1 [IC50=1.2(±1.1) pM] and NCI-N87 [IC50=1.2(±1.2) pM] target cells. In vivo, two 50-µg doses of E1-3 given three days apart cured all of the mice (N=8) bearing NCI-N87 xenografts (P=0.0005; Log-Rank). Tumors in the control group (PBMCs only) reached the endpoint (TV>1 cm3) with a median of 39.5 days. All mice remained tumor-free in the E1-3 group at 78 days. Conclusions: Trop-2 is an attractive target for T-cell-mediated killing of pancreatic, gastric and other epithelial cancers.