Human bispecific EGFRvIII and CD3 antibody engaging molecules
Inventors
Bigner, Darell D. • Sampson, John • Kuan, Chien-Tsun • CAI, Mingqing • CHOI, Bryan D. • Gedeon, Patrick C. • Pastan, Ira H.
Assignees
National Institutes of Health NIH • Duke University
Publication Number
US-10053514-B2
Publication Date
2018-08-21
Expiration Date
2034-07-09
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Abstract
We have constructed a polynucleotide encoding a bispecific antibody engaging molecule which has one arm that specifically engages a tumor cell which expresses the human EGFRvIII mutant protein on its surface, and a second arm that specifically engages T cell activation ligand CD3. The polynucleotide is codon optimized for expression in CHO cells. The subunits of the engaging molecules are organized to achieve greater efficiency. These are therapeutic agents.
Core Innovation
This invention relates to bispecific polypeptides designed for cancer therapy, specifically targeting cancers that express the EGFRvIII mutation. The bispecific molecule comprises a first human single chain variable region that binds specifically to the mutant EGFRvIII antigen on tumor cells and a second human single chain variable region that binds to the T cell activation ligand CD3. These two variable regions are arranged in series with specific linker sequences to enhance functionality and expression. The bispecific polypeptide can be encoded by a codon-optimized polynucleotide for efficient expression in Chinese Hamster Ovary (CHO) cells, representing therapeutic agents for directing T cell-mediated cytolytic activity towards EGFRvIII-expressing tumors.
The problem being addressed is the uniformly fatal nature of glioblastoma multiforme (GBM) and other tumors expressing EGFRvIII, which remain resistant to existing therapies such as surgery, radiation, and chemotherapy. Existing bispecific T-cell engaging molecules demonstrate potent tumor regression but are limited by the lack of tumor-specific targets frequently and homogeneously expressed. EGFRvIII is identified as a tumor-specific, constitutively active mutant receptor that is prevalent in GBM and other cancers, making it an ideal target. However, previous bispecific molecules have challenges such as murine-derived components which may induce neutralizing antibodies limiting repeated use.
To overcome these issues, this invention presents fully human bispecific T cell engaging molecules that incorporate human single chain variable fragments specific to EGFRvIII and to CD3. The molecules are constructed with optimized domain orders and linker sequences to promote secretion and function. The polynucleotides encoding these bispecific molecules are codon-optimized for expression in CHO cells. The molecules are selectively reactive, capable of engaging cytotoxic T cells to kill EGFRvIII-expressing tumor cells, demonstrated in vitro and in vivo by inhibition of tumor growth and induction of T cell-mediated cytotoxicity. Methods to generate such bispecific molecules include recombinant expression in various host cells and engineering of fully human variable regions from vaccinated patients or human antibody phage libraries to reduce immunogenicity.
Claims Coverage
The patent contains one independent claim that defines the bispecific polypeptide and its key features, and other dependent claims and methods that relate to polynucleotide encoding, production, and therapeutic use. There are four main inventive features in the independent claim covering the structural and functional aspects of the bispecific molecule and its linkers.
Bispecific polypeptide structure
The bispecific polypeptide comprises a first single chain human variable region binding specifically to EGFRvIII, made up of segments as set forth in SEQ ID NO: 5 and 6 in amino to carboxy order, in series with a second single chain human variable region binding to T cell activation ligand CD3, comprising segments as set forth in SEQ ID NO: 7 and 8 in amino to carboxy order.
Linker and spacer sequences between variable domains
The variable regions are connected via specific linker sequences, wherein a (G4S)3 sequence (SEQ ID NO: 9) intervenes between each segment, and a spacer polypeptide (G4S) (SEQ ID NO: 10) links the first and second single chain variable regions, optimizing molecular assembly and function.
Codon-optimized polynucleotide encoding
A polynucleotide encoding the bispecific polypeptide is defined, including a codon optimized sequence (SEQ ID NO: 1) for expression in CHO cells that comprises segments encoding the variable regions and linker sequences.
Methods of manufacture and therapeutic use
Methods include culturing a cell containing the polynucleotide to express and then collecting the bispecific polypeptide from cells or culture medium, and administering the bispecific polypeptide to a patient with an EGFRvIII-expressing tumor to induce a cytolytic T cell response against the tumor.
The claims define a bispecific human antibody molecule targeting EGFRvIII and CD3 with specific domain sequences and linkers, a corresponding codon-optimized encoding polynucleotide, methods for manufacturing the molecule via recombinant expression, and its therapeutic application in inducing T cell cytotoxicity against EGFRvIII-expressing tumors.
Stated Advantages
Provides a fully human bispecific T cell engaging molecule that reduces the risk of neutralizing antibody formation, allowing repeated administrations.
Specifically targets the tumor-specific EGFRvIII antigen that is prevalent and consistently expressed in multiple cancers, including glioblastoma multiforme.
Capable of efficiently recruiting and activating cytotoxic T cells to selectively kill EGFRvIII-expressing tumor cells.
Codon optimization enables efficient recombinant expression in CHO cells, facilitating production.
The bispecific T cell engaging molecules show dose-dependent inhibition of tumor growth in in vivo models, demonstrating therapeutic potential.
Documented Applications
Treatment of cancers expressing EGFRvIII, including glioblastoma multiforme, breast tumors, and lung tumors.
Use as therapeutic agents to induce cytolytic T cell responses targeting EGFRvIII-expressing tumor cells.
Production of bispecific molecules in recombinant host cells such as CHO cells for therapeutic protein manufacturing.
Parenteral administration for tumor therapy, including intravenous administration or direct delivery into body cavities or organs such as the brain.
Use in preclinical models such as immunocompromised mice bearing human tumor xenografts and human CD3 transgenic mice for efficacy and toxicity evaluation.
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