Revision as of 08:44, 18 September 2015

Single-chain variable fragment (Anti-EGFR)

Introduction: Single Chain Variable Fragment of Cetuximab

scFv (Single Chain Variable Fragment)

scFv(single-chain variable fragment) is a fusion protein that consist of variable region of heavy chain(VH) and light chain(VL) of antibody, and connected by a short linker. Here we use the amino acid sequence of GGSSRSSSSGGGGSGGGG as a linker to connect VH and VL.

Features of scFv:
1.specificity:Though remove of the constant regions and add a linker, scFv still maintain the specificity of the original immunoglobulin.
2.Convenient：ScFv is smaller than the entire antibody, so that the loading of production to E.coli could become lower.

Cetuximab

(International Nonproprietary Name (INN) : epidermal growth factor receptor (EGFR) inhibitor) is a chimeric (mouse/human) monoclonal antibody and it specific binds to target antigen epidermal growth factor receptor (EGFR).With high affinity it can prevent ligand binding and activation
We selected the single chain variable fragments (scFv) of monoclonal antibodies Cetuximab and named it Anti-EGFR

Epidermal growth factor receptor

EGFR is a transmembrane tyrosine kinase receptor that regulate the cell division and cell apoptosis.
EGFR is characterized by an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain containing the tyrosine kinase region followed by a carboxyl-terminal tail with tyrosine autophosphorylation sites.
EGFR is overexpressed on the cell surfaces of various solid tumors. Mutations in the gene encoding EGFR that lead to overexpression of this protein will lead to cells proliferate uncontrollably.

Mechanism:

1. Cetuximab inhibition

When Cetuximab binds to the extracellular domain of the EGFR,it may prevents the activation and subsequent dimerization of the receptor, inhibition in signal transduction and anti-proliferative effects. Moreover, this agent may inhibit EGFR-dependent primary tumor growth and metastasis.
They may occur by the decreasing in receptor activation and dimerization.

2.EGFR activation

Firstly, the ligand binding at the extracellular domain of EGFR will lead to the occurance of active homo- or hetero-dimers. Dimerization induces the activation of the tyrosine kinase(TK) domain, leading to autophosphorylation of the receptors on multiple tyrosine residues. This phosphorylation triggers recruitment of a range of adaptor proteins, , followed by a series of intracellular signaling cascades that finally will affect the cell proliferation, apoptosis, invasion, metastasis, and angiogenesis.

1.http://www.sciencedirect.com/science/article/pii/S0959804901002301
2.https://en.wikipedia.org/wiki/Cetuximab
3.[ ]M. Whirl-Carrillo, E.M. McDonagh, J. M. Hebert, L. Gong, K. Sangkuhl, C.F. Thorn, R.B. Altman and T.E. Klein. "Pharmacogenomics Knowledge for Personalized Medicine" Clinical Pharmacology & Therapeutics (2012) 92(4): 414-417

Experiment:

After receiving the DNA sequences from the gene synthesis company, we recombined each OmpA-N-scFv gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the OmpA-N-scFvs. The DNA sequence length of the OmpA-N-scFvs are around 1000~1200 bp. In this PCR experiment, the OmpA-N-scFv products size should be near at 1200~1400 bp. The Fig. showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.

Application of the part:

1.

cell staining experiment:
After creating the part of anti-EGFR，we are able to co-transform them with different fluorescent parts into our E. Cotector.
The next step is to prove that our co-transformed product have successfully displayed scFv of anti-EGFR and expressed fluorescent part.
To prove this, we have decided to undergo the cell staining experiment by using our E. Cotector to detect the EGFR in the cell lines.
Each type of E. Cotector has been co-transformed with two different fluorescent colors ---RFP and GFP

Procedure：
First of all, the main materials that we needed are red and green fluorescent of co-transform E.Coli with scFv of anti-EGFR, red green blue fluorescent E. coli without scFv and the cancer cell line – SKOV-3 that expressed EGFR, for staining used.
SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR, VEGF.
After injecting E.Coli into the wells, we had to shake the plate in darkness for 45minutes. After staining for 45 minutes, we will wash away the unbind E.Coli with PBS solution for a few times before observing the staining result under fluorescent microscope.

Below are our staining result：
Negative control:
As results,there is no green fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli .
There are red and green fluorescent anti-EGFR E.Cotectors stick on the cell’s surfaces as the anti-EGFR probes on E.Cotectors successfully detect and bind with EGFR.

2.

cell staining experiment:
After creating the part of scFv and transforming them into our E. Cotector, we were going to prove that our detectors have successfully displayed scFv of anti-EGFR. To prove this, we have decided to undergo the cell staining experiment by using our E. Cotector to detect the EGFR in the cell lines.

Procedure：
First of all, the main materials that we needed are red green blue fluorescent E.Coli with scFv of anti-EGFR, red and green fluorescent E. coli without scFv and the cancer cell line – SKOV-3 that expressed EGFR,for staining used. SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR.
After injecting E.Coli into the wells, we had to shake the plate in darkness for 45minutes. After staining for 45 minutes, we will wash away the unbind E.Coli with PBS solution for a few times before observing the staining result under fluorescent microscope.

Below are our staining result：
Negative control:
As results,there is no green fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli .

There are red fluorescent anti-EGFR E.Cotectors stick on the cell’s surface as the anti-EGFR probes on E.Cotectors successfully detect and bind with EGFR.

There are green fluorescent anti-EGFR E.Cotectors stick on the cell’s surface as the anti-EGFR probes on E.Cotectors successfully detect and bind with EGFR.

Modeling:

In the modeling part, we discover optimum protein production time by using the genetic algorithm in Matlab.
We want to characterize the actual kinetics of this Hill-function based model that accurately reflects protein production time.
When we have the simulated protein production rate, the graph of protein production versus time can be drawn (Fig.1) (Fig.2) (Fig.3). Thus, we get the optimum protein production time Compared with the simulated protein production rate of time, our experiment data quite fit the simulation.

From this graph, the protein expression reaches peak after growing about 15 hours. This means that the E. Cotector can have maximum efficiency at this point
From this graph, the protein expression reaches peak after growing about 18 hours. This means that the E. Cotector can have maximum efficiency at this point
From this graph, the protein expression reaches peak after growing about 15 hours. This means that the E. Cotector can have maximum efficiency at this point

Sequence and Features