Novel Humanized and Highly Efficient Bispecific Antibodies Mediate Killing of Prostate Stem Cell Antigen-Expressing Tumor Cells by CD8+ and CD4+ T Cells

Abstract

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3–anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8+ and CD4+ T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4+ T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.

A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

“…44 Human Embryonic Kidney cells HEK293T (ATCC CRL-11268) and the pharynx squamous cell carcinoma cell line FaDu (ACC 784) were kept in complete DMEM medium. 44 For cultivation of the epidermoid carcinoma cell line A431 (ATCC CRL-1555) and A431-LucC cells (see below) complete DMEM medium supplemented with 1 mM sodium pyruvate (Biochrom GmbH, Berlin, Germany) was used. To express the enzyme luciferase (Photinus pyralis) A431 cells were transduced with the open reading frame encoding the firefly luciferase gene as described previously.…”

“…44 The study including the consent form was approved by the local ethics committee of the university hospital of the medical faculty of "Carl Gustav Carus" TU-Dresden (EK27022006). Isolated T cells were cultured in RPMI 1,640 complete medium supplemented with 50 U/mL IL-2 (ImmunoTools, Friesoythe, Germany) at densities of 1-2 £ 10 6 cells/mL.…”

“…47 Purification of the his-tagged recombinant proteins from cell culture supernatant was performed by affinity chromatography on Ni-NTA columns (Qiagen, Hilden, Germany) according to previously published protocols. [44][45][46][47] For prokaryotic expression of the a-EGFR TM the E. coli strain SHuffle Ò T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET28a_a-EGFR TM using standard heat shock method. One single clone of genetically modified bacteria was grown over night C, 10,000g), the EGFR-specific TM was purified by Ni-NTA affinity chromatography.…”

“…[44][45][46][47] Recombinant Abs were detected by a mouse mAb directed to the his-tag (Qiagen) and a polyclonal rabbit a-mouse IgG conjugated with alkaline phosphatase (Dianova GmbH, Hamburg, Germany).…”

“…The binding of the a-EGFR TM to A431 or FaDu tumor cells was assessed by FACS analysis as described previously. 44 Briefly, 3 £ 10 5 cells were incubated with 20 ng/mL of the a-EGFR TM for 1 h. To determine affinity of the TM to EGFR, A431 or FaDu cells were incubated with increasing Ab concentrations ranging between 0.05 mg/mL and 320mg/mL. To detect specific TM binding, cells were subsequently stained with a mouse a-E5B9 mAb directed against the E5B9-tag of the TM and a PE-conjugated Goat F (ab') 2 Fragment a-mouse IgG (Fcg)-PE (Beckmann Coulter, Krefeld, Germany) as detection Ab.…”