Preparation And Function Of TIM-3 Single Domain Antibody

Cancer is one of the major diseases that seriously threaten human health.In recent years,cancer immunotherapy technology,represented by chimeric antigen receptor T cells(CAR-T)and immune checkpoint inhibitory antibodies,has brought revolutionary progress in cancer treatment.Immune checkpoints are a class of regulatory proteins expressed on the surface of immune cells(T cells,monocytes/macrophages).Suppressive ligands that are highly expressed on the surface of tumor cells can inhibit the activity of immune cells and help tumor cells escape the immune surveillance.Monoclonal antibodies targeting immune checkpoints can block the inhibition of tumor cells on immune cells and enhance the anti-tumor activity of the immune cells.At present,many monoclonal antibodies targeting CTLA-4 and PD-1 have been approved by FDA,and more immune checkpoints are being identified.These new immune checkpoints are important targets for the development of antibody drugs,and TIM-3(T cell immunoglobulin domain and mucin domain-3)is one of the promising candidates.TIM-3 was originally found to be expressed on CD4~+helper T cells(Th1)and CD8~+cytotoxic T cells(Tc1)that produce IFN gamma,and later on it turns out to be also expressed on Th17 cells,Treg cells and innate immune cells(DC,NK cells,monocytes).The natural ligand of TIM-3 is Galectin-9(beta-galactoside lectin protein),which is highly expressed in lymphoma,lung cancer,breast cancer,liver cancer and other cancer types.The binding of Galectin-9 to TIM-3 can lead to the exhaustion of T cells in tumor microenvironment and chronic infection.Monoclonal antibodies targeting TIM-3 can block this process.Traditional monoclonal antibodies have two antigen binding domains,VH and VL,and their molecular weights are relatively large.Minimized VH domain antibodies have many unique advantages,such as stronger tissue penetration,easier to be reformatted and to be used to construct multi-specific antibodies,etc.The most attractive domain antibodies in the R&D of antibody drugs are camel-derived VHH antibodies(also known as nanobodies),human and rabbit-derived VH domains.The major goal of the current project is to generate VH domain antibodies targeting TIM-3 by immunizing rabbits with recombinant TIM-3 protein and phage display technology,which will ultimately lead to the development of minimized TIM-3antibody drugs.The main results are as follows.1)The genes of TIM-3 and Galectin-9 were cloned and the expression plasmids of TIM-3 extracellular domain and Galectin-9 and lentiviral expression vectors of TIM-3full-length protein were constructed.TIM-3 extracellular domain and Galectin-9 protein were expressed and purified in human embryonic kidney cell HEK293F.A431 cells overexpressing TIM-3 full-length protein were constructed by lentiviral transduction.2)Using TIM-3 extracellular domain as immunogen,rabbits were immunized with titers being exceeded 100000.3)Using the spleen of immunized rabbits as resource,total RNA was extracted,and the phage displayed VH domain antibody library was constructed by reverse transcription and PCR amplification.Finally,five TIM-3 specific VH domain antibody sequences,named TIM3-R1,TIM3-R6,TIM3-R23,TIM3-29,TIM3-R53,were obtained by TIM-3 affinity panning.They can be classified to three major structural types(R1,R23,and R53)based on the sequence similarity.4)These monoclonal antibodies were expressed and purified in E.coli and mammalian cells.The specificity and affinity of TIM-3 monoclonal antibodies binding to recombinant and cell surface TIM-3 protein were determined by ELISA,Western Blot and Flow Cytometry.The Kd values(EC50)of the domain antibodies are ranged from 278 to 8740 nM as measured by ELISA,with R53 being the strongest binder that has a Kd value of 278 nM.The binding affinity of recombinant Galetin-9 to TIM-3 was204 nM as determined under the same conditions.The affinity of other monoclonal antibodies to TIM-3 was much lower than that of Galectin-9.Flowcytometry confirmed that monoclonal antibody R53 had obvious cell-binding activity.According to Western’s experiment,none of the five monoclonal antibodies could recognize denatured TIM-3 protein,indicating that they recognized the conformational epitope of TIM-3.5)Competitive ELISA assay showed that two TIM-3 domain antibodies(R23,R53)could block the binding of Galectin-9 to TIM-3.The blocking activity of R53 was stronger than that of R23,and both of the two antibodies displayed blocking potency in dose-dependent manner in the range of 1-200μg/ml.The other three antibodies failed to show blocking actibity.6)The effect of TIM-3 domain antibodies on the cytotoxicity of CAR-T cells was studied at the cellular level using CAR-T cells as a model.By activating PBMC cells and infecting PBMC cells with CAR virus,CAR-T cells were constructed.Flowcytometry analysis confirmed that CAR-T cells could express TIM-3.TIM-3antibodies were co-incubated with CAR-T cells and H9 tumor cells(A431 cell that stably expresses mesothelin)labeled with Luciferase.The results showed that domain antibody R53 significantly increased the cancer cell killing activity of CAR-T cell targeting mesothelin with moncloanl antibody YP158 by about 25%.7)The effect of domain antibody R53 on the anti-tumor activity of CAR-T cells in xenograft mice model was studied.Epidermal cancer H9 cells were subcutaneously inoculated in nude mice and treated with CAR-T cells,CAR-T cells combined with TIM3-R53 monoclonal antibody respectively.The growth rate of the tumors was monitored.It was found that R53 monoclonal antibody apparently increased the anti-tumor activity of CAR-T cells,and the tumor growth rate in the combination therapy group was much slower than the CAR-T monotherapy group.In conclusion,we have successfully generated domain antibodies that can block the binding of Galectin-9 to TIM-3.The properties and anti-tumor activities of these domain antibodies have been studied,and the resultant data will provide solid foundations fot the development of therapeutic antiodies targeting TIM-3 for cancer immunotherapy.