Construction Of Multifunctional Nano-antibody And The Study Of The Antitumor Mechanism

Since anti-CD20 antibody(rituximab)was approved by Food and Drug Administration(FDA)in 1997,more than dozens of mAbs have been used for cancer therapy.Even though monoclonal antibodies have achieved certain curative effect in cancer therapy,yet its therapeutic effect is mild.In order to improve the therapeutic effect,many strategies have been developed by conjugation with chemotherapeutic drugs,toxins,immune factors and radionuclides or preparation of multivalent antibodies by crossMAb technology.However,these formulations of antibodies have higher cytotoxicity,lower tumor accumulation,unknown mechanism and drug resistance.In recent years,nanomedicines have attracted much attention for significant tumor accumulation because of enhanced permeation retention effect(EPR).At present,target therapy based on nanotechnology have mainly focused on antibody-mediated targeting system.But few studies were reported about novel nanoscale antibody(nano-antibody,nAb)used by nanotechnology of structural analysis.In our previous study,type I and type II anti-CD20 mAbs were simultaneously conjugated to one nanocarrier to form CD20 mAbs nanoclusters.Compared with single mAbs,the nanoarrays showed stronger apoptosis effect.we believed that the additional apoptosis derived from extracellular crosslink of antigen.So we suspect that further crosslink or limitation of the CD20 molecule on the surface of target cell could promote cell apoptosis or not?In this study,we connected the type I anti-CD20 antibody to PEI of different molecular(PEIn-R,n = 1.8,10,25,50,750 kDa)to achieve different degrees of antigen crosslink.At the same time,the anti-CD20 mAbs were conjugated to long chain polymers(PI-4-R)with temperature-sensitive properties by chemical synthetic way.The temperature-sensitive polymers(poly(N-isopropylacrylamide),PNIPAM)shrinked with the increase of temperature which possesses a lower critical solution temperature(LCST),thus resulting in the further restriction of antigen.Different chain length PEIn-R and PI-4-R were successful synthetised and had similar structure of comb which could be confirmed by dynamic/static laser light scattering(DLLS/SLLS),transmission electron microscopy(TEM)and SDS-PAGE.Compared with nonconjugated PI-4,the LCST of PI-4-R increased nearly 2 ?,which mainly due to a large number of hydrophilic mAb ligands conjugated with the PI-4 chain,and the force of ionic increased.It also indirectly confirmed that the conjugation of anti-CD20 mAbs onto PI-4.About 250 and 300 antibodies were anchored on PEI750000-R and PI-4-R respectively which were confirmed by SLLS.Then,we detected the binding affinity and off-rate of nAb by second antibody labeling assay in Raji cells.Results demonstrated that there was no significant difference of binding activity between free Rituximab and nAb,but obvious decrease in off-rate of n Ab,which may give the reason of its effective antitumor effects.For vitro study,we separated human serum and peripheral blood lymphocytes to evaluate complement-dependent cytotoxicity(CDC)and antibody-dependent cellular cytotoxicity(ADCC)induced by nAb.Results showed that six kinds of nAb that we made exhibited similar effects as Rituximab in CDC and ADCC,which demonstrated that the nAb we prepared didn't change the structure and fuction of original mAb.When detecting the effect of nAb on programmed cell death(PCD)of cancer cells by flow cytometry,We found that compared with Rituximab,nAb achieved higher PCD,which was similar to the function of type II anti-CD20 antibody.Moreover,the PCD effect enhanced with the length of the chains of nano-antibody.Meanwhile,under the condition of the same antigen crosslink,the PCD effect induced by nano-antibody enhanced in higher temperature.Subsequently,we discussed and analyzed the mechanism of PCD by flow cytometry and confocal microscopy.When the antigen crosslink degree was further shifted or the antigen was limited,the fracture and diffusion of lysosomes became more significant and more cathepsin B was released from the lysosomes.Then,cathepsin B acted on the related protein of mitochondria and enhanced its permeability,which released cytochrome C.Casepase apoptosis pathway was activated and induced cell death.The lysosome-dependent apoptosis effect had been confirmed with Western Blot.In vivo,we further evaluated the therapeutic effect of nAb in mice(Scid)bearing subcutaneous tumor and lymphoma in situ.The results showed that the nAb group had higher survival rate,longer survival time and smaller tumor volume compared with Rituximab group suggesting that the PCD effect induced by nAb could play a role in vivo.In addition,the results of solid tumor experiments demonstrated that nAb increasingly accumulated in tumor via EPR and better therapeutic effect was achieved.To sum up,nano-antibody developed by nanotechnology can enhance the apoptosis effect via CDC,ADCC,and PCD,which could be further strengthened by the unique EPR effect of nanoparticles.Therefore,nano-antibody showed significant therapeutic effect toward tumor in vitro and in vivo.Our study will exsert guiding significance for clinical antitumor trial and design of new functional antibodies.