Targeting angiogenesis with shigalike toxin fused to vascular
- Slides: 44
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 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 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 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 gene produces four VEGFs isoforms. 5
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 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 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 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 3) receptors expressed on endothelial cells. 10
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 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 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 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 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 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 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 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
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
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 (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
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. Coli 26
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 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 • 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 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. 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 & 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
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 =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, 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 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 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 proteins in vitro 39
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 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 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 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
- Non vascular vs vascular plants
- Vascular and non vascular difference
- Non vascular plant reproduction
- Tumor angiogenesis
- Ibidi free sample
- Sprouting and intussusceptive angiogenesis
- Biochemical test
- Toxin neutralization test
- Toxin-coregulated pilus
- Bacteria toxin
- The teflon toxin
- Agedema
- Toxin neutralization test
- Erythrogenic toxin
- Botulinum toxin mechanism of action
- Cholera toxin
- Whats a fused sentence
- Fused coronals
- Epiphysis fused
- Permastore
- Comma splices and fused sentences exercise 1 answers
- Fused ring nomenclature
- Whats a comma splice
- The nine vertebrae fused to form two composite bone.
- Fused sentence.
- Fused curriculum
- Fused curriculum
- Rein sentence
- Bischler-napieralski synthesis
- Fused sentence
- Fused deposition modeling history
- How to dry ammonia gas
- Fused sentence definition
- Fused sentence
- Non finite subordinate clause
- Whats a fused sentence
- For upscale american families volvo
- Principles of market targeting
- Focused targeting strategy
- Through market segmentation companies divide large
- Creating value for target customers
- Resume jurnal
- Vertical targeting
- Rural market segmentation
- Sony market segmentation, targeting and positioning