Collected Imunoconjugate Targeting Tissue Factor On Tumor Cells For Immunotherapy

Background and ObjectiveChemotherapy as an important part of cancer treatment is different from surgeryand radiation therapy,which is an important means of comprehensive cancertreatment. Despite the use of multi-drug high-dosechemotherapy and other means tocontinuously improve the efficacy of cancer chemotherapy but for the personaltolerance and the impact of the dose-dependent toxicity of chemotherapy drugs, thetherapeutic effect of chemotherapy drugs is still limited, the therapeutic effects oftraditional chemotherapy drugs appears to be limited.Molecular targeted therapy is anewly developed approach for cancer therapy, the specific combination of themolecular structure specific expressed by the tumor cell, with high selectivity, lowtoxicity which traditional chemotherapy drugs do not have.With the higherperformance,molecular targeted therapeutic drugs have become a critical focus oncancer research.Preclinical studies revealed the processes of tumor angiogenesis, metastasis andinvasiveness are highly dependent on components of the blood coagulation cascade.One of the key proteins in coagulation is tissue factor （TF）. In addition, TF is alsoknown as a mediator of intracellular signaling events that can alter gene expressionpatterns and cell behavior，oncogenic transformation and, possibly there is a linkagewith Cancer Stem Cells，and its expression levels have been correlated with themetastatic potential of many types of tumor.In the course of the development of TF-targeted therapeutics, Hu and Garen havepreviously developed fVII（K341A）/IgG1Fc （Icon） immunoconjugatesforimmunotherapy of cancer.Since the binding of fVII to TF would initiate an extrinsic coagulation cascade,introduced a single mutation （K341A） to fVII peptidetoreduce its coagulation activity while retaining its binding activity to TF. however,animal experiments show the immunoconjugates with a certain influence coagulationin mice。And the recombinant adenoviral vector coding light chain of fVII and humanIgG1Fc showed good anti-angiogenic therapeutic effect in colorectal cancer animalmodel.In this study, molecular cloning techniques was used to construct theeukaryotic expression vector expressing hfVII-Lc and IgG1Fc then transferred toCHO-K1cells in vitro to get hfVII-LC--IgG1Fc immunoconjugates, in order toreduce the impact on coagulation and be safer than adenoviral vector.MethodsPart one The expression of TF in tumor cellsUsing the immunofluorescence method to detect the expression of TF in HT-29andNB4cell line.Part two hfVII-LC+hIgG1Fc protein preparation and purification1. Total RNA was extracted from human peripheral blood mononuclear cells andliver tissue respectively.2. hIgG1Fc, and hfVII-LC gene fragments were amplified using RT-PCR method.3. hfVII-LC PCR amplification products and pcDNA3.1（+） plasmid digested withrestriction endonuclease BamH I, EcoR I,T4DNA ligase reaction for1h, and thentransformed into DH5competent cells, PCR, restriction enzyme digestion andsequencing method was used for identification of positive clones of pcDNA3.1（+）-hfVII-LC, using the same method and the EcoR I and Xho I endonuclease toconstruct pcDNA3.1（+）-hfVII-LC+hIgG1Fc.4. Recombinant plasmid was transfected into CHO-K1cells by lipofectamine2000.and The transfectant clones were selected with G418.5. The positive monoclonal were grown in CHO-K1serum-free medium（SFM）Excel301and the SFM was collected.The hfVII-LC+hIgG1Fc protein wasaffinity purified using Ni-NTA resin.6. Then the immunoconjugate were identified by enzyme linked immunosorbent assay（ELISA） on tissue fctor（TF） affinity and specificity.Part three Screening of TF targeting peptides alternately from Phage DisplayPeptide Library1. Five rounds of panning were alternately conducted by targeting TF and HT-29cell lines which show the detectable TF expression.2. The30phage clones were assessed by enzyme-linked immunosorbent assay（ELISA）. DNA sequencing was provided for the phage clones.3. The affinity of synthetic peptides was verified with competitive inhibition ELISA.The experiment was repeated by the same way.ResultsPart oneImmunofluorescent staining of HT-29colon cancer cells and NB4cells.Strong TFexpression was found in HT-29and NB4cells but negative in the control.Part two1. Extracted total RNA from liver tissue and peripheral blood mononuclear cellsyrespectively by ultraviolet spectroscopy OD260/280in between1.8-2.0,indicating that the RNA of high purity, and RNA in agarose gel electrophoresiswith a clear, complete stripe.2. Han human liver tissue and lymphocytes as a template for amplification hfVII LCand hIgG1Fc DNA fragment confirmed by sequencing and exactly the same withseries gene bank reported.3. pcDNA3.1（+）-hfVII-LC+hIgG1Fc eukaryotic expression vectorUsing specific primers, the target gene fragment was amplified from liver tissueand lymphocytes, and insert it into the pcDNA3.1（+） eukaryotic expressionvector to construct pcDNA3.1（+）-hfVII-LC+hIgG1Fc recombinant vector. ByBamH I, EcoR I and Xho I three restriction endonuclease digestion, showing thatapproximately5.4kb,696bp and456bp stripe, the size of the three bands weredigested vector fragment and two target gene fragments. PcDNA3.1（+）- hfVII-LC+hIgG1Fc positive recombinant DNA sequencing, the sequencingresults the same with the Genebank, and with the encoding6×His tag proteinsequence. Of pcDNA3.1（+）-hfVII-LC the+hIgG1Fc eukaryotic expressionvector was constructed successfully.4. The detection of hfVII-LC of+hIgG1Fc mRNA expressionThe size of456bp hfVII-LC bands, size the the1152bp hfVII-LC+hIgG1Fcbands as well as the size of GAPDH327bp was amplified from total RNA ofCHO-K1-hfVII-LC+hIgG1Fc cells.CHO-K1cells transfected with emptyplasmid in the same RT-PCR amplification conditions and sample,no specificpurpose bands.5. After purification by Ni-NTA resin，the purity and molecular weight was detectedin SDS-PAGE gel.pure target protein molecular weight55-70KD was get fromthe collected serum-free medium, purified through Ni-NTA resin purification.His-tagged proteins was detected by western blot.6. ELISA assay hfVII-LC+hIgG1Fc protein with TF affinity:The detection of five different dilutions0,0.01,0.1,1and10g/mL hf-LC+hIgG1Fc and the TF binding capacity,450/630nm dual wave length weremeasured OD values:（parallel to the control hole average） for0.091±0.0012、0.283±0.0014、0.385±0.03、0.464±0.0116�'�0.765±0.0012respectively（P<0.05）. The higher the OD value the more combination of synthetic immuneconjugates with TF. The OD values are higher for the hole added immunecomplexes than none suggesting that the immune conjugates with TF affinity,and dose-dependented.Part three1. The phages were effectively enriched after five rounds of panning with therecovery improving from2.25×10^-4%to1.32×10-2%.2. In30individual phages, ELISA positive rate was76.7%.3. Five synthetic peptides were verified by ELISA, and the IC₅₀of each peptidesshowed3.25nM、6.72M、3.24×10³M、2.08×10²M and45.77M. Conclusion1. TF in a variety of tumors was abnormal expression also in HT-29and NB4celllines.2. Successfully constructed the eukaryotic hfVII-LC+hIgG1Fc fusion proteinexpression vector.3. hfVII-LC+hIgG1Fc immunoconjugate are obtained，which provide the basis forfurther study on cancer targeted therapy.4. Selected TF targeting peptides with high affinity.