Pharmaceutical companies, which are IQ DruSafe/3R-TPS members, explored novel ways to reduce reliance on non-human primate (NHP) use in nonclinical drug development. These companies conducted a survey on NHP use in toxicology studies during discovery and development of CD3-bispecific antibodies (BsAbs) for the treatment of cancer. The objectives of the survey were to collect data on and case study examples of both first-in-human (FIH)-enabling (≤1-month) and Phase II/III-enabling (3-month) NHP studies conducted with CD3-BsAbs. The survey addressed study design elements, e.g. number of dose groups, number of animals/dose group, recovery arms, etc. when a study is deemed warranted. Survey questions also focused on whether data from Phase II/III-enabling studies addressed patient risk that was not identified in FIH-enabling studies, and whether Phase II/III-enabling studies impacted regulatory decisions pertaining to clinical development. The survey findings enabled the development of a Weight of Evidence (WoE) approach to assess the utility of Phase II/III-enabling toxicity studies in understanding potential safety risks with CD3-BsAbs and informing later-stage molecule development. Both study design optimization and WoE approaches offer a path for reduced NHP use without compromising nonclinical safety assessment of CD3-BsAbs for oncology indications, as illustrated by several case examples.

Development of T cell engagers (TCEs) for oncology indications is regulated mainly by ICH S6 and S9. Investigational new drug applications are usually supported by a 1-month toxicology study in a relevant animal species. Before the start of registrational clinical trials, a longer-duration toxicology study, typically 3 months, is performed in the same species to support marketing. As longer-term studies with human-specific biologics in animal species can be hampered by development of anti-drug antibodies and TCEs are quick-acting molecules, we analyzed the value of 3-month toxicology studies for some of our TCEs. We present data from 1- and, where applicable, 3-month toxicology studies for 4 TCE programs and describe our interactions with regulatory agencies. In none of the cases did the 3-month studies reveal new information to influence further the respective clinical development plans. Considering 3Rs (Replacement, Reduction and Refinement) in pharmaceutical development, we highlight that 3-month studies with TCEs don't always offer additional safety insights. Therefore, alternative strategies should be evaluated and conduct of 3-month studies carefully considered in the light of 3Rs and discussed case-by-case with health authorities.

Abstract T cell engager (TCE) molecules are a targeted immune therapy that redirect a patient’s T cells to kill tumor cells. While several TCE molecules have now advanced to approval, clinical development has been challenging, especially in the solid tumor setting. There are few solid tumor surface antigens that are tumor-specific, and antigens with even low levels of expression in normal tissue can be recognized by a TCE and result in on-target off-tumor toxicity. To overcome this challenge, we have developed AMG 305, a first-in-class, dual targeting BiTE® (bispecific T cell engager) molecule that requires binding to both P-cadherin (CDH3) and mesothelin (MSLN) for potent cytotoxic activity. Immunohistochemistry analysis shows that CDH3 and MSLN are co-expressed in multiple solid tumor types. In normal tissues, immunostaining shows fewer tissues have overlapping co-expression than expression of just one or the other target. AMG 305 relies on an avidity-based approach to engage T cells to preferentially kill CDH3+MSLN+ tumor cells, with limited activity against normal cells that express only one of the target antigens. In vitro, AMG 305 induces potent cytotoxicity against tumor cells co-expressing CDH3 and MSLN, with greatly attenuated activity against cells expressing only CDH3 or MSLN. In vivo, AMG 305 demonstrates dose-dependent antitumor activity in a CDH3+MSLN+ xenograft tumor model. AMG 305 was clinically well-tolerated in a nonclinical safety study in cynomolgus monkey. Data from IND-enabling preclinical studies support initiation of the first-in-human study of AMG 305 in CDH3+MSLN+ solid tumors in 2023. Citation Format: Elizabeth Pham, Petra Lutterbuese, Petra Deegen, Natalie Mariano, Katja Matthes, Joachim Wahl, Pamela Bogner, Joan Lane, Kristin Lewis Wilson, Rodolfo Yabut, Virginie Naegele, Ines Ullrich, Stephanie Everts, Markus Muenz, Thomas Boehm, Sabine Stienen, Angela Coxon, Peter Kufer, Tobias Raum, Julie M. Bailis. AMG 305, a dual targeting BiTE®molecule with selective activity for solid tumors that co-express CDH3 and MSLN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND06.

Supplementary Video 4C and D from Selective Targeting and Potent Control of Tumor Growth Using an EphA2/CD3-Bispecific Single-Chain Antibody Construct

Supplementary Video 4I from Selective Targeting and Potent Control of Tumor Growth Using an EphA2/CD3-Bispecific Single-Chain Antibody Construct

Abstract Advanced gastric cancer remains a disease of high unmet medical need. In the United States, most patients present with symptomatic, incurable disease and prognosis is poor, with a 5-year survival rate of less than 10%. BiTE® (bispecific T cell engager) immune therapy activates a patient's own T cells to kill tumor cells and has the potential to overcome common mechanisms of therapy resistance. We generated fully human, half-life extended (HLE) BiTE® molecules against the tumor antigens MUC17 and CLDN18.2 for the treatment of gastric cancer. The mucin MUC17 is a protein normally found in the mucosal layer of intestinal epithelial cells that is delocalized and expressed in 45% of gastric tumors. The claudin CLDN18.2 is a protein normally found in the cellular tight junctions of gastric mucosa and intestinal epithelium that is delocalized and expressed in >60% of gastric tumors. AMG 199 (MUC17 HLE BiTE®) and AMG 910 (CLDN18.2 HLE BiTE®) show potent cytotoxic activity against gastric cancer cell lines that express MUC17 or CLDN18.2, respectively, in vitro, and promote significant tumor growth inhibition against established gastric tumor xenograft models in vivo. In preclinical non-human primate (NHP) toxicology studies, both molecules show evidence for BiTE® target engagement, including T cell activation and proliferation, but demonstrate different effects on target-expressing tissue. Weekly administration of AMG 199 is well tolerated in NHP with minimal findings in MUC17-expressing normal tissues. In contrast, treatment with AMG 910 led to direct cell killing of CLDN18.2-expressing gastric mucosal cells in NHP, a finding which was fully reversible once treatment was stopped. AMG 199 and AMG 910 may offer the potential to improve outcomes in advanced gastric patients worldwide. Citation Format: Julie M. Bailis, Petra Lutterbuese, Oliver Thomas, Kathrin Locher, John Harrold, Michael Boyle, Joachim Wahl, Shyun Li, Alexander Sternjak, Anja Henn, Christoph Dahlhoff, Virginie Naegele, Benno Rattel, Tobias Raum, Angela Coxon. Preclinical evaluation of BiTE®immune therapy targeting MUC17 or CLDN18.2 for gastric cancer [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 3364.

Individual or combinations of somatic mutations found in genes from colorectal cancers can redirect the effects of chemotherapy and targeted agents on cancer cell survival and, consequently, on clinical outcome. Novel therapeutics with mechanisms of action that are independent of mutational status would therefore fulfill a current unmet clinical need. Here the CEA and CD3 bispecific single-chain antibody MEDI-565 (also known as MT111 and AMG 211) was evaluated for its ability to activate T cells both in vitro and in vivo and to kill human tumor cell lines harboring various somatic mutations commonly found in colorectal cancers. MEDI-565 specifically bound to normal and malignant tissues in a CEA-specific manner, and only killed CEA positive cells. The BiTE® antibody construct mediated T cell-directed killing of CEA positive tumor cells within 6 hours, at low effector-to-target ratios which were independent of high concentrations of soluble CEA. The potency of in vitro lysis was dependent on CEA antigen density but independent of the mutational status in cancer cell lines. Importantly, individual or combinations of mutated KRAS and BRAF oncogenes, activating PI3KCA mutations, loss of PTEN expression, and loss-of-function mutations in TP53 did not reduce the activity in vitro. MEDI-565 also prevented growth of human xenograft tumors which harbored various mutations. These findings suggest that MEDI-565 represents a potential treatment option for patients with CEA positive tumors of diverse origin, including those with individual or combinations of somatic mutations that may be less responsive to chemotherapy and other targeted agents.

Abstract Colorectal cancer (CRC) cells can harbor somatic mutations with strong impact on cancer cell signaling that limit the effectiveness of both chemotherapy and targeted therapies including monoclonal antibodies and kinase inhibitors. Therefore, novel therapeutic modalities are needed that are not impacted by the presence of such genetic mutations. We have here evaluated a CEA/ CD3-bispecific BiTE antibody termed MEDI-565 (also known as MT111) for its ability to redirect cytotoxic T cells for lysis in co-cultures and in xenograft models of human CRC lines harboring oncogenic mutations. Redirected lysis of CEA-positive tumor cells by MEDI-565 occurred in a manner dependent on CEA surface antigen density but independent of exogenously added soluble CEA. Importantly, the potency of lysis was independent of the mutational status of genes commonly mutated or lost in colorectal and other cancers, including frequent mutations in KRAS, BRAF, PI3KCA, PTEN and TP53 genes. In mouse xenograft models, the ability of MEDI-565 to inhibit tumor growth was likewise independent of the mutational status of the human tumor xenograft models. Our findings suggest that MEDI-565 warrants further clinical investigation as a potential treatment option for patients with CEA-positive tumors harboring somatic mutations that are less responsive to traditional chemotherapy or targeted agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3524. doi:1538-7445.AM2012-3524

Abstract MEDI-565 (MT111) is a novel bispecific single-chain antibody of the BiTE (Bispecific T cell engager) class that transiently links carcinoembryonic antigen (CEA) on cancer cells with human CD3 on T cells. MEDI-565 specifically binds to human CEA with an affinity of 8.5 nM but not to any other member of the CEACAM family. In targeted expression studies on cryopreserved and formalin-fixed paraffin-embedded sections, MEDI-565 demonstrated membrane-localized binding specifically to epithelial cells in human cancerous tissues. The highest prevalence of MEDI-565 binding (∼90%) was in adenocarcinomas of gastrointestinal origin. In in vitro killing assays, MEDI-565 recruited T cells via the CD3 antigen (affinity of 310 nM) in a process that required concomitant binding to CEA positive tumor cells for T cell activation. As a consequence, T cells expanded, increased cell surface expression of the activation markers CD69 and CD25, and released perforin and granzymes. The release of cytotoxic granule content led to a Ca2+-dependent activation of pro-caspases and subsequent apoptosis of CEA-expressing tumor cells. Efficient target cell lysis by MEDI-565 was predominantly mediated by CD8+ T cells and occurred within a wide range of effector-to-target ratios (80:1 to 5:1) for T cells derived from various human donors. BiTE mediated killing was not affected by the presence of soluble CEA (≤5 µg/mL). The in vivo activity of MEDI-565 was investigated in subcutaneous xenograft models using immunocompromised SCID mice inoculated with mixtures of human T cells and human cancer cell lines. The antibody was eliminated from the serum with a half-life of a few hours following a single intravenous (IV) or subcutaneous (SC) injection. Despite a relatively short serum half-life, daily IV or SC bolus treatments over 5 days provided sufficient levels of exposure to inhibit the growth of CEA-positive tumors in a dose-dependent manner. Inhibition of growth was observed for tumors of different tissue origins and was dependent on the presence of human T cells and CEA expression by tumor cells. There were no MEDI-565-related in-life observations following treatment. These studies demonstrated that MEDI-565 has potent and selective anti-cancer activity in vitro and in vivo and provides evidence that MEDI-565 may be an effective monotherapy to treat CEA-expressing malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5625.

Carcinoembryonic antigen (CEA, CD66e) is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach.

MEDI-565 (MT111) is a bispecific single chain antibody of the BiTE (Bi-specific T-cell engager) class that transiently links carcinoembryonic antigen (CEA; CEACAM5; CD66e) on tumor cells and human CD3 on T cells. This results in the activation of T cells and the subsequent redirected lysis of target cells expressing CEA. MEDI-565 is being developed as a clinical candidate for the treatment of CEA-positive tumors. With the exception of the chimpanzee, MEDI-565 does not cross-react with CD3 orthologs. Therefore, as a potential tool for evaluating mechanistic safety in non-human primates, we constructed a biosimilar antibody, CyS111, which combines the CEA-specific component of MEDI-565 with one that binds cynomolgous CD3. This study characterizes the biological activity of CyS111 as it compares to MEDI-565 to determine its potential utility in assessing safety in a relevant species. CyS111 and MEDI-565 both demonstrated specific lysis of CEA-expressing target cells with similar half-maximal lysis (EC50) values and kinetics of lysis, despite moderate binding affinity differences for species-appropriate CD3 and CEA. However, CyS111 non-specifically activated T cells in the presence of CEA-negative target cells or in the absence of target cells entirely, a feature not shared by MEDI-565. In addition, CyS111 induced proliferation of both CD4+ and CD8+ T cells, whereas MEDI-565 induced CD8+ T cell proliferation with a slower kinetics, and only poorly induced proliferation of CD4+ T cells. Interestingly, aggregated forms of MEDI-565 displayed significantly enhanced potency of BiTE-mediated T cell activation and target cell lysis, whereas CyS111 aggregated forms did not. Taken together, these findings may have implications for the use of biosimilar surrogate molecules for evaluating mechanistic safety. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3247.

EpCAM (CD326) is one of the most frequently and highly expressed tumor-associated antigens known and recently has also been found on cancer stem cells derived from human breast, colon, prostate, and pancreas tumors. However, like many other tumor-associated antigens used for antibody-based immunotherapeutic approaches, EpCAM is expressed on normal tissues including epithelia of pancreas, colon, lung, bile ducts, and breast. To assess the therapeutic window of an EpCAM/CD3-bispecific single-chain antibody construct of the bispecific T-cell engager (BiTE) class, we constructed murine surrogate of MT110 (muS110) from single-chain antibodies specific for murine EpCAM and CD3 antigens. Immunhistochemical analysis showed that, with minor differences, the expression of EpCAM protein on a large variety of tissues from man and mouse was similar with respect to distribution and level. MuS110 exhibited significant antitumor activity at as low as 5 microg/kg in both syngeneic 4T1 orthotopic breast cancer and CT-26 lung cancer mouse models. Dosing of muS110 for several weeks up to 400 microg/kg by intraanimal dose escalation was still tolerated, indicating existence of a significant therapeutic window for an EpCAM-specific BiTE antibody in mice. MuS110 was found to have similar in vitro characteristics and in vivo antitumor activity as MT110, a human EpCAM/human CD3-bispecific BiTE antibody that currently is in formal preclinical development.

MuS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. A recent study has shown that muS110 has significant anti tumor activity at well-tolerated doses as low as 5 μg/kg in orthotopic breast and lung cancer models (Amann et al. in Cancer Res 68:143–151, 2008). Here, we have explored the safety profile of muS110 at higher doses. Escalation to 50 μg/kg muS110 caused in mice transient loss of body weight, and transient piloerection, hypomotility, hypothermia and diarrhoea. These clinical signs coincided with serum peaks of TNF-α, IL-6, IL-2, IFN-γ and IL-4, and an increase of surface markers for T cell activation. Because activation of T cells in response to BiTE antibodies is typically dependent on target cells, we analyzed mouse blood for the presence of EpCAM+ cells. Various mouse strains presented with a subpopulation of 2–3% EpCAM+ blood cells, mostly B and T lymphocytes, which was not detected in human blood samples. In vitro experiments in which the number of EpCAM+ cells in blood samples was either reduced or increased suggested that both T cell activation and cytokine release in response to muS110 was dependent on the number of target-expressing cells. In support for a role of EpCAM+ lymphocytes in the observed side effects, reduction of EpCAM+ blood cells in mice via a low-dose pre treatment with muS110 dramatically increased the tolerability of animals up to at least 500 μg/kg of the BiTE antibody. This high tolerability to muS110 occurred in the presence of non-compromised T cells. No damage to EpCAM+ epithelial tissues was evident from histopathological examination of animals daily injected with 100 μg/kg muS110 for 28 days. In summary, these observations suggest that side effects of muS110 in mice were largely caused by an acute T cell activation that was triggered by a subpopulation of EpCAM+ lymphocytes. Because humans have extremely low numbers of EpCAM+ cells in blood, this toxicity of an EpCAM-specific BiTE may be specific for mice.

2131 MEDI-538 (MT103) is a bispecific single-chain, T cell-engaging (BiTE) antibody that targets CD19+ B cells and recruits CD3+ cytotoxic T cells to lyse B cells. Ongoing Phase I clinical trials reveal that as a single agent administered by continuous intravenous (IV) infusions, MEDI-538 induces objective clinical responses and clears bone marrow infiltration in heavily pre-treated B cell non-Hodgkin’s lymphoma patients. In an effort to explore alternative routes of administration, we utilized animal models to evaluate the bioavailability, pharmacodynamic effects, and anti-tumor potency of MEDI-538 following subcutaneous (SC) administration. Bioavailability of MEDI-538 was determined following a single IV or SC bolus injection to cynomolgus monkeys and mice. A highly sensitive pharmacokinetic (PK) ELISA assay based on electrochemiluminescence was utilized to measure the amount of MEDI-538 present in each serum sample. Following SC delivery in mice, MEDI-538 concentrations reached a peak at 4-8 hours, exhibited dose-proportionality, and cleared from the blood at a similar rate as compared to IV administration of drug (t1/2 of 2-4 hours). Dose optimization studies showed that varying the dose concentration or volume for a given dose level did not effect the concentration of MEDI-538 in the serum after SC administration. Bioavailability of MEDI-538 delivered via a single SC dose in the mouse was 20-30% based on the mean area under the curve (AUC). In similar studies, PK parameters and bioavailability in cynomologus monkeys was comparable to that measured in mice. MEDI-538 also provided anti-tumor efficacy following SC administration in NOD/SCID mice that were engrafted with human B cell tumor cells mixed with human PBMC and that were capable of demonstrating human T cell-mediated killing of tumor cells. Pharmacodynamic effects were observed following SC administration of a hybrid mouse surrogate form of MEDI-538 (anti-human CD19 x anti-mouse CD3) to immunocompetent human CD19 transgenic knock-in mice. Treatment caused a rapid reduction in B cells and an increase in activated T cells in the spleen as measured by flow cytometry. These results demonstrated that SC delivery of MEDI-538 yielded comparable PK characteristics in multiple animal models, provided sufficient systemic levels for efficacy against tumor challenge in xenograft mouse models, and depleted B cells in an immunocompetent mouse model. Taken together, our data support the SC route of administration as an alternative method for delivery of MEDI-538.

Abstract The EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in malignant cells and thus provides opportunities for selective targeting of tumor cells. We describe here the development of a novel, bispecific single-chain antibody (bscAb) referred to as bscEphA2xCD3. This molecule simultaneously targets EphA2 on tumor cells and the T-cell receptor/CD3 complex on T cells and possesses structural and functional characteristics of the recently developed BiTE technology. An EphA2-specific single-chain antibody was selected for recognition of an epitope that is preferentially exposed on malignant cells based on the concept of epitope exclusion; this was fused to a CD3-specific single-chain antibody to generate bscEphA2xCD3. The resultant bscAb redirected unstimulated human T cells to lyse EphA2-expressing tumor cells both in vitro and in vivo. In separate experiments, efficient tumor cell lysis was achieved in vitro at drug concentrations ≤1 μg/mL, at a low T-cell effector-to-tumor target cell ratio (1:1), and with tumor cells that possess few available binding sites (2,400 per cell) for bscEphA2xCD3. Time-lapsed microscopy revealed potent cytotoxic activity of bscEphA2xCD3-activated T cells against monolayers of malignant cells but not against monolayers of nontransformed EphA2-positive cells except at the edges of the monolayer where the target epitope was exposed. BscEphA2xCD3 was also efficacious in human xenograft mouse models modified to show human T-cell killing of tumors. Together, our results reveal opportunities for redirecting the potent activity of cytotoxic T cells towards tumor cells that express selectively accessible epitopes and establish EphA2-specific bscAb molecules as novel and potent therapeutics with selectivity for tumor cells. [Cancer Res 2007;67(8):3927–35]

An important mode of action shared by human IgG1 antibody therapies is antibody-dependent cellular cytotoxicity (ADCC). ADCC relies on the interaction of the antibody’s Fc portion with Fc-gama receptors (FcγR) on immune effector cells. The anti-tumor activity of human IgG1 antibodies is frequently assessed in mouse models. Binding of human IgG1 to murine FcγRs is however of reduced affinity. We here show that ADCC of adecatumumab (MT201), a fully human IgG1 antibody specific for epithelial cell adhesion molecule (EpCAM/CD326), is drastically lower if human peripheral blood mononuclear cells are replaced by murine splenocytes as effector cells. When the variable domains of adecatumumab were genetically fused to a murine IgG2a backbone (yielding mu-adecatumumab), ADCC with murine effector cells was much improved, but at the same time significantly reduced with human effector cells. The serum half-lives of adecatumumab and mu-adecatumumab were determined in mice and dosing schedules established that gave similar serum trough levels during a 4-week antibody treatment. The anti-tumor activities of adecatumumab and mu-adecatumumab were then compared side-by-side in a lung metastasis mouse model established with a syngeneic B16 melanoma line expressing human EpCAM at physiologically relevant levels. Treatment of mice with mu-adecatumumab led to an almost complete prevention of lung metastases, while the human version of the antibody was much less active. This shows that adecatumumab has high anti-tumor activity when tested in a form that is better compatible with the species’ immune system. Moreover, our data suggest to routinely compare in mouse models human IgG1 and murine IgG2a versions of antibodies to properly assess the contribution of ADCC to overall anti-tumor activity.

C270 EpCAM (CD326) is a well studied tumor-associated antigen that is frequently expressed at high levels on a variety of human adeno and squamous cell carcinoma. Recently, EpCAM has also been found expressed on cancer stem cells from breast, colon, prostate and pancreas tumors. However, like many other tumor-associated antigens used for antibody-based immunotherapeutic approaches, EpCAM is also present on normal tissues including epithelia of pancreas, colon, lung, bile ducts and breast. Sequestration of EpCAM epitopes by associated membrane proteins and within intercellular boundaries of normal epithelia, and its overexpression on some tumors relative to normal tissue may explain the benign safety profile of some but not all EpCAM-directed antibody-based therapies. MT110 is an EpCAM-/CD3-bispecific single-chain antibody construct with high potency against EpCAM-positive human carcinoma lines. In vivo, eradication in immunodeficient mice of established tumors from human cell lines and of authentic tumor tissue of patients by low µg-doses of MT110 has been reported. Because MT110 is specific for human EpCAM and human CD3, we sought to assess the safety and therapeutic window of this class of bispecific antibodies with a biosimilar molecule. To this end, muS110 (for murine surrogate of MT110) was constructed from the variable domains of two monoclonal antibodies with specificity for mouse EpCAM and mouse CD3. In in-vitro assays, muS110 showed similar bioactivity with murine cells as MT110 with human cells. Immunhistochemical analysis revealed that, with some differences, the expression of EpCAM on a large variety of tissues from man and mouse was comparable with respect to tropism and intensity. MuS110 exhibited highly significant anti-tumor activity in both syngeneic 4T1 orthotopic breast cancer and CT26 lung cancer mouse models. Administration of muS110 for several weeks at doses exceeding by far the efficacious dose levels was well tolerated, indicating existence of a significant therapeutic window for the EpCAM-specific BiTE molecule. The dose-limiting toxicity of muS110 was related to cytokine release but not to damage of EpCAM-expressing normal tissues. These data show that EpCAM-specific BiTE molecules can distinguish EpCAM expressed on tumor cells from the EpCAM expressed on normal tissue, suggesting that a therapeutic window may also exist for the human-specific BiTE MT110, which is in formal preclinical development for the treatment of patients with EpCAM-expressing adenocarcinoma.