Study On The Factors Of Safety And Efficacy Of Novel Antibody-drug Conjugates

Tumor has become a maj or disease endangering human health.According to the data from national cancer center,about 10,000 people were diagnosed with tumor every day in China.Meanwhile,the mortality rate of tumor in China was higher than the global average.Although there are many potent cytotoxic drugs,anti-tumor cytotoxic drugs are difficult to distinguish small differences between tumor cells and normal cells,resulting in strong side effects.Monoclonal antibody(mAb)can specifically recognize tumor cell surface antigen;however,the efficacy of single use is unsatisfactory.Antibody-drug conjugates(ADC),which couple mAb and cytotoxic drug together by chemical bond,fully take advantage of the specificity of mAb and the high anti-tumor activity of cytotoxic drug to achieve the efficiency and low-toxic anti-tumor effects.Theoretically,ADC remained stable in blood circulation;when ADC identify tumor surface antigens,the cytotoxic drugs are released after internalization of the ADC into tumor cells.There are hundreds of ADC being developed now,but only four are listed.Mylotarg(?)(Gemtuzumab ozogamicin),one of the listed ADC,was withdrawn because of the poor safety and failure to effectively prolong patient survival,and it was re-listed until a reasonable clinical medicine regimen was proposed.Security problems are ubiquitous in ADC,which are mainly embodied in two aspects:1.Cytotoxic drugs are released outside tumors due to the insufficient stability of linker,2.ADC are internalized into normal cells due to the insufficient specificity of antigens.In addition,the heterogeneous of ADC generated by non-specific conjugation method lead to greatly increase the difficulty of clinical safe medication.According to the problems above,this research aims to prove effective strategies to improve the safety of ADC and promote the application of ADC in tumor therapy.B-lymphocyte CD20 is known as a tumor antigen that highly expressed on the surface of most malignant B cells.Firstly,CD20 was selected as the antigen and Ofatumumab(OFA)was chose as vector.Sortase A-mediated transpeptidation was used to specifically conjugate triple glycine-modified MMAE to the carboxyl terminal of the heavy chain of OFA to generate homogeneous ADC(OFA-HL-MMAE and OFA-HL-vcMMAE),and the DAR and conjugate site of MMAE were determined by HPLC and MS.Both OFA-HL-MMAE and OFA-HL-vcMMAE showed good anti-tumor activity in vitro on CD20+tumor cells.OFA-HL-vcMMAE,at 5 mg/kg dose,can eliminate xenograft tumors with mean volume ?400 mm3 rapidly while no obvious toxicity was observed until at 20 mg/kg dose.All aboves showed that highly homogeneity of ADC was beneficial to safety improvement.Valine-citrulline(vc)linker are highly stable in human plasma and can be effectively digested by intracellular cathepsin.Introducing vc linker between OFA-HL and MMAE can significantly improve the anti-tumor activity of the ADC without affecting their safety.Herein,we used CD20+tumor cells as antigen carriers and OFA-HL and OFA-HL-MMAE as examples to establish a new quantitative bioanalysis method of ADC based on flow cytometry.The new method using in situ antigens not only avoids the problem of soluble membrane protein expression in traditional ADC bioanalysis,but also improves the authenticity of ADC bioanalysis as a supplementary approach.Sortase A-mediated conj ugation,application of vc linker and the specific recognition between OFA and CD20 endow OFA-HL-vcMMAE with safe and effective anti-tumor activity in the research.Tumor-associated antigens(TAA),such as CD20,are expressed in normal cells and highly expressed in tumor cells.Therefore,the TAA-targeted ADC can unavoidably damage the antigen-positive normal cells,which is one of the main reasons for the inadequate safety of ADC.Tumor specific antigens(TSA)are only expressed in tumor cells,which can effectually discriminate tumor cells from normal cells.But,only a small part of TSA is located on the cell membrane,most of them are located inside the cell.Intracellular proteins can be processed into antigenic peptides,which are able to be presented to the cell surface by MHC I molecules to form the MHC-peptide complex(pMHC),and pMHC can be specifically recognized by T cell receptors(TCR)to mediate the T-cell immune response.NY-ESO-1 is a cancer/testis protein that is highly expressed in a wide range of tumor types.Although NY-ESO-1 is normally expressed in male germ line cells,due to the lack of MHC I in male germ line cells,the peptides degraded from NY-ESO-1 and presented by HLA(human MHC)can become a TSA.In order to further improve the safety of ADC,we attempted to utilize NY-ESO-1157-165/HLA-A*02(The pMHC formed by HLA-A*0201 and peptide at position 157 to 165 of NY-ESO-1)as antigen and high affinity and specificity TCR(1G4113)as vector to generate ADC.First,we successfully expressed soluble 1G4113 in IgGl constant region fusion form,which laid the foundation for the development of TCR-based ADC.Then,sortase A was employed to generate site-specific MMAE and 1G4113 conjugations(164113-vcMMAE),and 1G4113-vcMMAE showed highly selective activity against NY-ESO-1157-165/HLA-A*02 positive tumor cells.Although the safety of 1G4113-vcMMAE was satisfactory,its anti-tumor activity was not ideal.Many researches have proved that the expression level of a certain pMHC is very low,so does NY-ESO-1157-165/HLA-A*02;when we increased the expression level of NY-ESO-1 157-165/HLA-A*02,the anti-tumor activity of 1G4113-vcMMAE was increased markedly.In addition,we found that the affinity and endocytosis of 1G4113-vcMMAE were not the main factors for its poor anti-tumor activity,suggesting that the antigen expression level may be the main limited factor for the anti-tumor activity of 1G4113-vcMMAE.Choosing tumor-specific pMHC as the target greatly improved the tumor specificity of ADC that endowed ADC with high safety.However,the low expression level of tumor specific pMHC may lead to the inadequate accumulation of cytotoxic drugs in tumor cells,resulting insufficient anti-tumor activity of ADC.For the better apply tumor-specific pMHC to the development of ADC,we tried to improve the activity of cytotoxic drugs to compensate for the limitation caused by the inadequate expression of antigens.Since it is difficult to find a suitable small molecular cytotoxic drug with significantly stronger activity than MMAE,we turn our attention to immune cells.T cells,especially cytotoxic T cells,have very high anti-tumor activity,and a single active T cell could effectively kill tumor cell.Therefore,we attempted to use T cells as cytotoxic drug and T cell based bispecific molecules as vector to study the effect of cytotoxic drug activity on pMHC-targeted ADC.First,click chemo-enzymatic conjugation approach was used to generate 1G4113-5-CD3,which composed with anti-CD3 antibody and 1G4113-5.1G4113-5-CD3 could recognize T cells by anti-CD3 antibody and carry T cells to NY-ESO-1157-165/HLA-A*02 positive tumor cells by 1G4113-5,and then T cells could kill tumor cell after activation.Encouragingly,1G4113-5-CD3 could kill A375 cells with extremely low antigen expression level and avoid killing antigen negative K562-A2 cells.What's more,when T cells were pre-activated,the anti-tumor activity of them mediated by 1G4113-5-CD3 was observably increased.T cells showed specific and potent anti-tumor activity when mediated by 1G4113-5-CD3,which indicated that the anti-tumor activity of ADC can be significantly increased by enhancing the activity of cytotoxic drugs.Cytokines,like TNF-? and IFN-?,have been shown to stimulate the expression of MHC I molecule,and then increase the expression level of pMHC on the surface of tumor cells,which might enhance the anti-tumor activity of ADC.Staphylococcal enterotoxin C2(SEC2)is a classical superantigen,which can tremendously activate T cells at very low dosage.SEC2 has been used clinically as a supplementary therapeutic agent for tumor treatment for many years in China.The non-specific activation of T cells stimulated by SEC2 can release massive amounts of cytokines,which can promote the expression of NY-ESO-1157-165/HLA-A*02 on cell surface and further enhance the anti-tumor activity of 1G4113-based ADC.In addition,the non-specific activation of T cells stimulated by SEC2 can up-regulate the anti-tumor activity of 1G4113-5-CD3.Hence,combination of SEC2 with tumor specific pMHC-target ADC or bispecific molecules not only combined the anti-tumor activity of two drugs,but also achieved the effect of 1+1>2.Finally,we proved that SEC2 can cross the intestinal epithelium in an immunologically integral form,maintaining T cells-stimulated and anti-tumor activity but with reduced systemic toxicity,which provided reference for better clinical application of SEC2.Being directed against the main problems of ADC security,this research demonstrated that sortase A-mediated conjugation can generate highly homogeneous ADC,introducing vc linker between antibodies and cytotoxic drugs can reduce the out-tumor release of cytotoxic drugs without affecting their release in tumor cells,and targeting tumor specific pMHC can improve the tumor specificity of ADC.Combining these three advantages,the safety of ADC can be significantly improved,but the activity of ADC was reduced.Next,we improved the activity of ADC by increasing the activity of cytotoxic drugs and the expression level of pMHC while preserving the safety of ADC.In addition,this study successfully achieved the soluble expression of TCR and complete the preliminary evaluation of the druggability of TCR-based protein drugs,and demonstrated the oral administration ability of SEC2,proving more strategies for tumor precision medicine.