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AMG 794 is a half-life extended BiTE® immune therapy targeting the oncofetal antigen Claudin 6 (CLDN6). AMG 794 redirects T cells to kill CLDN6-expressing tumor cells and is being developed for the treatment of non-small cell lung cancer (NSCLC) and epithelial ovarian cancer (EOC). CLDN6 is a compelling tumor antigen that is expressed during embryonic and fetal development, transcriptionally silenced in adult tissues, and re-expressed on the surface of NSCLC and EOC cells. By immunohistochemistry, CLDN6 staining of the cell membrane was observed in 27% of non-squamous NSCLC (n = 63) and 69% of EOC (n = 92) samples, the majority of which were of the high-grade serous ovarian cancer subtype. Expression of CLDN6 protein was not detected in most normal adult tissues, with rare CLDN6 immunostaining limited to individual cells in the pituitary, pancreas, small intestine, kidney, and female reproductive organs. AMG 794 is a fully human BiTE® molecule that binds both human and cynomolgus monkey CLDN6 and CD3. AMG 794 binds human CLDN6 and CD3 with equilibrium dissociation constant (KD) of 13 nM and 36 nM, respectively. In vitro, AMG 794 redirects human T cells to kill CLDN6-expressing cancer cells with a half-maximal lysis concentration (EC50) of 2.6 ± 1.1 pM to 127.4 ± 53.4 pM. Consistent with the mechanism of action of BiTE® immune therapy, AMG 794 induces T cell activation and transient production of cytokines in co-cultures of T cells and CLDN6-expressing tumor cells. Remarkably, AMG 794 binding and cytotoxic activity is selective for CLDN6 over other claudin family proteins, despite high homology in the extracellular loops with CLDN9. Weekly dosing of AMG 794 significantly inhibited the growth of established lung and ovarian xenograft tumors in immunocompromised mice injected with human T cells. Anti-tumor activity was associated with an increase in tumor-infiltrating T cells. AMG 794 was well tolerated in a one-month repeat-dose toxicology study in cynomolgus monkey, with evidence for target engagement. The potent, selective activity of AMG 794 for CLDN6-expressing NSCLC and EOC cells, together with an acceptable nonclinical safety profile, supported the advancement of AMG 794 into clinical development. A first-in-human study to explore the safety, tolerability, pharmacokinetics, and anti-tumor activity of AMG 794 in patients with CLDN6-positive advanced/metastatic non-squamous NSCLC or EOC will be enrolling patients in March 2022. Citation Format: Elizabeth Pham, Anja Henn, Beate Sable, Joachim Wahl, Kip Conner, Katja Matthes, Shivani Gupta, Rodolfo Yabut, Famke Aeffner, Kristin Lewis Wilson, Jonas Anlahr, Christoph Dahlhoff, Vijay Kale, Matthias Friedrich, Tobias Raum, Peter Kufer, Angela Coxon, Sabine Stienen, Julie M. Bailis. AMG 794, a Claudin 6-targeted half-life extended (HLE) bispecific T cell engager (BITE®) molecule for non-small cell lung cancer and epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5202.

Multiple myeloma (MM) is a cancer of the antibody-producing plasma cells (PC). MM is invariably fatal due to frequent disease relapse and/or treatment refractoriness, and therapies that provide deeper and more durable responses are needed. BiTE® (Bispecific T Cell Engager) molecules are immunotherapy agents that redirect a patient’s T cells to lyse tumor cells by simultaneously engaging a tumor associated antigen (TAA) on cancer cells and CD3ε on T cells. Clinical activity has been observed in MM using BiTE® molecules and other T cell engagers that target B-cell maturation antigen (BCMA). BCMA is an attractive MM TAA due to its broad prevalence and restricted normal tissue profile, mainly PCs and plasmablasts. However, heterogenous expression in MM cancer cells, potential antigen loss, and the presence of high levels of soluble BCMA in MM patient sera present challenges that may prevent BCMA-only-targeted therapies from achieving their full potential. To address these issues, we generated a BiTE® molecule capable of engaging BCMA and a second MM TAA, CS1 (also known as SLAMF7). This BiTE® molecule also contains an Fc-based domain to provide half-life extension (HLE). Here, we evaluated the in vitro and in vivo properties of this BCMA-CS1 HLE BiTE® molecule. The BCMA-CS1 HLE BiTE® molecule had nanomolar binding affinity for human BCMA, CS1, and CD3ε and the non-human primate (NHP) orthologues. In vitro, we observed picomolar activity against human MM cell lines with a range of BCMA and CS1 expression in a T cell dependent cellular cytotoxicity (TDCC) assay using human T cells or NHP peripheral blood mononuclear cells. The BCMA-CS1 HLE BiTE® molecule retained TDCC activity in the presence of soluble BCMA up to 2500 ng/mL as well as against human MM cells engineered to express only BCMA or CS1. In vivo, this BiTE® molecule inhibited tumor growth in a MM xenograft model. We also evaluated the BCMA-CS1 HLE BiTE® molecule in NHP over 15 days (IV dosing; intra-animal dose escalation from 60→240 μg/kg or 180→540 μg/kg on days 1 and 8). We observed hallmarks of BiTE® molecule activity in all groups, including transient decreases in circulating lymphocytes and moderate increases in cytokines like MCP-1. We measured the PC-specific transcripts BCMA and J-chain in NHP bone marrow and blood as surrogates for PC levels using ddPCR. These transcripts were reduced in both treatment groups (≥90%) with the strongest effects occurring in the 180→540 μg/kg group. Lastly, we showed that CS1 is highly and broadly expressed in MM patient samples and is restricted to a few normal hematopoietic cell types including PCs, NK cells, T cells, and some monocytes. These data suggest that the BCMA-CS1 HLE BiTE® molecule has potent in vitro and in vivo activity and may provide therapeutic benefit for MM patients by expanding the population of MM cancer cells that can be eliminated by a BiTE® molecule while overcoming common mechanisms that can impair BCMA-only-targeted MM therapies. Citation Format: Elizabeth T. Andrews, Stephanie C. Casey, Mohammad Farhad Amani, Grit Lorenczewski, Mozhgan Farshbaf, Lisa Winkel, Matthias Klinger, John M. Harrold, Famke Aeffner, Ana Goyos, Matthias Friedrich, Tara Arvedson, Matthew G. Chun. Evaluation of a dual-targeting BCMA-CS1 HLE BiTE® molecule for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6251.

Acute myeloid leukemia is a grievous illness. BiTE® (bispecific T cell engager) molecules redirect T cells by engaging CD3 and a tumor-associated antigen (TAA). These molecules have shown clinical efficacy but one mechanism of resistance is loss of a single TAA. We hypothesized that a BiTE® molecule targeting >1 TAA could reduce relapse frequency. FLT3 and CD123, were selected for a dual-targeting BiTE® (dBiTE࣪) molecule.A half-life extended (HLE) CD123-FLT3 dBiTE࣪ molecule was evaluated in vitro, in mouse xenografts, and in non-human primate (NHP) tolerability studies. The molecule had nM affinity for human and NHP FLT3, CD123, and CD3, and pM efficacy in cytotoxicity assays using human T cells or NHP PBMCs. The molecule achieved 100% killing against single-positive (sp) cells (CRISPR-generated isogenic cell lines) at potencies like those of double-positive (dp) cells (7.4 ±. 4.1 pM FLT3sp, 7.1 ±. 3.8 pM CD123sp and 3.4 ±. 1.5 pM dp, n=3). In a mouse xenograft model, the molecule induced significant activity and extended survival > 3 weeks (1.0, 0.1, and 0.01 mg/kg (p ≤ 0.001)). In mice with sp tumors, survival benefit could not be calculated due to high survival, demonstrating that both arms of the molecule are active. In NHP, the molecule had a half-life of 52 hours (0.3 or 3 µg/kg). FLT3 mRNA levels, a marker of FLT3-expressing cells, decreased in blood following dosing. Repeat dosing was not tolerated, and cytokine release was observed. Some cytokines were reduced while others increased. CD123 is reportedly expressed on endothelial cells (ECs), with increased expression in inflammatory conditions. An immunohistochemical survey found that CD123 is expressed on human and NHP monocyte/macrophages and ECs with limited distribution in lymphoid tissues and lamina propria of the gut. We hypothesized that cytokine release following administration may result in increased CD123 expression and in turn, further increased cytokine levels with repeat dosing. CD123 was detected on primary human umbilical vein ECs (HUVECs). In co-cultures of HUVECs and T cells, a CD123 BiTE® molecule induced expression of CD123 on HUVEC cells at concentrations that elicited redirected lysis (12.5 pM), T cell activation, and cytokine secretion. To better understand BiTE®-induced upregulation of CD123 on ECs, HUVECs were cultured with supernatants (SN) from a TDCC assay or recombinant IL-3, IL-6, TNFα, or IFNγ. Assay SN and TNFα induced >2-fold CD123 expression on HUVECs, but not on CD123-negative primary human pulmonary microvascular ECs. These data demonstrate that CD123 expression on ECs was increased upon exposure to a CD123 mono-targeting BiTE® molecule, potentially through BiTE®-induced secretion of TNFα. Additional studies are ongoing.In sum, a CD123-FLT3 HLE dBiTE࣪ molecule was active against both dp- and sp-positive target cells in vitro and in vivo. Careful selection of TAA for dBiTE࣪ molecules is necessary to increase efficacy and maintain safety. Citation Format: Rebecca Goldstein, Christine Karbowski, Anja Henn, Petra Deegen, Joachim Wahl, Katja Matthes, Christoph Dahlhoff, Brooke Rock, Sabrina Benchaar, Katie Hsu, Brandy Alexander, Matthias Friedrich, Joan Lane, Xiaoting Wang, Jonas Anlahr, Markus Muenz, Tobias Raum, Tara L. Arvedson. Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6313.

BiTE® (bispecific T cell engager) immune therapy consists of two connected single-chain variable fragments, one targeting T-cells via CD3, and one targeting the tumor via a tumor-associated antigen. Given their short half-life, these molecules are administered by continuous intravenous (iv) infusion to ensure tumor accumulation. To extend plasma half-life, they are conjugated to an Fc domain, resulting in a molecule with an extended pharmacokinetic profile. To evaluate their biodistribution in a syngeneic mouse model, a molecule targeting murine mesothelin and murine CD3 (muMSLN HLE BiTE®), and a non-targeting control HLE BiTE® molecule were radiolabeled with the positron emission tomography (PET) isotope zirconium-89 (89Zr). METHODS: The control and muMSLN HLE BiTE® were labeled with 89Zr. Immunocompetent BALB/c mice were engrafted in the lower mammary fat pad with mesothelin-positive murine mammary carcinoma cell line 4T1. PET imaging was performed at 1, 3, 5, 7 and 9 days after 50 µg 89Zr-muMSLN HLE BiTE® was administered by iv infusion (n = 8). Next, biodistribution of 10 µg (n = 6), 50 µg (n = 6) and 200 µg 89Zr-muMSLN HLE BiTE® (n = 5) and 50 µg 89Zr-control HLE BiTE® (n = 6) was compared via PET imaging on day 1, 3 and 5. PET-scans were quantified by mean standardized uptake value (SUVmean). Tracer injections were 4 MBq. To compare uptake in multiple groups, an analysis of variance followed by a post-hoc Tukey's multiple comparison test was performed. Values are expressed as mean ± standard deviation. RESULTS: PET imaging following 50 µg 89Zr-muMSLN HLE BiTE® dosed iv revealed a blood elimination half-life of 63.4 hours. Uptake in tumor, spleen, thymus and liver were visible from 3 days post injection (pi). On day 5, tumor uptake was highest (SUVmean = 1.50 ± 0.2) with a tumor to blood ratio of 1.9 ± 0.3 and a spleen to blood of 1.7 ± 0.2. Heart SUVmean 5 days pi revealed that 10 μg 89Zr-muMSLN HLE cleared faster from the blood (0.5 ± 0.1) than the 50 μg (0.8 ± 0.1, P < 0.01) and the 200 μg dose group (0.8 ± 0.1, P < 0.01). Tumor SUVmean was higher in 50 µg (1.5 ± 0.2) than the 10 µg dose group (1.2 ± 0.1, P < 0.01), but similar to the 200 μg dose group (1.3 ± 0.1, P = 0.28). Spleen uptake 5 days pi was dose-dependent (SUVmean: 10 µg = 1.6 ± 0.2; 50 µg = 1.3 ± 0.1; 200 µg = 0.8 ± 0.1, P < 0.01). Spleen and tumor SUVmean 5 days pi of 50 µg 89Zr-muMSLN HLE BiTE® were higher than 50 µg 89Zr-control HLE BiTE® (spleen = 0.5 ± 0.1, P < 0.01; tumor = 0.8 ± 0.1, P < 0.01), while blood SUVmean of 50 µg 89Zr-control HLE BiTE® was similar (0.6 ± 0.1, P = 0.12). CONCLUSION: PET imaging with a 89Zr-labeled muMSLN HLE BiTE® in a tumor-bearing syngeneic mouse model revealed a long blood half-life, specific uptake in tumor and spleen, and dose-dependent pharmacokinetics. Taken together, the HLE BiTE® molecule demonstrates an extended pharmacokinetic profile over previously evaluated small, canonical, BiTE® molecules. Citation Format: Frans V. Suurs, Grit Lorenczewski, Julie M. Bailis, Sabine Stienen, Matthias Friedrich, Elisabeth G.E. de Vries, Derk Jan A. de Groot, Marjolijn N. Lub-de Hooge. PET imaging shows dose-dependent pharmacokinetics of a 89Zr-labeled mesothelin/CD3 half-life extended bispecific T-cell engager molecule in a syngeneic mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2769.

BACKGROUND Bispecific T-cell engagers (BiTE®) harness the immune system against cancer. BiTE® antibody constructs are small proteins of ~53 kDa existing of two connected single-chain variable fragments. MuS110, a BiTE® with affinity for murine CD3 (KD= 2.9 nM) and murine EpCAM (KD= 21 nM), was radiolabeled with positron emission tomography (PET) isotope zirconium-89 (89Zr) to study its pharmacokinetics and involvement of the immune system in an immunocompetent mouse model bearing a syngeneic tumor. METHODS MuS110 and two control BiTE® antibody constructs (hyS110 and AMG110) were radiolabeled with 89Zr. HyS110 has affinity for murine CD3 and human EpCAM, AMG110 for human CD3 and human EpCAM. The human and murine variants of EpCAM and CD3 are not cross-reactive. Either immunocompetent BALB/c or immunodeficient nude BALB/c mice were all orthotopically engrafted in the lower mammary fat pad with EpCAM-positive murine breast cancer cell line 4T1. PET-imaging was performed at 0.5, 3, 6, 24, 48 and 72 h after intravenous (iv) administration of 10 µg 89Zr-muS110. Distribution of 10 µg 89Zr-muS110, 89Zr-hyS110 and 89Zr-AMG110 was assessed 24 h after administration in BALB/c and nude BALB/c mice with PET-imaging and ex-vivo biodistribution. In addition, a group of BALB/c mice received 10 µg muS110 iv daily for 5 days followed by 10 µg 89Zr-muS110 or 10 µg 89Zr-AMG110 and PET-imaging 24 h after tracer administration. Values are expressed as median (interquartile range). RESULTS PET-imaging revealed fast renal clearance of 89Zr-muS110 in the BALB/c mice resulting in a blood half-life of 0.93 h (one-phase decay). Tumor- and spleen-to-blood ratios increased to 3.7 (3.0 to 4.5) and 9.3 (7.5 to 11.1) after 72 h. Ex-vivo biodistribution 24 h after tracer administration showed 89Zr-muS110 and 89Zr-hyS110 accumulation in the spleen with 8.2 % injected dose per gram (ID/g) and 8.7 %ID/g, in comparison to 89Zr-AMG110 (2.3 %ID/g; vs 89Zr-muS110 P0.05). In nude BALB/c mice spleen and mesLN uptake of 89Zr-muS110 was lower than in BALB/c mice (spleen: 3.4 vs 8.2 %ID/g, P0.05). 89Zr-muS110 uptake was lower in spleen and mesLNs following 5 days of 10 µg muS110 iv compared to control mice (spleen: 4.2 vs 8.2 %ID/g, P CONCLUSION Distribution of BiTE® 89Zr-muS110 is predominantly mediated by the affinity for CD3, resulting in uptake in lymphoid tissues. Citation Format: Frans V. Suurs, Derk J. de Groot, Urszula M. Domanska, Grit Lorenczewski, Sabine Stienen, Matthias Friedrich, Elisabeth G. de Vries, Marjolijn N. Lub-de Hooge. PET-imaging of 89Zr-labeled bispecific T-cell engagers in syngeneic tumor bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1134.

Oncostatin M has been characterized as a potent growth inhibitor for various tumor cells. Oncostatin M–treated glioblastoma cells cease proliferation and instigate astrocytal differentiation. The oncostatin M–induced cell cycle arrest in G1 phase is characterized by increased level of the cyclin-dependent kinase (CDK) inhibitory proteins p21Cip1/Waf1/Sdi1 and p27Kip1. Induction of p21 protein corresponds to increased mRNA level, whereas p27 accumulates due to increased stability of the protein. Interestingly, stabilization of p27Kip1 occurs even in S phase, showing that p27 stabilization is a direct consequence of oncostatin M signaling and not a result of the cell cycle arrest. Degradation of p27 in late G1 and S phase is initiated by the ubiquitin ligase complex SCF-Skp2/Cks1. Oncostatin M inhibits expression of two components of this E3 ligase complex (Skp2 and Cks1). Although combined overexpression of Skp2 and Cks1 rescues p27 degradation in S phase, it can not override p27 accumulation in G1 phase and cell cycle arrest by oncostatin M. In addition to increasing Cdk inhibitor level, oncostatin M also impairs cyclin A expression. Cyclin A mRNA and protein level decline shortly after oncostatin M addition. The accumulation of two CDK inhibitor proteins and the repression of cyclin A expression may explain the broad and potent antiproliferative effect of the cytokine. (Cancer Res 2006; 66(13): 6530-9)

T cell engaging bispecific antibody constructs (BiTE®), such as blinatumomab which targets CD19-positive cells, have shown great promise for treating certain CD19-positive hematological malignancies. Blinatumomab comprises a single chain Fv (scFv) that binds CD19 and a scFv that targets the T cell CD3 protein. The molecular weight of this “canonical” BiTE® is ~ 55 kDa, making it susceptible to kidney-mediated clearance and resulting in a short serum half-life (~ 4 hours). To maintain effective serum concentrations, canonical BiTE® antibody constructs must be administered by continuous IV (cIV) infusion. While there are many advantages associated with cIV administration (e.g., safety and uniform PK profile), patient convenience could be enhanced if the BiTE® antibody construct were compatible with once-weekly administration. To achieve this, the serum half-life of the BiTE® antibody construct would need to be extended. A canonical BiTE® targeting CD33 (AMG 330) is currently being evaluated in a phase I clinical trial. Like blinatumomab, AMG 330 is dosed cIV. To extend the serum half-life of AMG 330 and enable once-weekly dosing, several approaches were evaluated including fusion of AMG 330 to human albumin and Fc-containing moieties. Each of these half-life extended (HLE) constructs was evaluated in vitro, in mouse xenograft models and in non-human primates. In vitro assays evaluated 1) binding to both human and cynomolgus CD33 and CD3 proteins, and 2) cytotoxicity using human and cynomolgus target and effector cells. In each of these assays the canonical and HLE BiTE® antibody constructs demonstrated similar activity: single-digit nM binding and single digit pM cytotoxicity. Canonical and HLE BiTE® antibody constructs were subsequently evaluated in an orthotopic mouse model in which MOLM13 cells were administered IV and activated human T cells were administered IP two days later. The Fc-based HLE BiTE® antibody constructs provided a similar survival advantage when administered Q4D or Q5D as the canonical BiTE® when administered QD. However, the albumin fusion–based HLE BiTE® was less efficacious when administered Q4D than the QD- administered canonical BiTE®. Lastly, the PK/PD relationship was evaluated for each of the constructs in non-human primates. The serum half-lives varied from 6 hours for the canonical BiTE® to 44-167 hours for the HLE BiTE® antibody constructs. Each of the HLE BiTE® antibody constructs showed on-target depletion of CD33-positive monocytes and neutrophils in the blood and depletion of CD33-positive cells in the bone marrow. These data demonstrate that half-life extended BiTE® antibody constructs can be generated that retain comparable in vitro and in vivo activity as a canonical BiTE® and achieve a serum half-life compatible with once weekly dosing. Citation Format: Tara L. Arvedson, Mercedesz Balazs, Pamela Bogner, Kurt Black, Kevin Graham, Anja Henn, Matthias Friedrich, Patrick Hoffmann, Roman Kischel, Peter Kufer, Ralf Lutterbuese, Markus Muenz, Tobias Raum, Benno Rattel, Karen Rex, Dan Rock, Oliver Thomas, Joachim Wahl, Andreas Wolf, Angela Coxon. Generation of half-life extended anti-CD33 BiTE® antibody constructs compatible with once-weekly dosing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 55. doi:10.1158/1538-7445.AM2017-55

Bispecific T cell engager (BiTE®) antibody constructs redirect T cells to induce lysis of tumor cells. The anti-CD19/CD3 BiTE® Blincyto® can deplete target cells in both the blood and tissue compartment in B cell malignancies, suggesting that the BiTE® mechanism of action will also be effective against solid tumors. The tumor differentiation antigen mesothelin (MSLN) is an attractive target for the BiTE® approach. MSLN is highly expressed in >80% of ovarian and pancreatic tumors and mesothelioma. Expression of MSLN in normal tissues appears restricted to mesothelial cell surfaces such as the pleural, pericardial, and peritoneal layer. Here, we report the preclinical characterization of an anti-MSLN/CD3 BiTE® antibody construct with extended half-life. The BiTE® antibody construct binds MSLN and CD3 with low nM affinity and has low pM cytotoxic activity against MSLN-positive ovarian, pancreatic and lung cancer cell lines in vitro. Activity of the anti-MSLN/CD3 BiTE® antibody construct is observed at low effector to target cell ratios, and in cancer cells that express low levels of MSLN. This BiTE® antibody construct is also cytotoxic to cell lines that are resistant to chemotherapy. In mice, significant tumor growth inhibition of an established ovarian tumor xenograft model was achieved by IV dosing of the anti-MSLN/CD3 BiTE® antibody construct every 5 days. Pharmacokinetic evaluation of the anti-MSLN/CD3 BiTE® antibody construct in non-human primates demonstrated a half-life of 4-10 days. The potency and specificity of the anti-MSLN/CD3 BiTE® antibody construct, together with projected ability to dose once a week IV in humans, supports development of this BiTE® for treatment of MSLN-positive tumors. Citation Format: Alexander Sternjak, Fei Lee, Joachim Wahl, Dan Rock, Oliver Thomas, Sabine Stienen, Ralf Lutterbuese, Patrick Hoffmann, Tobias Raum, Peter Kufer, Benno Rattel, Angela Coxon, Matthias Friedrich, Julie Bailis. Preclinical evaluation of a BiTE® antibody construct with extended half-life that targets the tumor differentiation marker mesothelin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3630. doi:10.1158/1538-7445.AM2017-3630

Bispecific T-cell engaging (BiTE®) antibody constructs connect T cells to target-positive cells to induce cytotoxicity. Clinical proof of concept was achieved in B cell malignancies with the anti-CD19 BiTE® Blincyto®, and several BiTE® antibody constructs are currently in development for solid tumor indications. With the goal of developing a BiTE® for ovarian cancer, we focused on targeting the Folate Receptor (FOLR1). FOLR1 mRNA and protein are highly expressed in >80% of ovarian tumor samples. The FOLR1 protein localizes to the cell membrane of ovarian tumor epithelial cells, whereas in normal tissues such as kidney tubules expression is restricted to the apical cell layer. We generated human FOLR1-BITE® antibody constructs that cross react to cynomolgus monkey and here present the preclinical characterization of these molecules. FOLR1-BiTE® antibody constructs had low nanomolar binding affinity for FOLR1 but did not bind to other FOLR family members. The FOLR1-BiTE® antibody constructs recognized diverse epitopes of the FOLR1 protein. In vitro, binding of FOLR1-BiTE® to CD3 on T cells and FOLR1 on tumor cells led to upregulation of CD25, CD69 and GITR on T cells, as well as release of cytokines including TNF, IFNγ, IL-6 and MCP-1. T cell activation by FOLR1-BiTE® was strictly target cell-dependent. Using time lapse imaging, we demonstrated that T cells were recruited to cancer cells expressing FOLR1 to form a cytolytic synapse and induce target cell lysis. Cytotoxicity mediated by FOLR1-BiTE® antibody constructs occurred even at very low target expression levels (1000 receptors per cell), and was not impacted by the presence of soluble receptor at levels found in ovarian cancer patients. FOLR1-BiTE® antibody constructs also mediated redirected lysis of cell lines that were resistant to standard of care chemotherapy. The in vivo activity of the FOLR1-BiTE® was explored in mouse xenograft studies where immunocompromised mice were implanted with mixtures of human T cells and human cancer cell lines. Daily intraperitoneal administration of the BiTE® inhibited tumor growth, even though the serum half-life of the BiTE® was just a few hours. Citation Format: Julie M. Bailis, Jessica N. Orf, Alexander Sternjak, Brian Belmontes, Ryan Case, Natalia Grinberg, Wesley Chang, Beth Hinkle, Matthias Friedrich. Cellular mechanism of action of bispecific T-cell engager (BiTE®) antibody constructs targeting Folate Receptor 1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4999.