Dual specific immunotoxin for brain tumor therapy

Inventors

Bigner, Darell • Kuan, Chien-Tsun • Pastan, Ira H. • PEAGRAM, Charles

Assignees

UNITED STATES GOVERNMENT HEALTH AND HUMAN SERVICES (NIH), Secretary of, Department of • National Institutes of Health NIH • Duke University

Abstract

We tested the in vitro and in vivo efficacy of a recombinant bispecific immunotoxin that recognizes both EGFRwt and tumor-specific EGFRvIII receptors. A single chain antibody was cloned from a hybridoma and fused to toxin, carrying a C-terminal peptide which increases retention within cells. The binding affinity and specificity of the recombinant bispecific immunotoxin for the EGFRwt and the EGFRvIII proteins was measured. In vitro cytotoxicity was measured. In vivo activity of the recombinant bispecific immunotoxin was evaluated in subcutaneous models and compared to that of an established monospecific immunotoxin. In our preclinical studies, the bispecific recombinant immunotoxin, exhibited significant potential for treating brain tumors.

Core Innovation

The invention relates to a recombinant bispecific immunotoxin that recognizes both the wild-type epidermal growth factor receptor (EGFRwt) and the tumor-specific EGFR variant III mutant (EGFRvIII). A single-chain variable region antibody was cloned from a hybridoma producing monoclonal antibody D2C7 and fused to a cytotoxic agent, specifically a form of Pseudomonas exotoxin A with a C-terminal KDEL peptide, which increases retention within cells and enhances cytotoxicity. The binding affinity and specificity of the immunotoxin for both EGFRwt and EGFRvIII were characterized using surface plasmon resonance and flow cytometry, demonstrating high affinity binding to both receptors. The efficacy of this bispecific immunotoxin was tested in vitro for cytotoxicity against various tumor cell lines expressing EGFRwt and/or EGFRvIII and in vivo in mouse xenograft tumor models, showing significant potential for brain tumor treatment.

The problem addressed by the invention is the limited effectiveness of existing therapies for glioblastoma multiforme (GBM) and other malignant gliomas, which are aggressive brain tumors with poor prognosis despite surgery, radiation, and chemotherapy. Current treatments improve median survival by only a short duration, creating a critical need for more effective therapeutic approaches. While monoclonal antibodies targeting either EGFRwt or the tumor-specific EGFRvIII have been explored, tumors often express both antigens, and resistance to single-target therapies can occur. There is therefore a need for antibodies or immunotoxins that can simultaneously target both EGFRwt and mutant EGFRvIII to improve specificity and therapeutic efficacy against brain tumors.

The invention further provides methods of treating tumors in humans by administering a single-chain variable region antibody derived from the D2C7 monoclonal antibody fused to Pseudomonas exotoxin A, which binds with high affinity (at least 5×10^8 M−1) to both EGFRwt and EGFRvIII. This dual specificity allows for selective killing of tumor cells expressing either or both receptors. The immunotoxin can be administered by various routes, including direct delivery to the central nervous system, and can be utilized for treating a range of tumors that express EGFR or EGFRvIII, including glioblastomas, astrocytomas, breast tumors, and head and neck tumors.

Claims Coverage

The patent includes one independent claim that covers a method of treating tumors using a dual-specificity single chain variable region antibody immunotoxin. The main inventive features describe the antibody's binding properties, its derivation from D2C7, its fusion with a cytotoxic agent, and specific administration modes and tumor types.

The claim coverage focuses on the use of a bispecific single chain variable region antibody immunotoxin derived from monoclonal antibody D2C7, which binds both EGFRwt and EGFRvIII with high affinity and is fused to Pseudomonas exotoxin A for targeted tumor cell killing. The claims further cover specific tumor types, routes of administration, fusion protein construction, and defined antibody sequences, emphasizing a novel method for treating tumors expressing either or both EGFR antigens.

Stated Advantages

Documented Applications