An optimized IgG-based B7-H3xCD3 bispecific antibody for treatment of gastrointestinal cancers.

T cell-based immunotherapy has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions remain rare, particularly in gastrointestinal cancers like colorectal cancer (CRC). B7-H3 is overexpressed in multiple cancer entities including CRC on both tumor cells and tumor vasculature, the latter facilitating influx of effector cells into the tumor site upon therapeutic targeting. We generated a panel of T cell-recruiting B7-H3xCD3 bispecific antibodies (bsAbs) and show that targeting a membrane-proximal B7-H3 epitope allows for a 100-fold reduction of CD3 affinity. In vitro, our lead compound CC-3 showed superior tumor cell killing, T cell activation, proliferation, and memory formation, whereas undesired cytokine release was reduced. In vivo, CC-3 mediated potent antitumor activity in three independent models using immunocompromised mice adoptively transferred with human effector cells with regard to prevention of lung metastasis and flank tumor growth as well as elimination of large established tumors. Thus, fine-tuning of both target and CD3 affinities as well as binding epitopes allowed for the generation of a B7-H3xCD3 bsAbs with promising therapeutic activity. CC-3 is presently undergoing good manufacturing practice (GMP) production to enable evaluation in a clinical "first-in-human" study in CRC.

A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL.

BACKGROUND: In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs. METHODS: We developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo. RESULTS: CLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts. CONCLUSIONS: CLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.

Bispecific Antibodies in Prostate Cancer Therapy: Current Status and Perspectives.

Prostate carcinoma (PC) is the second most common cancer in men. When the disease becomes unresponsive to androgen deprivation therapy, the remaining treatment options are of limited benefit. Despite intense efforts, none of the T cell-based immunotherapeutic strategies that meanwhile have become a cornerstone for treatment of other malignancies is established in PC. This refers to immune checkpoint inhibition (CI), which generally reinforces T cell immunity as well as chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) that stimulate the T cell receptor/CD3-complex and mobilize T cells in a targeted manner. In general, compared to CAR-T cells, bsAb would have the advantage of being an "off the shelf" reagent associated with less preparative effort, but at present, despite enormous efforts, neither CAR-T cells nor bsAbs are successful in solid tumors. Here, we focus on the various bispecific constructs that are presently in development for treatment of PC, and discuss underlying concepts and the state of clinical evaluation as well as future perspectives.

Bispecific NKG2D-CD3 and NKG2D-CD16 fusion proteins for induction of NK and T cell reactivity against acute myeloid leukemia.

BACKGROUND: Monoclonal antibodies (mAbs) mediate their effects in great part by inducing ADCC of NK cells, and multiple efforts aim to increase this function by engineering mAbs optimized Fc-parts. Even more potent antitumor immunity can be induced by strategies to stimulate T cells with their profoundly higher effector potential. However, upon increased immunostimulatory potential, the necessity to target highly tumor-specific antigens becomes critically important to reduce side effects. METHODS: We here report on bispecific fusion proteins (BFP) that target ligands of the immunoreceptor NKG2D (NKG2DL), which are widely expressed on malignant cells but generally absent on healthy tissue. They consist of the extracellular domain of NKG2D as targeting moiety fused to Fab-fragments of CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) antibodies. RESULTS: NKG2D-CD16 displayed increased affinity to the FcγRIII on NK cells compared to engineered Fc-parts, which are contained in optimized mAbs that presently undergo clinical evaluation. In line, NKG2D-CD16 induced superior activation, degranulation, IFN-γ production and lysis of acute myeloid leukemia (AML) cell lines and patient AML cells. NKG2D-CD3 in turn potently stimulated T cells, and comparison of efficacy over time revealed that NKG2D-CD16 was superior upon short term application, while NKG2D-CD3 mediated overall more potent effects which manifested after longer times. This can be attributed to treatment-induced proliferation of T cells but not NK cells. CONCLUSIONS: Taken together, we here introduce novel "antibody-like" BFP that take advantage of the highly tumor-restricted expression of NKG2DL and potently activate the reactivity of NK cells or T cells for immunotherapy of AML.