| Recent studies have shown that tumor microenvironment（TME）has a more profound influence on the tumor growth and metastasis.The TME provides crucial contributions to the licensing of cancer hallmark capabilities and disease progression.Factors and cells that do not support tumor growth are usually down regulated or mitigated in TME.One mechanism that mediates immune escape has been identified in cancer.It was reported that tumor cells and TME lost co-stimulatory molecules but overexpressed inhibitory molecules.High expression levels of co-inhibitors on tumor cells,known as immune checkpoint molecules,were significantly associated with distal metastasis and advanced TNM stages.Coinhitibory ligands and receptors had been identified including B7-H1（programmed death-1 ligand-1）,B7-DC（programmed death-1 ligand-2）,PD-1（programmed death-1）,B7-H3,and B7-H4.Aberrant B7-H1,B7-H3 and B7-H4 expression were associated with adverse pathologic features and poor outcome by which malignant tumors might evade the host immune response.Within the tumor microenvironment,regulatory cells and expression of immune checkpoint molecules could promote tumor growth and metastasis.Checkpoint inhibition therapy did not kill tumor cells but targeted at T cells to restore anti-tumor ability.Immune checkpoint blockade showed great potential in clinical trials.CTLA-4and PD-1 are critical immune checkpoint molecules and antibodies directed against PD-1/B7-H1（PD-L1）pathway have already shown their clinical efficacy.FDA approved PD-1 antibodies for the treatment of several solid tumors including urothelial carcinoma,head and neck cancer,Hodgkin’s lymphoma,renal cell carcinoma,non-small cell lung cancer and melanoma.Clinical trials suggested that PD-L1 expression and tumor-infiltrating lymphocytes were significant for predicting response to immune checkpoint blockade.Several studies of anti-PD-1 therapeutic antibodies in multiple tumor types,including lung cancer,malignant melanoma and gastric cancer,suggested that PD-L1 expression was associated with a likelihood of better efficacy of PD-1 blockade antibody.Anti PD-1response rate and median PFS were significantly improved among patients with a PD-L1 negative tumor,indicating that PD-L1 was associated with response to anti-PD-1therapy and clinical outcomes.PD-1 therapeutic mAbs discovery along with PD-L1predictive biomarker are necessary to reflect the level of PD-L1 expression before planned treatment.PD-L1 expression by IHC was approved by FDA as one selection criterion for anti PD-1 therapy.PD-L1 test could be performed to determine patients likely to respond to PD-1 inhibitors.However,scoring of immunohistochemistry for PD-L1 and TILs has not yet been standardized.PD-L1 antibodies as companion diagnostics haven’t been validated in China.The PD-1 treatment strategy needed to be evaluated by PD-L1 IHC staining with high sensitivity and specificity.Moreover,a Phase 1 study was designed to characterize the safety and tolerability of B7-H3antibody and preclinical data of B7-H4 therapeutic antibody was also revealed recently.Expression of B7-H3 and B7-H4 could be a predictive biomarker in cancer immunotherapy.Much investigation was made to find the correlation between B7-H3 and B7-H4 as well as their expression level in tumor microenvironment.B7-H3 and B7-H4 belong to B7 family and exert negative signal to fine tune immune responses.B7-H3 and B7-H4protein were not detectable in normal tissues while broadly expressed in a variety of tumor types,including melanoma,breast cancer,prostate cancer and colorectal cancer.Combined expression of B7-H3 and B7-H4 is of importance as independent predictors of a poor prognosis in cancer patients.It was also confirmed that we should consider B7-H1,B7-H3 or B7-H4 and tumor infiltrating lymphocytes when using PD-1 antibody.According to the different characteristics of tumor microenvironment,cancer was classified into four subgroups:T1(B7-H1pos with TILs driving adaptive immune resistance),T2（B7-H1 negative with no TIL indicating immune ignorance）,T3(B7-H1pos with no TIL indicating intrinsic induction)and T4（B7-H1 negative with TIL indicating the role of other suppressors in promoting immune tolerance）.T1 group had the best response rate and prognosis among patients receiving anti PD-1 therapy.B7-H1,B7-H3 and B7-H4 expression in cancer tissues can be heterogeneous in staining intensity and have variable distribution throughout an individual tissue.It will be useful developing anti-human B7-H1,B7-H3,B7-H4 and CD8 mAbs suitable for immunohistochemical staining in formalin-fixed tissue sections with reliable clarity and intensity.IHC detection in a single tissue sample will systematically determine the expression pattern of B7-H1,B7-H3 and B7-H4 molecules at different stages of tumor development and its correlation with immune cell subsets in tumor microenvironment,as well as clinic pathological parameters.Relationships analysis among B7-H1,CD8and Foxp3 were performed to examine the dynamic interactions that occur within the tumor microenvironment,which could be significant to determine the immune classification（immunoscore）for gastric cancer.These m Abs are potentially useful as companion diagnostic agents to analyze B7-H1,B7-H3,B7-H4 and CD8 expression in the clinical setting while the expression pattern in the corresponding stage will be important to clarify tumor immune escape mechanism.Part I Anti-human B7-H1,B7-H3,B7-H4 and CD8 monoclonal antibodies for tumor IHC testB7-H1,B7-H3,B7-H4 and CD8 antibodies were used to study tumor prognosis and clinical diagnosis by immunohistochemical staining.A group of B7-H1,B7-H3,B7-H4 and CD8 monoclonal antibodies were identified by flow cytometry and ELISA.Normal tissues（including tonsils,spleen,embryonic tissues,etc.）,lung cancer and colorectal cancer tissues were collected and analyzed for B7-H1,B7-H3,B7-H4 and CD8 expression.First of all,immunofluorescence and flow cytometry showed that anti-human B7-H1,B7-H3 and B7-H4 monoclonal antibodies specifically bind with transgenic cells or tumor cells highly expressing B7-H1,B7-H3 and B7-H4.And immunohistochemical staining of B7-H1 verified its high level in tonsil and spleen.B7-H1 was expressed in embryonic lung/stomach/colon tissues.Normal spleen and tonsil showed CD8expression.Both cytoplasm and membrane of cells from spleen and tonsil could be stained as CD8 positive.It was also found that a large number of CD8+T cells gathered in colorectal cancer with a high degree of lymphocyte infiltration.In order to establish multiplex immunohistochemical staining method,PD-1,CD8 and Foxp3 was detected in tonsils.PD-1 and CD8 were found on the cell membrane and Foxp3 was mainly expressed in the nucleus.In this project,B7-H1,B7-H3,B7-H4 and CD8 monoclonal antibodies were developed for IHC detection providing a consistent and objective assessment for tumor diagnosis and prognosis on a multi-analysis basis.Part IIExpression and clinical significance of immune molecules B7-H1/B7-H3/B7-H4 in gastrointestinal carcinomaThis study intended to evaluate B7-H1,B7-H3,B7-H4 and CD8 expression pattern in colorectal cancers and B7-H1 expression and the status of Foxp3+,CD8+cells in gastric cancers.Based on the IHC assay,we would find the possible association between lymphocyte infiltration,B7-H1/B7-H3/B7-H4 immunoexpression and clinical parameters.B7-H1,B7-H3,B7-H4 and CD8 monoclonal antibodies were developed suitable for immunohistochemical staining in formalin-fixed colorectal tissue sections achieving a high level of sensitivity and specificity.Immunohistochemical method was used to examine the expression of B7-H1 and number of Foxp3+,CD8+cells in 267 cases of gastric cancer.The study indicated B7-H1 was highly expressed in the cytoplasm and membrane of cancer cells.B7-H1,B7-H3 expression were upregulated in colorectal adenoma specimens while B7-H4 molecule was only expressed in cancer tissues.We also studied B7-H1,Foxp3 and CD8 levels in gastric cancer and found that less CD8 expression in gastric cancer was associated with more possibility that patient would have lymph node metastasis.Distant metastasis was likely to happen with high Foxp3 or low CD8expression.B7-H1,B7-H3,B7-H4 and CD8 were highly expressed in colorectal cancer tissues and showed differential expression levels during disease progression.Expression of B7-H1,Foxp3 and CD8 in gastric tissues was related to patient’s tumor growth,lymph node metastasis and and distant metastasis status.Part III Humanization of anti PD-1 monoclonal antibodies and the characteristic feature study in vitroAnti-human PD-1 chimeric monoclonal antibody and humanized PD-1 antibodies were completed and expressed in 293F cell line.The PD-1 mAbs were tested to investigate the binding ability and whether they inhibit PD-1/PD-L1 signal on immune cells response.Heavy chain variable region（VH）and light chain variable region（VL）of the PD-1monoclonal antibody were obtained by PCR.The VH and VL parts were linked with a specific expression vector carrying human Fc region to construct chimeric mAbs.Based on antibody library sequencing,CDR grafting was applied to make the humanized PD-1monoclonal antibodies.Affinity and binding assay were carried out by Octet and Biacore.The PD-1 mAbs were co cultured with PBMC to see the blocking effect on cell activation and proliferation.5 PD-1 chimeric antibodies（MHc2.1,MHc3.2,MHc5.1,MHc6.1 and MHc7.3）specifically recognized L929/PD-1 cells,among which hu5.1,hu7.3 were under humanization.The 2 antibodies hu5.1 and hu7.3 recognized different epitopes from the commercial humanized PD-1 antibody 5C4.Antibody/antigen binding and dissociation analysis showed hu7.3 had a higher affinity and binding ability.PBMCs from healthy donor were separated and cultured adding PD-1 antibodies in vitro showing that hu7.3antibody could block PD-1/PD-L1 signal on activated lymphocytes and promote cell proliferation and IFN-γsecretion in a concentration dependent way.In this study,5 anti PD-1 chimeric antibodies（MHc2.1,MHc3.2,MHc5.1,MHc6.1 and MHc7.3）were successfully constructed.Two of them（hu5.1 and hu7.3）were humanized.These m Abs are potentially useful to study PD-1/PD-L1 regulation on immune cells and further development as a tumor targeting drug.Tumor immune escape and the initiation of metastasis are critical steps in malignant progression of tumors immune.Highly expressed checkpoint molecules B7-H1,B7-H3 and B7-H4 were served by tumor cells to acquire escape mechanisms and evade host immunity in the tumor microenvironment.The blockade of immune checkpoints that prevents cancer cells from turning off T cells represents a major recent breakthrough in the treatment of malignant diseases.Immune checkpoint antibodies of promising clinical responses and immunohistochemistry（IHC）-based companion diagnostics were necessary for developing a diverse repertoire of precise therapeutic approaches with safety and efficacy.We successfully developed a series of anti-human B7-H1,B7-H3 and B7-H4 and CD8 monoclonal antibodies.B7-H1 and B7-H3 were up regulated in colorectal polyps while B7-H4 was only observed in the development of cancer and was related to infiltrating immune cells.We established a multiplex immunohistochemical staining method in order to achieve an accurate localization of B7-H1,B7-H3 and B7-H4 in tumors by IHC-based antibodies.High B7-H1 or Foxp3 expression with low CD8+cells in gastric cancers indicated lymph node metastasis and tumor progress.Anti PD-1antibodies including chimeric and humanized were obtained.And we utilized a eukaryotic cell to express antibodies with high yield and less cost.The antibodies could recognize PD-1 antigen and showed that it blocked PD-1/PD-L1 signal to promote PBMCs activation and proliferation in vitro.This project not only provided immune checkpoint antibodies with potential diagnostic value but also antagonistic PD-1 mAbs to further explore in vivo efficacy of checkpoint blockade and clarify the mechanism of tumor immune escape. |
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