Abstract BiTE antibodies are designed to transiently connect target cells with T cells of patients for induction of potent redirected lysis of cancer cells. Clinical proof-of-concept has been obtained with CD19/CD3-bispecific BiTE antibody blinatumomab (MT103), which showed a high response rate in patients with relapsed/refractory non-Hodgkin's lymphoma and acute B-lymphocytic leukemia. An EpCAM/CD3-bispecific BiTE antibody called MT110 currently is in a dose-escalating phase 1 study in patients with lung or gastrointestinal cancers, and BiTE antibodies targeting carcinoembryonal antigen (CEA), EGFR, CD33 or melanoma chondroitin sulfate proteoglycan (MCSP) are in earlier stages of development. Here, we have generated a series of novel BiTE antibodies for cancer therapy that are based on our novel BiTE antibody platform, which is human in sequence and cross-reactive between human and non-human primate antigens. One set of target antigens selected for new BiTE generation is also targeted by conventional monoclonal antibodies that are in various stages of clinical development. These are mesenchymal-epithelial transition factor (cMet), insulin-like growth factor receptor type I (IGFR-1), prostate-specific stem cell antigen (PSCA), carboanhydrase type IX (CAIX) and Her2/neu. These targets are all expressed by cancer cells of solid tumors, and some also by their so-called cancer stem cells. By targeting fibroblast activating protein alpha (FAP-alpha), a first BiTE antibody was generated that not only targets sarcoma cells but also stromal fibroblasts, which constitute the stromal tissue of many human cancers. By targeting endosialin (CD248), a first BiTE antibody was generated targeting cancer blood vasculature. We show that various single-chain antibodies specific for the seven target antigens cMet, IGFR-1, FAP-alpha, PSCA, CAIX, Her2/neu and endosialin could in each case be used to construct novel BiTE antibodies. All BiTE antibodies showed bispecific binding, cross-reactivity between human and Cynomolgus monkey antigens, and, most importantly, potent redirected lysis of CHO cells expressing the respective target antigen. This shows that new T cell-engaging BiTE antibodies can be made specific for a great variety of potential tumor-associated and also for stromal antigens. 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 2434.

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.

2404 BiTE antibodies are emerging as new class of uniquely T cell-engaging antibodies. A CD19/CD3-bispecific BiTE antibody called MT103/MEDI-538 has shown objective complete and partial responses in relapsed non-Hodgkin’s lymphoma (NHL) patients in an ongoing phase 1 study, providing clinical proof-of-concept for this new class of antibody-derived therapeutics. A number of other BiTE antibodies are in clinical and pre-clinical development targeting EpCAM (CD326), CEA and receptor tyrosine kinase EphA2. As a novel approach for treatment of acute myeloid leukemia (AML) and melanoma, we have constructed new BiTE antibodies with similar in-vitro potency and other properties as observed for more advanced clinical and pre-clinical BiTE antibody candidates.
 One BiTE antibody for treating AML is specific for CD33, a target validated by the immunotoxin gemtuzumab ozogamicin. Even with current treatment regimens, only about 20 percent of AML patients survive five or more years. The high efficacy of MT103 in NHL suggests that also other blood borne malignancies such as AML will be responsive to the therapeutic principle of BiTE antibodies.
 The other BiTE antibody is specific for melanoma-associated chondroitin sulfate proteoglycan (MCSP), which is among the best characterized melanoma surface antigens. Melanoma occasionally well respond to T cell-based therapies, such as adoptive T cell transfer or vaccination, but are frequently limited by immune escape mechanism, such as loss of MHC class I expression, to which BiTE antibodies are insensitive.
 CD33- and MCSP-specific BiTE antibodies of human sequence were selected as preclinical lead candidates based on their potency of redirected target cell lysis, and a superior profile of biophysical and pharmaceutical characteristics. CD33- and MCSP-specific BiTE antibodies showed redirected lysis by human peripheral T cells of CD33- and MCSP-expressing cell lines, respectively, at half-maximum concentrations as low as 5 pg/ml (90 femtomolar). Lysis was highly specific because it did not occur with CD33- or MCSP-negative cell lines, or with a BiTE antibody solely sharing the anti-CD3 portion with CD33- and MCSP-specific BiTE antibodies. CD33- and MCSP-specific BiTE antibodies are being investigated for safety in ongoing animal studies.
 Preclinical candidates for both CD33-specific and MCSP-specific BiTE antibodies may provide highly efficacious approaches for the treatment of AML and melanoma, respectively, both of which are diseases with a high need for new treatment options.

La presente invention concerne un polypeptide comprenant un premier domaine de liaison humain capable de se fixer a un epitope de la chaine (epsilon) du CD3 humain ou d'un primate autre que le chimpanze, un second domaine de liaison capable de se fixer au recepteur EGF, au recepteur Her2/neu ou a l'IgE d'un humain et/ou d'un primate autre que le chimpanze, ainsi qu'un procede destine a la production de ce polypeptide. L'invention concerne egalement des acides nucleiques codant pour le polypeptide, des vecteurs comprenant ces acides nucleiques et des cellules hotes comprenant le vecteur. Dans un autre aspect, l'invention concerne une composition pharmaceutique comprenant ledit polypeptide et des applications medicales du polypeptide.