AbstractmRNA expression patterns of genes for metabolic key-enzymes (sucrose phosphate synthase [SPS], phosphoenolpyruvate carboxylase [PEPC], pyruvate kinase, ribulose 1,5-bisphosphate carboxylase/oxygenase, glutamine synthetase 1, and glutamine synthetase 2) were investigated in leaves of rice plants grown at two nitrogen (N) concentrations. The relative gene expression patterns were similar in all leaves except for 9 th leaf, in which mRNA levels were generally depressed.Though an increased N concentrations prolonged the expression period of each mRNA, it did not affect the relative expression intensity of any mRNA in a given leaf. SPS V max , SPS limiting activity, PEPC activity, and carbon flow were examined using 14 CO 2 . The ratio between PEPC activity and SPS V max was higher in leaves developed at the vegetative growth stage (vegetative leaves: 5 th and 7 th 1 leaf) than in leaves developed after the ear primordia formation stage (reproductive leaves: 9 th and flag leaf). PEPC activity and SPS V max decreased with declining leaf N concentration. The 14 C photosynthate distribution to the amino acids fraction was higher in vegetative than in reproductive leaves when compared for the same leaf N status. Thus, at high PEPC activity to SPS activity ratio, more 14 C photosynthate was distributed to the amino acid pool, whereas at higher SPS activity, more 14 C was channeled into the carbohydrate fraction. Thus, leaf ontogeny was an important factor controlling photosynthate distribution to the Nor C-pool, respectively, regardless of leaf N status.
Background TIM-3 and CD39 are co-expressed in tumor-infiltrating T cells and antigen-presenting cells and considered to act as negative regulators of anti-tumor immunity. TIM-3 has multiple ligands including galectin-9, phosphatidylserine, CEA-CAM-1 and HMGB1. Although galectin-9 induces apoptosis in T cells via TIM-3, most of the TIM-3 antibodies under clinical development only partially block the galectin-9-binding to TIM-3. Adenosine is an immunosuppressive metabolite that suppresses T and NK cells. Inhibition of ATP-hydrolysis by purinergic ectoenzyme CD39 may recover anti-tumor immunity through reducing adenosine level in tumor microenvironment. We report herein BP1210, a novel TIM-3 biparatopic antibody (BpAb), that blocks the binding of multiple ligands including galectin-9 and BP1212, a bispecific antibody (BsAb) against TIM-3 and CD39, that blocks TIM-3-signaling and adenosine-mediated immune suppression. Methods The antibodies against TIM-3 and CD39 were cloned from mice immunized with recombinant proteins. Optimal combinations of clones were selected by functional assay for BpAb and BsAb, then the CDRs were grafted to human framework in effector null scFv-Fc format with knobs-intoholes mutations. Binding affinities and blocking/inhibiting activities were analyzed using recombinant proteins and cells endogenously expressing TIM-3 or CD39. The proliferation and cytokine production of T cells induced by antibodies were determined using human peripheral blood mononuclear cells (PBMCs). Anti-tumor efficacy of BP1210 was investigated in tumor models in huTIM-3 knock-in mice or in NOG mice transplanted with PBMCs. Results BP1210 BpAb that fully blocks the interaction of TIM-3 to both galectin-9 and phosphatidylserine, enhanced the activation of T cells stimulated by the antigen. i.e., BP1210 increased the population of IFNg + T cells, the production of cytokines, and suppressed the apoptosis of T cells, which are higher than other TIM-3 antibodies that fully blocks the binding to phosphatidylserine but not to galectin-9. in vivo studies showed that BP1210 retards the tumor growth and enhances the tumor-growth suppression by anti-PD-L1 antibody. BP1212 BsAb preferentially bound to the CD39 + TIM-3 + cells (exhausted T cells and APCs) and effectively blocked the enzymic activity of CD39 and binding to TIM-3 ligands in these cells. BP1212 augmented the proliferation and cytokine production of T cells, suggesting that BP1212 enhances the T cell immunity even in immunosuppressive tumor microenvironment. Conclusions BP1210 and BP1212 show the advantages over conventional TIM-3 and CD39 antibodies in T cell-mediated tumor immunity. Our engineered antibodies with novel combinations of antibody clones against immune checkpoints will provide new therapeutic options of tumor immunotherapy. Ethics Approval The present study was approved by the Institutional Ethics Committee of BrightPath Biotherapeutics Co., Ltd.(approved number: ERD-01). Animal studies were approved by the Institutional Animal Care and Use Committee (ap...
BackgroundT cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is a part of modules expressed on dysfunctional or exhausted T cells as well as dendritic cells and has emerged as a target for several therapeutic antibodies that are under clinical development. Co-blockade of TIM-3 and PD-1 results in tumor regression in preclinical models and improves anticancer T cell responses in patients with advanced cancers. TIM-3 has been reported to have multiple ligands including galectin-9, phosphatidylserine, CEACAM-1 and HMGB1, which bind to different regions on the extracellular domain of TIM-3. Most of the TIM-3 antibodies developed to date are intended to inhibit phosphatidylserine that binds to the pocket in TIM-3 immunoglobulin V domain. Galectin-9 binds to carbohydrate motifs on the opposite side of phosphatidylserine-binding site in immunoglobulin V domain and thereby induces cell death in TIM-3+ T cells. We report herein novel antibodies that block TIM-3 binding to multiple ligands including these two important ligands simultaneously.MethodsAnti-TIM-3 antibodies were generated by immunizing mice with a purified recombinant TIM-3 protein and TIM-3-expressing mammalian cell line. Phage display libraries were constructed using cDNAs of splenocytes and lymph node cells of the immunized mice, then subjected to the biopanning using recombinant TIM-3 proteins. After analyzing specificities and affinities to the TIM-3 protein, scFvs obtained were classified by epitope bin and inhibitory effects on TIM-3 binding to the multiple ligands. The scFvs were converted to scFv-Fc to generate biparatopic (bispecific) antibodies.ResultsAt least five classes of TIM-3 antibodies were obtained, and each class was grouped into different epitope bins and has unique inhibitory profiles for multiple ligands of TIM-3. Their biparatopic (bispecific) forms were produced from the scFv clones and subjected to the analyses of TIM-3 binding, inhibition of ligand binding, and immune activation. As expected, the biparatopic antibodies that recognize two different epitopes showed higher affinity and specificity to TIM-3 than monospecific forms. A lead biparatopic antibody that block the binding of TIM-3 to galectin-9 and phosphatidylserine showed remarkable potency on T cell activation, protection from exhaustion and apoptotic cell death of T cells as well as more potent anti-tumor efficacy.ConclusionsThis study demonstrates the successful development of a novel biparatopic antibody that blocks the binding of TIM-3 to phosphatidylserine and galectin-9 simultaneously. The antibody shows the advantages over conventional TIM-3 antibodies in reducing T cell exhaustion and potentially manipulated for the development of human monoclonal antibodies for therapeutic treatment of cancer.
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