Targeting angiogenesis with shiga-like toxin fused to vascular endothelial growth factor

1. Targeting angiogenesis with shiga-like toxin fused to vascular endothelial growth factor Osama O. Ibrahim, Ph.D Consultant Biotechnology Gurnee IL. USA

2. Angiogenesis • Angiogenesis is the growth of new blood vessels from pre-existing blood vessels. • In healthy adult organisms angiogenesis occurs only in few specialized processes. • Angiogenesis occurs in pathological processes such as the growth of primary and metastatic tumor lesions.

3. Angiogenesis(Cont.) • Angiogenesis is a complex process involving: - Enzymatic degradation of basement membranes of local vanules. - Chemo-tactic migration and proliferation of endothelial cells. - Synthesis of new basement membranes and recruitment of auxiliary cells.

4. Angiogenesis(Cont.) • Interaction of angiogenic factors with cellular receptors regulate angiogenesis: - Angiogenesis is controlled by positive and negative regulators. - Angiogenic factors secreted by tumor cells initiate new vessels formation. - Vascular endothelial growth factors (VEGFs) have emerged as important stimulators of angiogenesis.

5. Vascular endothelial growth factors (VEGFs) • Alternative splicing of RNA transcribed from single VEGF gene produces four VEGFs isoforms.

6. VEGF receptors(KDR/ FLK-1) • VEGF receptor ( KDR/FLk-1)is a receptor tyrosine kinase with tissue distribution restricted primary to endothelial cells. Extracellular ligand binding domain Hydrophobic trans membrane Cytoplasmic membrane

7. Effects of VEGF/VEGFR binding dimerization and activation • - PI3k = phosphateidlinositol-3 kinase. • Akt/PkB= serine-threonine protein kinase. • P38MAPK = P38mitogen activated protein kinase. • Raf= serine-threonine protein kinase. • MEK = tyrosine-threonine kinase (MAPK2) • - ERK = Extracellular signal regulated kinase. Vascular permiability Endothelial cell survival Endothelail cell migration Endo.cell. proliferation • Rini BL, Small EJ. J.Clin Oncol. 2005,23:1028-1043

8. Shega- like toxins(SLTs) • SLTs are produced by the bacteria entero-hemorrhagic E.coli • SLTs are the causative agents of hemolytic uremic syndrome (HUS). • HUS is characterized by renal failure, thrompo-cytopenia, and hemolytic anemia. • Kidneys, pancreas and brains in HUS patients contain swollen and detached endothelial cells.

9. SLT-I structure • SLT-I is a 70 KDa oligomer that consists of one A-subunit and five B-subunits. • The 32 KDa catalytic A-subunit consists of two fragments(A1 and A2) connected by protease cleavage site. • The B-subunit bind to a carbohydrate receptor expressed on endothelial cells. Arg248-Val-Ala-Arg251

10. SLT-I receptor • The B-subunit of SLT-I binds with high affinity to globotriosyl-ceramide (Gb3) receptors expressed on endothelial cells.

11. SLT-I uptake and processing • SLT-I is endocytosed in clathrin-coated pits and delivered to lysosomal compartment. • In the lysosomal compartment the A-subunits is cleaved into A1 and A2 fragments by the protease furin. • Released A1 fragments are trans-located to the cytosol. Lysosomal compartment

12. SLT-I mechanism of action • The A1 fragment has N-glycosidase activity. • The A1cleaves single adenosine in the position 4324 of 28S rRNA in the 60S ribosome unit. • The cleavage inhibits the binding of the EF-I /aminocyl- tRNA complex to 60S rRNA unit, therefore blocking protein synthesis and causing endothelial cell death. Cytosol

13. Hypothesis • Fusing the A-subunit or A1 fragment of shiga-like toxin (SLT) to vascular endothelial growth factor (VEGF) will produce a highly cytotoxic fusion protein (VEGF/SLT) that will selectively target endothelial cells at site of angiogenesis.

14. Rationale • Endothelial cells are extremely sensitive to SLTs. • Anticipate that SLTs fused to VEGF will preferentially target endothelial cells with a high density of KDR/FLK-1 Receptors. • Fusion SLT to VEGF instead of chemical conjugation may better preserve the receptor binding activity of VEGF.

15. Targeting angiogenesis • Targeting tumor vasculature is an attractive approach. • The goal is to attack the endothelial cells in order to destroy the tumor vascular system and starve the tumor. • How can the tumor vasculature be attacked without damaging normal vasculature? (specific target).

16. Advantage of VEGF as a ligand for targeting angiogenesis • The tissue distribution of VEGF receptor KDR/FLk-1, is restricted primarily to endothelial cells. • Proliferating endothelial cells express higher levels of KDR/FLK-1 than quiescent endothelial cells. • Over expressing of KDR/FLK-1receptor can be utilized for selective targeting of proliferated endothelial cell at sites of angiogenesis.

17. Research goals • Develop SLT and VEGF fusion protein molecules that will selectively target and inhibit the growth of cells over-expressing the KDR/FLk-1 receptor. • Demonstrate the VEGF/SLT fusion proteins retains biochemical activity of the individual toxin and legend moieties. • Demonstrate selectivity of VEGF/SLT fusion proteins to endothelial cells expressing KDR/FLk-1.

18. Experimental design • Construct plasmids for the expression of VEGF121 fusion proteins containing the entire A-subunit or A1 fragment of SLT-I. • Express and purify VEGF121/A and VEGF121 /A1 fusion proteins from E.coli. • Evaluate the biochemical activities of VEGF/SLT fusion proteins. • Evaluate the growth inhibitory activity and selectivity of VEGF/SLT fusion proteins in vitro.

19. Construct plasmid for the expression of VEGF121/A and VEGF121/A1

20. SLT-I donor plasmid pJB144 • SLT-I was sub cloned from bacteriophage HB19 and inserted into EcoRI and PstI restriction site of pTZ18 • The constructed plasmid contain 2kb holotoxin DNA of SLT-I (1.2kb A and 0.8 kb B-subunits)

21. Construction of SLT Primers SLT-A (L) SLT-A1 (S)

22. PCR amplification of DNA encodingSLT-A holotoxin and A1 fragment 0.87 kb (A) 0.6 kb (A1) SLT DNA Extracted from E. coli DH5α

23. Construction ofVEGF121/A and VEGF121/A1 E.coli DH5 SLT-I A (L): 879 bp = 293 aa SLT-I A1(S): 591 bP = 197 aa

24. Construction of primers for VEGF121/A and VEGF121/A1 DNA BgIII BgIII

25. PCR screening of VEGF121/A and VEGF121/A1 constructs carbenicillin Kanamycin • 2,4,8 16 & 9 VEGF121/A DNA fragments (1.25 kb). • 4,9 & 10,13 VEGF121/A1 DNA fragments (0.97kb). • Marker SLT-A DNA fragments (0.87 kb) .

26. Express and purify of VEGF121/A and VEGF121/A1 fusion proteins from E.Coli

27. Mechanism of protein expression by pET vectors in host cells of E coli BL21 (DE3) pLysS The figure illustrates the elements which control the transcription recombinant genes inserted into pET vectors • IPTG induction results in high level of T7RNA polymerase. • T7RNA polymerase drive the transcription of the target gene on pET plasmids. • Un induced cells controlled by pLysS / E plasmid encoding T7 lysozyme.

28. Construction of fusion proteins expressed from pET-29a and pET-32a plasmids VEGF121/A = 51KD (L) VEGF121/A1 = 42 kD (S)

29. Expression of VEGF121/A and VEGF121/A1 from pET-29a vectors • Clone No. 13 expressed 42 kDa VEGF121/A1 fusion protein. • Clone Nos. 18 & 9 are negative.

30. Expression of VEGF121/A and VEGF121/A1 from pET-32a vectors Clone No. 10,9 & 4 expressed 42 kDa VEGF121/A1 fusion protein Clone No. 2,4,8 &16 expressed 51 kDa VEGF121/A fusion protein.

31. Distribution of fusion proteins between soluble and insoluble forms in E.coli lysates pET29-a pET-32a pET-32a 50 % of the 42 kDa is insoluble 100 % of 51 kDa is insoluble

32. Kinetics of VEGF121/A1fusion Soluble & Insoluble protein induction at 250 & 370 C S= soluble I= Insoluble Fusion proteins were induced more rapidly at 370C

33. VEGF121/A recovery after dialysis with different buffers at pH 9.0.

34. Evaluate the biochemical activities of VEGF121/A and VEGF121/A1 fusion proteins

35. S-tag assay for the concentration of VEGF121/A1 and VEGF121/A after dialysis S-Tag =15 a.a. S-protein .=104 a.a. Ribonuclase = 119a.a Substrate is Poly-C S-tag rapid assay kit

36. Inhibition of luciferase translation by VEGF/SLT fusion proteins N-glycosidase activity in VEGF121/A1, VEGF121/A and fusion proteins inhibited protein synthesis of luciferase enzyme in vitro system compared to sVEGF121 and buffer solution. [Rabbit reticulocyte lysate system]

37. Kinetics and dose dependent of luciferase inhibition by VEGF/A & VEGF/A1 Purified VEGF121/A incubated at 0.8nM & 8.0 nM conc. In rabbit reticulocyte in vitro with 1ug firefly mRNA as reporter gene Purified VEGF121/A1 incubated at 0.8nM to 80 nM conc. In rabbit reticulocyte in vitro with 1ug firefly mRNA as reporter gene

38. Incubation of /KDR autophosphorylation by VEGF/SLTfusion proteins Immunoblot system • VEGF121/A1 fusion protein induced KDR autophosphorelation at concentration as low as 2.5nm which is comparable with recombinant VEGF165 • VEGF121/A was not active in this assay.

39. Evaluate the inhibitory activity and selectability of VEGF121/A and VEGF121/A1 fusion proteins in vitro

40. Growth inhibition of 293/KDR by VEGF /SLT fusion proteins Higher inhibition • VEGF121/A1 fusion protein significantly inhibited 293/KDR cells. • VEGF121/A fusion protein did not affect 293/KDR cell growth. • The parental cell 293 [human embryonic kidney cell line] showed not affect.

41. Biological activities of VEGF /SLTfusion proteins • VEGF121/A and VEGF121/A1 fusion proteins inhibit protein translation in a cell-free system. • VEGF121/A1 fusion proteins induced autophosphorylation of KDR/FLK-1 receptors. • VEGF121/A1 fusion proteins induced selectively inhibit growth of KDR/FLK-1 expressing cells.

42. Conclusions • Plasmid encoding VEGF121 fused to the A subunit or A1fragment of SLT-I were constructed. • Fusion proteins expressed in E.coli (DE3) pLysS cells were recovered in highly purified form from cell inclusion bodies. • VEGF121/A1 fusion protein are biochemically and biologically active. • VEGF121/A1 inhibit the growth of human embryonic kidney cell 293/KDR.

43. Summary • The biological activities demonstrated for VEGF/SLT fusion proteins in vitro suggest that they can be applied to selectively inhibit angiogenesis in vitro. • The result from this project provide a basis to develop VEGF/SLT fusion proteins for therapeutic application against humanpathologies that depend on angiogenesis.

44. Thank you for your attention