The Role Of TIM-3 In Tumor Immunity And The Mechanisms Of Action

The First Part:The Expression of TIM-3 in Tumor Microenvironment and the Anti-Tumor Mechanisms of Targeting TIM-3Background:Recently,tumor immunotherapy has achieved remarkable clinical results.Although the immune checkpoint blockade strategy for cytotoxic T lymphocyte antigen 4(CTLA-4)and programmed death 1(PD-1)has shown great success,the majority of cancer patients have not yet benefit from this new therapy.In addition to PD-1 and CTLA-4,a variety of immunomodulatory molecules are expressed in tumor infiltrating lymphocytes(TIL).T cell immunoglobulin mucin domain 3(TIM-3)has shown to mediate immune tolerance in mouse models of infection,alloimmunity,autoimmunity,and tumor immunity.Previous studies found that TIM-3 is highly expressed on regulatory T cells(Tregs)in patients with non-small cell lung cancer(NSCLC),and that TIM-3 expression on CD4+T cells is associated with poor clinical outcome in cancer patients.Therefore,targeting TIM-3 is a new strategy to further improve current immunotherapy.Although a large number of experimental data showed that TIM-3 has immunosuppressive function in vivo,the specific mechanism is still unclear.In order to effectively target TIM-3 for tumor immunotherapy,further in-depth research of mechanisms is needed.These studies will provide ideas for the rational design of new combination therapies for other immune checkpoint blockades.Objective:To study the expression of TIM-3 in the tumor microenvironment and its significance;the therapeutic effect and mechanisms of TIM-3 monoclonal antibody combined with PD-1 monoclonal antibody in various mouse tumor models;the mechanism of tolerance in immunotherapyMethods:Flow cytometry was performed to determine the expression of TIM-3 in tumor infiltrating immune cells in tumor-bearing mice.The activation,proliferation,and effector molecules of TIM-3+subpopulations in tumor infiltrating lymphocytes were determined.The total number of mitochondria mass in TIM-3+or TIM-3" subpopulations of tumor infiltrating lymphocyte was determined using the Mitotracker Deep Red kit.The cytoxicity of TIM-3+or TIM-3-CD8+subsets were examined by cytolytic assay.Treat tumor-bearing mice with TIM-3 monoclonal antibody in combination with PD-1 monoclonal antibody and the growth curve or the overall survival or the number of lung metastatic nodules were observed.The population distribution and phenotypic changes of tumor infiltrating lymphocytes were detected by flow cytometry at 24 hours or 96 hours after antibodies treatments.According to the phenotypic changes of tumor-infiltrating lymphocytes in tumor-bearing mice,an improved tumor immunotherapy protocol was designed.The TIM-3.PD-1 and LAG-3 monoclonal antibodies were designed to treat tumor-bearing mice simultaneously,and the growth curves of mice were observed.Flow cytometry and cytolytic assay were performed to detect the distribution and phenotypic changes of tumor infiltrating lymphocyte in tumor-bearing mice after treatment with triple-combined antibodies.Results:We found that TIM-3 is highly expressed in tumor infiltrating CD4+,CD8+,Treg,type 1 dendritic cells and type 1 tumor associated macrophages.Especially on Treg,the percentage of TIM-3+subset is about 80%.TIM-3+subpopulations in CD4+and CD8+T cells highly express activation molecules CD44,OX40 and cell proliferation-related molecule Ki67,but express low levels of naive molecules such as CD62L,IL7R.In addition,the TIM-3+conventional CD4+and CD8+TILs express higer levels of IFN-y and granzyme B when compared to TIM-3-TIL.Although PD-1+TIM-3+TILs have been described as exhausted T cells,PD-1+TIM-3+TILs have slightly higher number of mitochondrial mass compared to PD-1-TIM-3-TILs.Seahorse assay showed that PD-1+TIM-3+TILs have similar level of oxygen comsuption rate but higer extracellular acidification rate compared to PD-1-TIM-3-TILs,indicating PD-1+TIM-3+TILs are more activated.Cytolytic assay also showed that PD-1+TIM-3+TILs are more potent effector T cells compared to PD-1-TIM-3-subset.Compared with PD-1 monoclonal antibody treatment alone,TIM-3 mAb combined with PD-1 mAb significantly inhibited the tumor growth in mouse intestinal cancer MC38 model.TIM-3 mAb combined with PD-1 mAb could significantly prolong the overall survival of tumor bearing mice in ID8 model,but the treatment with PD-1 mAb alone did not have any effect.In the mouse breast cancer model 4T1,neither treatment of PD-1 mAbs or TIM-3 mAbs alone or their combination did affect tumor growth rate.Treatment with PD-1 mAb alone did not reduce the lung metastasis in this model.In contrast,TIM-3 mAb combined with PD-1 mAb could significantly reduce the number of lung metastatic nodules.With flow cytometry analysis,we found that TIM-3 mAb combined with PD-1 mAb significantly enhanced the proliferation and effector function of CD4+and CD8+T cells at 24 hours or 96 hours.At the same time,we found that another immunoinhibitory receptor molecule,LAG-3,was significantly up-regulated 24 or 96 hours after TIM-3 and PD-1 mAbs combination therapy.Treatment with the combination of TIM-3.PD-1 and LAG-3 mAbs could further improve the efficacy of immunotherapy and the improved therapeutic effect was due to the increase of the cytolytic function and the percentage of granzyme B expressed by CD8+T cells.ConclusionTIM-3 is an activation marker of tumor infiltrating T cells.TIM-3 is a hallmark of activated T cells in our syngeneic transplant tumor model.The TIM-3+PD-1+ population is not an exhausted but a functional T cell population.The TIM-3 mAb can be combined with the PD-1 mAb to significantly improve the immunotherapeutic efficacy of the PD-1 mAb alone in multiple tumor models,overcoming the resistance to PD-1 mAb therapy in some tumors.TIM-3,PD-1,LAG-3,GITR and other immunoregulatory receptors expressed on T cells are cross-regulated to control hyperactivated T cells in the tumor microenvironment.The combination of TIM-3,PD-1 and LAG-3 mAbs can further improve the therapeutic effect of TIM-3 and PD-1 mAbs combination.The Second Part:Functional Study of a Humanized TIM-3 Monoclonal AntibodyBackground:TIM-3 has been considered to be a negative regulator of anti-tumor immune responses.Several features of TIM-3 make it an ideal target for next generation immunotherapy.First,the selective expression of TIM-3 on T cells in tumors makes it more precisely treated by targeting tumor infiltrating T cells,which can reduce non-specific toxicity.In addition,downstream signal of TIM-3 is quite different from that of CTLA-4 and PD-1.thus these molecules can be targeted simutaneously.Third,several preclinical studies have shown that TIM-3 and PD-1 mAbs have additive antitumor activities.Thus,specific expression and intracellular signal transduction make it great target for TIM-3 blockade therapy or in combination with current PD-1 and CTLA-4 based tumor immunotherapies.Our previous data indicated that TIM-3 is expressed on tumor infiltrating CD4+and CD8+T cells in multiple types of tumors.We have also demonstrated that anti-PD-1 and anti-TIM-3 combination therapy overcomes the resistance of PD-1 mAb alone in a variety of mouse tumor models including colorectal cancer,ovarian cancer and breast cancer models.Treatment of TIM-3 and PD-1 mAbs combination significantly enhanced the effector function of CD8+T cells.These data indicated that TIM-3 has great potential for clinical application as a new target for tumor immunotherapy.There is an urgent need to develop antibodies against human TIM-3 to improve the predicament of existing tumor immunotherapy.Objective:To develop monoclonal antibodies targeting human TIM-3;to screen for monoclonal antibodies with high affinity and which can block the binding of TIM-3 to its ligand;to study the effect of humanized antibodies on T cells in vitro;to study the anti-tumor function of antibodies in humanized systemsMethods:We constructed cell lines stably expressing human or monkey TIM-3 IgV domain.These cell lines were used to screen for hybridoma clones that express high affinity TIM-3 antibodies by Flow cytometry.We also established in vitro functional assays for TIM-3 mAbs.The first is the phosphatidylserine(PtdSer)binding assay.We induced apoptotic mouse splenocytes to expose PtdSer by UV irradiation in vitro and confirmed the exposure of the PtdSer on cell surface by flow cytometry.We then used flow cytometry to determine the blocking ability of TIM-3 antibody on the binding of TIM-3 to its ligand PtdSer.We also established a T cell functional assay for TIM-3.We stimulated T cells to induce expression of TIM-3 in vitro.We then re-stimulated the TIM-3-expressing T cells in the presence or absence of TIM-3 antibody.The cytokines were measured by ELISA.To evaluate the antitumor function of the anti-human TIM-3 monoclonal antibody in humanized systems using a humanized mouse model of human colorectal cancer,four experimental groups were used i.e.control IgG group,the anti-human TIM-3 monoclonal antibody alone group,the anti-human PD-1 monoclonal antibody alone group or the anti-human TIM-3 combined with anti-human PD-1 monoclonal antibodies group.Tumor growth was recorded,and growth curves were compared.Results:We successfully constructed cell lines that overexpress human or monkey TIM-3 IgV domains,and by several rounds of fluorescence activated cell sorting(FACS)we obtained stable cell lines.The hybridoma clones that are capable of binding human or monkey TIM-3 IgV domains were screened by stable TIM-3 IgV domain expressing cell lines,and clones with strongest binding ability were selected for further development.The variable region of genes encoding immunoglobin heavy and light chains of the selected hybridoma clones were cloned and humanized and then expressed in a commercial expression system.At the same time,we induced apoptotic cells in vitro and verified the exposure of phosphatidylserine on the surface of apoptotic cells.We found that the antibody we screened was able to effectively block the binding of the TIM-3 fusion protein to its ligand phosphatidylserine.In the functional study of antibody,we found that in vitro stimulation of T cells can induce the expression of TIM-3,the proportion of CD4+TIM-3+T cells could reach more than 48%,and the proportion of CD8+TIM-3+T cells was above 77%.When these T cells were re-stimulated,we found that our anti-human TIM-3 monoclonal antibody candidate 1 significantly increased the expression of IFN-y secreted by T cells.In the in vivo functional study of candidate 1,we used the humanized mouse model NSG to inoculate human colorectal cancer cell line HT29 after adoptively transferring of human PBMC.We found that anti-PD-1 antibody Nivolumab was able to slow down tumor growth slightly.Compared with Nivolumab treatmemt group,the combination of candidate 1 and Nivolumab significantly inhibited tumor growth.Conclusion:We have successfully screened a monoclonal antibody that has high affinity and is effective in blocking the binding of TIM-3 to its ligand phosphatidylserine.We perfomed a functional assay on the most sensitive monoclonal antibody,candidate 1,which demonstrated that candidate 1 can enhance the effector function of T cells in vitro and synergizes with Nivolumab to exert more potent anti-tumor function in vivo.The Third Part:Study of the function of TIM-3 on regulatory T cellsBackground:Regulatory T(Treg)cells play an important role in maintaining immune tolerance and preventing autoimmunity,autoinflammatory diseases and immunopathology during tissue infection and injury.Treg cell is a critical component in cellular immunity and is a key factor in maintaining the proper balance of immune responses.Foxp3-deficient mice and humans who bear defective Foxp3 alleles develop severe systemic and organ-specific autoimmune diseases and lymphoproliferative disorders.Treg cell also plays a crucial role in inhibiting anti-tumor immune responses and maintaining transplant tolerance.Treg cells also exist in healthy tissues,but at a lower number.In different tissues,Treg cells express different chemokine receptors and adhesion molecules.But the mechanism of how Treg cells are enriched and maitained in the tissues is not clear.In tumor tissues,Treg cells can account for 30%to 50%of CD4+T cells.Studies have shown that tumor infiltrating Treg cells upregulate a variety of immune regulatory receptors such as CTLA-4,ICOS,TIM-3,GITR,etc.,as well as the inhibitory cytokines such as interleukin-10(IL-10),transforming growth factor beta(TGF?),etc.Previous report showed that TIM-3 is specifically upregulated on tumor-infiltrating Treg cells but not on Treg cell in peripheral blood in non-small cell lung cancer patients.Recently,there have been reports that TIM-3+Treg cells have a stronger suppression function.In the first part of my study,we also further confirmed that tumoral Treg specifically up-regulated the expression of TIM-3 in our mouse models.In addition,TIM-3 was also confirmed to be expressed in tissue Treg.However,the role of TIM-3 in Treg that mediates tumor immune tolerance is not clear.Objective:In order to further understand the molecular mechanisms underlying TIM-3+Treg cell function,we will perform expression profiling studies comparing TIM-3+Treg and TIM-3-Treg isolated from head and neck squamous cell carcinoma.We will generate Treg-specific TIM-3 deficient mice.We will then determine the effect of TIM-3 deletion specifically in Treg on tumor growth.We will further study the role of TIM-3 in the function of tumor infiltrating Treg cells.Methods:To study the molecular basis of TIM-3+Treg cells,we sorted TIM-3=Treg and TIM-3-Treg cells from human head and neck squamous cell carcinoma by FACS,and we used Affymetrix Human Transcriptome Array 2.0 Gene chip to perform global gene expression analysis to examined the differentially expressed genes between two Treg subsets.Then we generated one allele of TIM-3 floxed mice(TIM-3f/+)and one allele of TIM-3 knockout mice(TIM3+/-)by CRISPR technology.We then crossed TIM-3f/+,TIM3+/-and Foxp3YPF-Cre mice to generate TIM-3+/-Foxp3Cre and TIM-3f/-Foxp3Cre mice.We confirmed Treg-specific TIM-3 deletion by flow cytometry.We inoculated TIM-3f/-Foxp3Cre mice and TIM-3+/-Foxp3Cre control mice with immunogenic intestinal cancer MC38 tumor cells or melanoma B16 tumor cells which are of poor immunogenicity.We subsequently monitored tumor growth in these mice.We also analyzed the phenotypic changes of tumor infiltrating lymphocytes in tumor microenvironment by flow cytometry.We sorted Treg cells from tumor tissues ex vivo by FACS and set up Treg micro suppression assay to study the effect of TIM-3 deletion on Treg suppression function.Results:We FACS purified TIM-3+Treg and TIM-3-Treg cells from human head and neck squamous cell carcinoma.We then performed gene expression analysis by using Affymetrix Human Transcriptome Array 2.0 gene chip.TIM-3+Treg and TIM-3-Treg cells have a large number of differentially expressed genes.TIM-3+Treg cells highly express genes that are also found upregulated in the effector T cells,and TIM-3-Treg cells highly express naive T cell markers.We then generated TIM-3+/-and TIM-3f/-+mice and by breeding with Foxp3YPF-Cre mice,we finally obtained TIM-3+/-Foxp3Cre and TIM-3f/-Foxp3Cre genotypes of mice.These mice were used to study the function of TIM-3 on Treg.The expression of TIM-3 on Treg in tumor tissues was detected by flow cytornetry,and its absence on tumor Treg cells was confirmed in TIM-3f/-Foxp3Cre mice.By examining the growth of mouse tumors in TIM-3f/-Foxp3Cre mice,we confirmed that TIM-3 deficiency in Treg resulted in inhibited tumor growth in immunogenic MC38 tumor.However,TIM-3 deletion in Treg cells did not affect the progression of tumors in B16 model.By analyzing the phenotypic changes on tumor infiltrating immune cells by flow cytometry,we found that after TIM-3 was deleted in Treg,tumor infiltrating immune cells were increased.In addition,Th1 immune responses were also increased.By establishing a Treg microsuppression assay in vitro,we found that Treg cells from TIM-3f/-Foxp3Cre tumor-beraing mice had a decreased suppressive function,indicating that TIM-3 is required for the suppression function of tumoral Treg cells.Conclusion:Gene chip expression profiling of human tumoral Treg cells showed that TIM-3+Treg cells have many characteristics of effector T cells,therefore are effector Treg cells suppression function of Treg is dependent on TIM-3.In MC38 tumor model,the loss of TIM-3 expression in Treg led to a decrease in immune suppression in the tumor microenvironment and inhibited tumor growth.The loss of TIM-3 expression in Treg also resulted in an increase of the percentage of total lymphocytes in the tumor.Thus,TIM-3 is very important for the suppression function of Treg,and specific targeting of TIM-3 in Treg can greatly improve the efficacy of tumor immunotherapy.