Targeting angiogenesis with shigalike toxin fused to vascular

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Targeting angiogenesis with shiga-like toxin fused to vascular endothelial growth factor Osama O. Ibrahim,

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

Angiogenesis is the growth of new blood vessels from pre-existing blood vessels. u In

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

Angiogenesis (Cont. ) u Angiogenesis is a complex process involving: - Enzymatic degradation of

Angiogenesis (Cont. ) u 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. 3

Angiogenesis (Cont. ) u Interaction of angiogenic factors with cellular receptors regulate angiogenesis: -

Angiogenesis (Cont. ) u 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. 4

Vascular endothelial growth factors (VEGFs) u Alternative splicing of RNA transcribed from single VEGF

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

VEGF receptors (KDR/ FLK-1) Extracellular ligand binding domain Hydrophobic trans membrane Cytoplasmic membrane u

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

Effects of VEGF/VEGFR binding dimerization and activation Endothelial cell survival Endothelail cell migration Vascular

Effects of VEGF/VEGFR binding dimerization and activation Endothelial cell survival Endothelail cell migration Vascular permiability - PI 3 k = phosphateidlinositol-3 kinase. - Akt/Pk. B = serine-threonine protein kinase. - P 38 MAPK = P 38 mitogen activated protein kinase. - Raf = serine-threonine protein kinase. Endo. cell. proliferation - MEK = tyrosine-threonine kinase (MAPK 2) - ERK = Extracellular signal regulated kinase. v Rini BL, Small EJ. J. Clin Oncol. 2005, 23: 1028 -1043 7

Shega- like toxins (SLTs) SLTs are produced by the bacteria entero-hemorrhagic E. coli u

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

SLT-I structure Arg 248 -Val-Ala-Arg 251 SLT-I is a 70 KDa oligomer that consists

SLT-I structure Arg 248 -Val-Ala-Arg 251 SLT-I is a 70 KDa oligomer that consists of one A-subunit and five Bsubunits. u The 32 KDa catalytic A-subunit consists of two fragments(A 1 and A 2) connected by protease cleavage site. u The B-subunit bind to a carbohydrate receptor expressed on endothelial cells. u 9

SLT-I receptor u The B-subunit of SLT-I binds with high affinity to globotriosyl-ceramide (Gb

SLT-I receptor u The B-subunit of SLT-I binds with high affinity to globotriosyl-ceramide (Gb 3) receptors expressed on endothelial cells. 10

SLT-I uptake and processing Lysosomal compartment u u u SLT-I is endocytosed in clathrin-coated

SLT-I uptake and processing Lysosomal compartment u u u SLT-I is endocytosed in clathrin-coated pits and delivered to lysosomal compartment. In the lysosomal compartment the A-subunits is cleaved into A 1 and A 2 fragments by the protease furin. Released A 1 fragments are trans-located to the cytosol. 11

SLT-I mechanism of action Cytosol u u u The A 1 fragment has N-glycosidase

SLT-I mechanism of action Cytosol u u u The A 1 fragment has N-glycosidase activity. The A 1 cleaves single adenosine in the position 4324 of 28 S r. RNA in the 60 S ribosome unit. The cleavage inhibits the binding of the EF-I /aminocyl- t. RNA complex to 60 S r. RNA unit, therefore blocking protein synthesis and causing endothelial cell death. 12

Hypothesis u Fusing the A-subunit or A 1 fragment of shiga-like toxin (SLT) to

Hypothesis u Fusing the A-subunit or A 1 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. 13

Rationale Endothelial cells are extremely sensitive to SLTs. u Anticipate that SLTs fused to

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

Targeting angiogenesis Targeting tumor vasculature is an attractive approach. u The goal is to

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

Advantage of VEGF as a ligand for targeting angiogenesis The tissue distribution of VEGF

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

Research goals Develop SLT and VEGF fusion protein molecules that will selectively target and

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

Experimental design Construct plasmids for the expression of VEGF 121 fusion proteins containing the

Experimental design Construct plasmids for the expression of VEGF 121 fusion proteins containing the entire A-subunit or A 1 fragment of SLT-I. u Express and purify VEGF 121/A and VEGF 121 /A 1 fusion proteins from E. coli. u Evaluate the biochemical activities of VEGF/SLT fusion proteins. u Evaluate the growth inhibitory activity and selectivity of VEGF/SLT fusion proteins in vitro. 18 u

Construct plasmid for the expression of VEGF 121/A and VEGF 121/A 1 19

Construct plasmid for the expression of VEGF 121/A and VEGF 121/A 1 19

SLT-I donor plasmid p. JB 144 Ø SLT-I was sub cloned from bacteriophage HB

SLT-I donor plasmid p. JB 144 Ø SLT-I was sub cloned from bacteriophage HB 19 and inserted into Eco. RI and Pst. I restriction site of p. TZ 18 Ø The constructed plasmid contain 2 kb holotoxin DNA of SLT-I (1. 2 kb A and 0. 8 kb B-subunits) 20

Construction of SLT Primers SLT-A (L) SLT-A 1 (S) 21

Construction of SLT Primers SLT-A (L) SLT-A 1 (S) 21

PCR amplification of DNA encoding SLT-A holotoxin and A 1 fragment 0. 87 kb

PCR amplification of DNA encoding SLT-A holotoxin and A 1 fragment 0. 87 kb (A) 0. 6 kb (A 1) SLT DNA Extracted from E. coli DH 5α 22

Construction of VEGF 121/A and VEGF 121/A 1 E. coli DH 5 SLT-I A

Construction of VEGF 121/A and VEGF 121/A 1 E. coli DH 5 SLT-I A (L): 879 bp = 293 aa SLT-I A 1(S): 591 b. P = 197 aa 23

Construction of primers for VEGF 121/A and VEGF 121/A 1 DNA Bg. III 24

Construction of primers for VEGF 121/A and VEGF 121/A 1 DNA Bg. III 24

PCR screening of VEGF 121/A and VEGF 121/A 1 constructs carbenicillin • 2, 4,

PCR screening of VEGF 121/A and VEGF 121/A 1 constructs carbenicillin • 2, 4, 8 16 & 9 • 4, 9 & 10, 13 • Marker Kanamycin VEGF 121/A DNA fragments (1. 25 kb). VEGF 121/A 1 DNA fragments (0. 97 kb). SLT-A DNA fragments (0. 87 kb). 25

Express and purify of VEGF 121/A and VEGF 121/A 1 fusion proteins from E.

Express and purify of VEGF 121/A and VEGF 121/A 1 fusion proteins from E. Coli 26

Mechanism of protein expression by p. ET vectors in host cells of E coli

Mechanism of protein expression by p. ET vectors in host cells of E coli BL 21 (DE 3) p. Lys. S The figure illustrates the elements which control the transcription recombinant genes inserted into p. ET vectors 1. IPTG induction results in high level of T 7 RNA polymerase. 2. T 7 RNA polymerase drive the transcription of the target gene on p. ET plasmids. 3. Un induced cells controlled by p. Lys. S / E plasmid encoding T 7 lysozyme. 27

Construction of fusion proteins expressed from p. ET-29 a and p. ET-32 a plasmids

Construction of fusion proteins expressed from p. ET-29 a and p. ET-32 a plasmids VEGF 121/A = 51 KD (L) VEGF 121/A 1 = 42 k. D (S) 28

Expression of VEGF 121/A and VEGF 121/A 1 from p. ET-29 a vectors •

Expression of VEGF 121/A and VEGF 121/A 1 from p. ET-29 a vectors • Clone No. 13 expressed 42 k. Da VEGF 121/A 1 fusion protein. • Clone Nos. 18 & 9 are negative. 29

Expression of VEGF 121/A and VEGF 121/A 1 from p. ET-32 a vectors Clone

Expression of VEGF 121/A and VEGF 121/A 1 from p. ET-32 a vectors Clone No. 10, 9 & 4 expressed 42 k. Da VEGF 121/A 1 fusion protein Clone No. 2, 4, 8 &16 expressed 51 k. Da VEGF 121/A fusion protein. 30

Distribution of fusion proteins between soluble and insoluble forms in E. coli lysates p.

Distribution of fusion proteins between soluble and insoluble forms in E. coli lysates p. ET 29 -a p. ET-32 a 50 % of the 42 k. Da is insoluble p. ET-32 a 100 % of 51 k. Da is insoluble 31

Kinetics of VEGF 121/A 1 fusion Soluble & Insoluble protein induction at 250 &

Kinetics of VEGF 121/A 1 fusion Soluble & Insoluble protein induction at 250 & 370 C S= soluble I= Insoluble Fusion proteins were induced more rapidly at 370 C 32

VEGF 121/A recovery after dialysis with different buffers at p. H 9. 0. 33

VEGF 121/A recovery after dialysis with different buffers at p. H 9. 0. 33

Evaluate the biochemical activities of VEGF 121/A and VEGF 121/A 1 fusion proteins 34

Evaluate the biochemical activities of VEGF 121/A and VEGF 121/A 1 fusion proteins 34

S-tag assay for the concentration of VEGF 121/A 1 and VEGF 121/A after dialysis

S-tag assay for the concentration of VEGF 121/A 1 and VEGF 121/A after dialysis S-Tag =15 a. a. S-protein . =104 a. a. Ribonuclase = 119 a. a Substrate is Poly-C S-tag rapid assay kit 35

Inhibition of luciferase translation by VEGF/SLT fusion proteins N-glycosidase activity in VEGF 121/A 1,

Inhibition of luciferase translation by VEGF/SLT fusion proteins N-glycosidase activity in VEGF 121/A 1, VEGF 121/A and fusion proteins inhibited protein synthesis of luciferase enzyme in vitro system compared to s. VEGF 121 and buffer solution. [Rabbit reticulocyte lysate system]

Kinetics and dose dependent of luciferase inhibition by VEGF/A & VEGF/A 1 Purified VEGF

Kinetics and dose dependent of luciferase inhibition by VEGF/A & VEGF/A 1 Purified VEGF 121/A incubated at 0. 8 n. M & 8. 0 n. M conc. In rabbit reticulocyte in vitro with 1 ug firefly m. RNA as reporter gene Purified VEGF 121/A 1 incubated at 0. 8 n. M to 80 n. M conc. In rabbit reticulocyte in vitro with 1 ug firefly m. RNA as reporter gene 37

Incubation of /KDR autophosphorylation by VEGF/SLT fusion proteins Immunoblot system • VEGF 121/A 1

Incubation of /KDR autophosphorylation by VEGF/SLT fusion proteins Immunoblot system • VEGF 121/A 1 fusion protein induced KDR autophosphorelation at concentration as low as 2. 5 nm which is comparable with recombinant VEGF 165 • VEGF 121/A was not active in this assay. 38

Evaluate the inhibitory activity and selectability of VEGF 121/A and VEGF 121/A 1 fusion

Evaluate the inhibitory activity and selectability of VEGF 121/A and VEGF 121/A 1 fusion proteins in vitro 39

Growth inhibition of 293/KDR by VEGF /SLT fusion proteins Higher inhibition • VEGF 121/A

Growth inhibition of 293/KDR by VEGF /SLT fusion proteins Higher inhibition • VEGF 121/A 1 fusion protein significantly inhibited 293/KDR cells. • VEGF 121/A fusion protein did not affect 293/KDR cell growth. • The parental cell 293 [human embryonic kidney cell line] showed not affect. 40

Biological activities of VEGF /SLT fusion proteins VEGF 121/A and VEGF 121/A 1 fusion

Biological activities of VEGF /SLT fusion proteins VEGF 121/A and VEGF 121/A 1 fusion proteins inhibit protein translation in a cell-free system. u VEGF 121/A 1 fusion proteins induced autophosphorylation of KDR/FLK-1 receptors. u VEGF 121/A 1 fusion proteins induced selectively inhibit growth of KDR/FLK-1 expressing cells. u 41

Conclusions Plasmid encoding VEGF 121 fused to the A subunit or A 1 fragment

Conclusions Plasmid encoding VEGF 121 fused to the A subunit or A 1 fragment of SLT-I were constructed. u Fusion proteins expressed in E. coli (DE 3) p. Lys. S cells were recovered in highly purified form from cell inclusion bodies. u VEGF 121/A 1 fusion protein are biochemically and biologically active. u VEGF 121/A 1 inhibit the growth of human embryonic kidney cell 293/KDR. u 42

Summary u u The biological activities demonstrated for VEGF/SLT fusion proteins in vitro suggest

Summary u u 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 human pathologies that depend on angiogenesis. 43

Thank you for your attention 44

Thank you for your attention 44