The Design Of Anti-tumor Drugs Based On Miniaturized And Functionalized Antibodies

Cancer is one of the greatest threats to human health in the 21st century.Over the last several decades,several new technologies have revolutionized cancer treatments.As a milestone among therapeutic methods,antibody-based drugs have achieved remarkable success in clinical cancer treatment.Thus far,over 80 antibody-based drugs have been approved by the Food and Drug Administration(FDA).Although,antibody-based drugs take an important role in the therapy of cancer,the limitations of the traditional antibody drugs have been exposed gradually with the development of clinical study.The limitations mainly reflected in the following aspects:(1)Typical antibody-based drugs are mainly IgG formats with the molecular weight of 150 kDa,having the characteristics of lower exposure amounts in tumor site,the difficult of penetrating into the tumor core and the heterogeneous distribution in tumor site.These characteristics might lead to poor efficiency and relapse;(2)The long half-life of antibody drugs also increases the possibility of 'on target off tumor'effect,which lead to narrow therapeutic windows;(3)Most antibodies drugs on the market target tumor-associated antigens(TAAs),which are surface markers expressed on tumor cells.However,several studies have also shown that a low level of TAAs are expressed on normal cells,which may trigger 'on-target off tumor'effects.Therefore,the clinic has great demand on the targets like the tumor specific antigens(TSAs),which are specifically expressed on tumor cells.Mutant peptide/major histocompatibility complex(pMHC)are the typical representative of TSAs,with the character of lower expression in tumor cells.That's why pMHC targets could not be exploited to monoclonal antibody or antibody drug conj ugates,which have a requirement for the amount of antigen(>3000).With the development of molecular biology technology and genetic engineering,the formats of antibody drugs are not limited to typical IgG and various forms of miniaturized antibodies have sprung up.In order to improve the anti-tumor activity of miniaturized antibodies,miniaturized antibodies try to conjugate with toxins or with the immune cells to develop to functional antibodies(antibody drug conjugates and bispecific antibody).In view of the limitations of antibody drugs in the clinical treatment,our study try to solve these problem in the perspective of miniaturized and functional antibody drugsIn this study,a commercialized anti-CD20 monoclonal antibody,ofatumumab(OFA),is selected as a research object.We construct four antigen binding fragment-based antibody fragments:Fab,2F(ab),Fab-CH2mut and Fab-CH3mut.After preliminary evaluation of these antibody fragments,we choose Fab and Fab-CH3mut for further study.We generate three site-specific monomethyl auristain E(MMAE)-conjugated ADC/AFDCs(Fab-vcMMAE,Fab-CH3mut-vcMMAE and OFA-vcMMAE)by Sortase A mediated transpeptidation.Compared with OFA-vcMMAE,the two AFDCs maintain most of the binding affinity and the ability of internalization.In vitro studies reveal that Fab-vcMMAE(2.92 nM)and OFA-vcMMAE(1.31nM)have almost identical IC50 values against CD20-positive cell lines,while Fab-CH3-vcMMAE(16.87 nM)have a lower anti-tumor activity.In vivo studies show that Fab-vcMMAE has a significantly higher maximum tolerated dose(MTDs),a 30-fold shorter half-life,and slightly lower antitumor activity within the MTDs than OFA-vcMMAE.The distribution study show that the Fab has higher penetration rates into the tumors than OFA in a xenograft model.Additionally,no obvious difference in tumor drug accumulation is found between the Fab and OFA groups after the penetration process,but the Fab-CH3mut group exhibit less tumor drug accumulation,possibly contributing to the inferior anti-tumor activity of Fab-CH3mut-vcMMAE in vivo.This might attribute to the monomer form of Fab-CH3mut.Our result demonstrate that AFDCs have the potential in the therapy of cancer and could make up for the drawbacks of the traditional antibody drugs.What's more,our results can also provide guidance for the modification of miniaturized antibodiesAFDCs with poor pharmacokinetics,may result in poor anti-tumor activity.In order to improve the anti-tumor activity of AFDCs,we can choose to improve their pharmacokinetics or couple with stronger toxins.Auristain and maytansine with the IC50 of pM range,are currently used toxin,and the stronger ones are hard to obtain.The human immune cells have great potential in cancer treatment,so we try to replace the toxin of the AFDCs to OKT3 antibody,which target the T cell and obtain the T cell dependent bispecific antibody.The anti-tumor activity of bispecific antibodies do not relay on the internalization property of antibodies,so antibodies with poor internalization property such as OFA or even with no internalization property can be develop to bispecific antibodies.We conjugate Fab-OKT3 with Fab-CD20 by chemo-enzymatic method,which contain the advantages of higher coupling efficiency,correct assembly efficiency and easy to purification.Our results demonstrate that 2F(ab')-CD20-CD3 antibody incubated with positive tumor cells could trigger T cell activation,proliferation and killing of tumor cells specifically.The in vitro results show that 2F(ab')-CD20-CD3 has EC50 values of 2.75 pM and 2.62 pM aganist CD20-positive cell line Ramos and Daudi.The in vivo studies show that 2F(ab')-CD20-CD3 could obviously suppress the tumor growth with the administration dosage of 1mg/kg.This results are comparable with the activity of the typical IgG format bispecific antibody(rituximab)targeting to CD20.Furthermore,the lack of Fc region of 2F(ab')-CD20-CD3 with faster penetration property can avoid side effects due to the nonspecific binding of Fc.Though,Fab-vcMMAE and 2F(ab')-CD20-CD3 antibody exhibit high anti-tumor activity,the CD20 antigens express on normal B cells,which may produce on target off tumor effect.We choose neoantigen such as pMHC as research object to improve the safety issue of antibody drugs.MAbs or ADCs show poor efficiency on neoantigen such as pMHC,with lower expression level.T cell can be effectively activated by 10?100 antigens and exert the ability of killing tumor cells.So T cell dependent bispecific antibody is an effective supplement of the therapy of antibody-based drugs.In this study.we choose KRAS G12V/HLA-A02-01 as a research target,and generate 2F(ab')-2G1?CD3 bispecific antibody by chemo-enzymatic method.We preliminary evaluate the properties and the in vitro anti-tumor activity of 2F(ab')-2G1-CD3.The result show that 2F(ab')-2G1-CD3 has an obvious killing effect towards positive tumor cell lines SW480(KARS G12V/HLA-A0201)with an EC50 of 31.5 nM,while has no anti-tumor activity towards negative tumor cell lines A375(KRAS wild/HLA0201),K562-A2(KARS wild/HLA0201)and K562-A3(KARS wild/HLA0301).In the in vivo experiment,IgG format 2G1×CD3 bispecific antibody show an excellent anti-tumor activity at the administration dosage of lmg/kg for three times,which indicate the anti-tumor potential of 2F(ab')-2G1-CD3.Our results provide a new thought for the treatment of neo-antigen with low expression.In conclusion,we obtain the novel antibody fragment drugs with high activity and safety,based on the miniaturization and functionalization of traditional antibody.Our results demonstrate that antibody fragment drug conjugates and miniaturized bispecific antibody can solve the restrictions of typical antibody drugs due to the large molecular form,target characteristics and antigen number.