1. A novel bispecific ligand-directed toxin designed to simultaneously target EGFR on human glioblastoma cells and uPAR on tumor neovasculature
Brandon Kats

2. Introduction
Glioblastoma multiforme (GBM) is the most common of all primary intracranial malignancies
Targeted toxins, which are catalytic protein toxins directed by a ligand that recognizes the over-expressed markers on cancerous cells, are becoming more widely used for GBM treatment because glioblastoma rarely metastasizes outside of the cranium

3. BLT’s
BLT’s or bispecific ligand directed toxins are made by linking two of these ligands to a toxin
They increase targeting capability and potency by targeting two types of cells but reduce toxicity

4. EGFATFKDEL
EGFATFKDEL is one of these BLT’s designed to target epidermal growth factor receptors (EGFR) and urokinase receptors (uPAR)
It is then linked to exotoxin A (PE38) which is a known anticancer toxin
EGFR promotes processes responsible for tumor growth and progression, including proliferation, angiogenesis, invasion, metastasis, and inhibition of apoptosis and uPAR is found on the endothelial neovasculature that sustains tumors’ high metabolic demand
So by targeting these two receptors it destroys both solid tumors and the tumor-associated neovasculature

5. Construction of EGFATFKDEL and EGFATFKDEL 7mut
Synthesis and assembly of hybrid genes encoding the single-chain EGFATFKDEL was accomplished using DNA-shuffling and DNA cloning techniques.
To create an EGFATFKDEL molecule with decreased immunogenicity eight amino acids representing the seven major epitopes on PE38 were mutated decreasing immune response to the second generation drug by 80-90%

6. The human glioblastoma cell lines U87 MG, U118 MG, U373 MG, and T98G, human umbilical vein endothelial cells (HUVECs), and Raji cancerous B cells, were used as a control cell line
Cultures were incubated and grown and then tested in the lab as well as being transferred to test mice

7. In Vitro Study
In the lab the cancer cultures were placed in well plates and then dosed with EGFATFKDEL 7mut. After 72 hours the cultures were treated so that individual cells could be counted.
In untreated wells cancerous cell counts ranged from cpm while those treated had none.
Further tests were conducted to determine whether EGFATFKDEL 7mut actually killed the cancer and it was found that it does.

8. In Vivo Study
Mice were injected with 3 × 106 U87-luc cells. Once tumors reached approximately 0.03 cm3 (day 6), mice were divided into groups and treated with EGFATFKDEL 7mut, ATFKDEL, or 2219ARLKDEL.
Treatment groups were given 2μg of toxin intratumorally 4 times a week for 5 weeks
Tumor size was measured using a digital caliper, and volume was determined as a product of length, width, and height

9. Results
EGFATFKDEL was tested against U-87 cells as well as endothelial cell lines separately to determine if it would kill the cells but also target and kill the neovasculature.
Both tests showed that EGFATFKDEL was the most effective at not only destroying the tumor but keeping the cancerous cells from reproducing by limiting blood supply to the tumor

10. Selective activity of EGFATFKDEL against glioblastoma cells in vitro

11. EGFATFKDEL mutation
Tests were performed on mice to determine if the mutated EGFATFKDEL performed better than unmutated.

12. In vivo efficacy of EGFATFKDEL 7mut against U87-luc tumors
In vivo efficacy of EGFATFKDEL 7mut against U87-luc tumors. Weekly images show the luciferase activity of U87-luc tumors in individual mice during and after intratumoral treatment

13. Discussion
Treatments worked and four of the eight mice treated with EGFATFKDEL 7mut survived past day 110 – 70 days after treatment had ended. This shows that the treatment is durable.