| Current immune checkpoint blockade strategies have been successful in treating certain types of solid cancer.However,checkpoint blockade monotherapies have not been successful against most hematologic malignancies including multiple myeloma and leukemia.There is an urgent need to identify new targets for development of cancer immunotherapies.LILRB1,an immunoreceptor tyrosine-based inhibitory motif(ITIM)-containing receptor,is widely expressed on human immune cells,including B cells,monocytes and macrophages,dendritic cells(DCs),subsets of natural killer(NK)cells and T cells.The ligands of LILRB1,such as MHC class I molecules,activate LILRB1 and transduce a suppressive signal,which inhibits the immune responses.High expression of MHC Class I molecules on tumor cells was shown to act as an immune escape mechanism for tumors through direct interaction with immune effectors.Studies have confirmed that the MHC Class I and LILRB interaction could be an ideal immune checkpoint.However,it is not clear whether LILRB1 blockade can be effectively used for cancer treatment,especially for the treatment of hematologic malignancies.Here,in this thesis,we demonstrated that LILRB 1,as a checkpoint inhibitor,can be targeted to turn "on" NK cells in vivo for cancer treatment.In brief,this thesis details the major findings in two parts.The first part of study explains why we focus on LILRB 1 blockade in natural killer cells and we generated a penal of 48 anti-LILRB 1 rabbit monoclonal antibodies by screening single memory B cell clones,cloning antibody genes,epitope binning and characterize the representative mAbs selected from the 12 epitope bins.We measured the LILRB 1 expression on NK cells from cancer patients to determine whether LILRB 1 is up-regulated on NK cells from cancer patients,when compared with NK cells from healthy donors.The percentage of LILRB 1 expressing NK cells is significantly higher in patients with persistent multiple myeloma after treatment than that in healthy donors.Similarly,the percentage of LILRB1 positive NK cells is also significantly higher in peripheral blood from patients with late stage prostate cancer when compared with that of healthy donors.We also found that majority of the cancer cell lines expressed MHC class I molecules and stimulated the activation of LILRB1 reporter cells.Exceptions were tumor cells,which did not express MHC class I molecules on cell surface.These results suggest that the ability of cancer cells to activate the LILRB1 reporter cells is positively correlated with their MHC class I expression levels.These results suggest that LILRB1 signaling in NK cells may be activated by tumor cells with high expression of MHC Class I,thus induce immunosuppression that includes suppression of NK cell cytotoxic activities.Hence,developing anti-LILRB 1 specific mAbs,especially the antagonistic antibodies,could rescue this suppression.A total of 229 anti-LILRB 1 specific single B cell clones were generated from rabbit B cell culture wells and assessed by ELISA for their binding capacity to LILRB 1 expressed on surface of LILRB 1 NFAT-GFP reporter cells which express GFP when the surface LILRB 1 is cross-linked.We selected 60 binders for further cloning and IgG expression in HEK293 cells.Of the 60 antibodies,48 exhibited EC50 values in the low nanomolar range(0.01-3 nM).To group the 48 mAbs by their binding epitopes,sandwich epitope binning assay was performed by a Bio-Layer Interferometry(BLI)assay using Octet RED96.Twelve epitope bins were identified among the 48 high affinity binders.Twelve representative.mAbs from the 12 epitope bins were selected for further characterization which include:ⅰ.affinity as measured by Octet RED96;ⅱ.binding domain on LILRB 1 using Fc fusion proteins with different domains of LILRB 1;and ⅲ.cross-reactivity to other LILRA and LILRB family members.Some antibodies in Bin1,Bin7,Bin8 and Bin9 showed antagonistic activity in the LILRB1 NFAT-GFP reporter assay.All antagonistic mAbs bind to the D1D2.In contrast,stalk region is necessary for agonistic antibodies interacting with LILRB1.In the second part,we focused on a novel antagonistic LILRB1 specific monoclonal antibody B1-#176 and showed that LILRB1 blockade by#176 increased the tumoricidal activity of NK cells against several types of liquid tumor cells,in vitro and in vivo.Humanization of#176 was successfully performed.Among all the antagonistic antibodies developed in this study,B1-#176 is the only one exhibiting specific and high affinity LILRB1 binding,and potent LILRB1 blocking activity.To better understand the mechanism of B1-#176 in blocking LILRB1 activation,we next determined its binding epitope.B1-#176 showed specific binding to the D1D2 region of LILRB1.We made a series of amino acid mutations in the D1D2 region of LILRB1 to determine key amino acids for B1-176 binding,and two amino acids(R84 and Y76)were found to contribute to LILRB1 interacting with B1-#176.Rabbit/human chimeric antibody C176 with N297A in the glycosylation site was constructed for further anaylsis.We assessed the in vitro efficacy of anti-LILRB1 antibody#176 using the NK cell line NKL and primary NK cells from health donors or tumor patients.Our results showed that B1-C176 N297A antibody increased the cytotoxic activity of NKL against MM cell lines KMS27,OPM2 and RPMI8226,and the T cell leukemia line Jurkat.Interestingly,we found that pre-B leukemia cell line 697 and Burkitt lymphoma cell line Raji were resistant to NKL cytotoxicity even when treated with anti-LILRB1 antibody.To sensitize the 697 cells and Raji,we overexpressed MICA on 697 and Raji cells.As expected,overexpressing MICA on the cancer cell surface stimulated the cytotoxic activities of NKL cells against these cells and anti-LILRB1 antibody further increased the cytotoxic activities of NKL cells.These results suggest that LILRB1 blockade and NKG2D activation on NKL cells synergistically increase their cytotoxic activity.Anti-LILRB1 antibody increased the cytotoxic activities of primary LILRB1+NK cells from healthy donor against several MHC Class I positive liquid tumor cell lines,but not against Daudi cells,which do not express the LILRB1 ligand MHC class I.We tested the in vivo function of anti-LILRB1 antibody in NSG mice.NKLresistant 697 cell and NKL-sensitive 697 MICA mixtures were injected together with NKL cells into the peritoneal cavity of NSG mice.Our results showed that antiLILRB 1 antibody increased the in vivo cytotoxic activity of NKL cells against 697MICA cells.We further assessed the efficacy of anti-LILRB1 antibody to prevent multiple myeloma development in a xenograft mouse model.The mice that received NKL cells and anti-LILRB1 antibody had significantly lower disease burden and longer survival than did mice that received NKL cells and control IgG or mice that received only phosphate buffered saline(PBS).Taken together,our results indicate that blocking LILRB1 signaling on immune effector cells such as NK cells may represent a novel strategy for development of anticancer immunotherapy.Anti-LILRB1 antagonistic antibody B1-#176 is a promising antibody drug candidate for rescuing the immune suppressions caused by LILRB 1 activation. |
