Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as ‘vasculogenic mimicry’ (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody–endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear β-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases

Production of genetically engineered antibodies in myeloma cells: Design, expression, and applications

The criteria for producing recombinant antibodies in myeloma cells are discussed. Heavy- and light-chain genes are cloned into expression vectors with different selectable markers and transfected simultaneously into recipient cells. Expression vectors that contain not only the elements required for gene expression but features that facilitate the exchange of variable or constant region genes have been designed. Obtaining variable region genes of interest has been greatly facilitated by the advent of the polymerase chain reaction. Murine variable region genes may be joined to human constant regions, whose domain structure has been exploited by the design of cloning “cassettes.” In these, unique restriction sites placed between constant region exons allow for domain duplication, deletion, or exchange. Immunoglobulin fusion proteins, in which antigen specificities have been fused to heterologous proteins, have been produced successfully. Certain antibody properties have also been altered by site-directed mutagenesis. These manipulations not only have provided useful insights into structure-function relationships in antibodies, but enable the production of antibodies with altered or novel properties

Genetically engineered antibodies and their application to brain delivery

Techniques of genetic engineering and expression have been applied to the production of antibodies in a variety of expression systems. Combinatorial libraries produced in bacteriophage may present an alternative to animal immunization as a source of antigen-binding specificities. Transfectomas which express genetically engineered antibody genes provide one approach to overcoming some of the limitations inherent in classical monoclonal antibodies. Novel antibodies have been produced with a variety of modifications: as chimeric antibodies, as ‘humanized’ antibodies, with catalytic groups, as bifunctional or fusion proteins and as functional fragments such as Fabs or Fvs. The domain structure of the antibody is favorable to such manipulation; the novel proteins often retain their antibody-derived activity and acquire new properties as well. Chimeric and CDR-grafted antibodies have been effective in immunotherapy, but problems of immunogenicity remain. Careful analysis and comparison of effector functions among immunoglobulin isotypes may be applied to the design of effective therapeutic antibodies. In addition, antibody combining specificities can be joined with non-immunoglobulin sequences thereby providing properties not usually found in antibodies. In particular, antibodies fused with the growth factors insulin-like growth factor(IGF)-1, IGF-2 and transferrin have shown increased uptake into the brain parenchyma. These fusion proteins provide a family of reagents with many potential applications