Dual specific immunotoxin for brain tumor therapy
Inventors
Bigner, Darell D. • Kuan, Chien-Tsun • Pastan, Ira H. • Pegram, Charles
Assignees
United States Government Health And Human Services (nih), Secretary of, Department of • Duke University • US Department of Health and Human Services
Publication Number
US-10072084-B2
Publication Date
2018-09-11
Expiration Date
2029-04-06
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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 dual specific immunotoxin that recognizes both the wild-type epidermal growth factor receptor (EGFRwt) and the tumor-specific EGFR variant III mutant (EGFRvIII). This is exemplified by the development of a recombinant bispecific immunotoxin derived from a single chain variable region antibody cloned from a hybridoma producing monoclonal antibody D2C7, fused to a form of Pseudomonas exotoxin A containing a C-terminal KDEL peptide which increases retention within cells. The bispecific immunotoxin exhibits high binding affinity and specificity to both EGFRwt and EGFRvIII proteins as demonstrated by in vitro binding assays and cytotoxicity tests and shows significant anti-tumor efficacy in vivo in subcutaneous tumor models, outperforming monospecific immunotoxins targeting only one form of EGFR.
The problem being addressed is the need for more effective therapeutic agents for treating glioblastoma multiforme and other brain tumors. Current treatments, including surgery, radiation, chemotherapy, and temozolomide, provide limited improvement in patient survival. Monoclonal antibodies and single-chain variable fragments targeting tumor-associated antigens such as EGFRwt or EGFRvIII individually have limitations, including tumor heterogeneity and the potential for resistance when targeting only one antigen. Therefore, there is a continuing need for therapies that can simultaneously target both EGFRwt and EGFRvIII expressed on tumor cells, thereby increasing tumor cell killing and improving therapeutic efficacy.
Claims Coverage
The patent contains two independent claims focusing on bispecific therapeutic antibodies and fusion proteins targeting both EGFRwt and EGFRvIII with high binding affinity and coupled to a cytotoxic agent.
Dual specificity and high-affinity binding
The invention provides therapeutic single-chain variable region antibodies and fusion proteins that bind with a binding affinity of at least 5×10⁸ M⁻¹, as measured by surface plasmon resonance, to both EGFR found on normal human cells and the tumor-specific EGFRvIII mutant.
Fusion with cytotoxic Pseudomonas exotoxin A
The single-chain variable region antibody is covalently linked, optionally as a fusion protein, to a cytotoxic agent which is a form of Pseudomonas exotoxin A, often including a C-terminal KDEL peptide to increase intracellular retention and enhance cytotoxicity.
Disulfide-stabilized antibody structure
The single-chain variable region antibody and the fusion proteins are engineered to be disulfide stabilized for improved folding and stability.
Antibody sequence specificity
The antibody comprises a VH sequence as shown in SEQ ID NO: 1, a VL sequence as shown in SEQ ID NO: 2, and complementarity determining regions (CDR1, CDR2, and CDR3) as shown in SEQ IDs 3 to 8, derived from monoclonal antibody D2C7 clone hybridoma source.
The claims cover therapeutic bispecific antibodies and fusion proteins that target both EGFRwt and EGFRvIII with high affinity, are linked to cytotoxic Pseudomonas exotoxin A (optionally including a KDEL retention signal), and are disulfide stabilized, with specific sequences derived from the D2C7 monoclonal antibody.
Stated Advantages
The bispecific immunotoxin increases killing of tumor cells by binding both EGFRwt and EGFRvIII antigens expressed on tumor cells, thereby enhancing cytotoxicity compared to monospecific antibodies.
Fusion with the KDEL peptide improves intracellular retention of the toxin, increasing its cytotoxic potency.
Disulfide stabilization of the single-chain variable region antibody increases folding efficiency and stability of the immunotoxin.
The invention provides a single therapeutic agent effective against heterogeneous tumor populations expressing both EGFRwt and EGFRvIII, potentially reducing the need for multiple therapies.
The immunotoxin exhibits significant in vivo anti-tumor activity in preclinical models, demonstrating potential for effective brain tumor therapy.
Documented Applications
Treatment of brain tumors, including glioblastoma multiforme, that express EGFRwt and/or EGFRvIII.
Use in in vitro diagnostic methods to determine the proportion of tumor cells expressing EGFRwt and EGFRvIII to guide therapeutic planning.
Potential treatment of other tumors expressing EGFRwt and EGFRvIII, such as breast, head and neck, astrocytoma, and lung cancer.
Use of the immunotoxin via compartmental delivery methods, including delivery to the brain or tumor resection cavity, to target tumors while limiting cytotoxicity to normal tissues.
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