Programmed Death Ligand-1 Binding Research Articles

HMGB1 promotes neutrophil PD-L1 expression through TLR2 and mediates T cell apoptosis leading to immunosuppression in sepsis

Neutrophils and T lymphocytes are closely related to occurrence of immunosuppression in sepsis. Studies have shown that neutrophil apoptosis decreases and T lymphocyte apoptosis increases in sepsis immunosuppression, but the specific mechanism involved remains unclear. In the present study, we found Toll-like Receptor 2 (TLR2) and programmed death-ligand 1 (PD-L1) were significantly activated in bone marrow neutrophils of wild-type mice after LPS treatment and that they were attenuated by treatment with C29, an inhibitor of TLR2. PD-L1 activation inhibits neutrophil apoptosis, whereas programmed death protein 1 (PD-1)activation promotes apoptosis of T lymphocytes, which leads to immunosuppression. Mechanistically, when sepsis occurs, pro-inflammatory factors and High mobility group box-1 protein (HMGB1) passively released from dead cells cause the up-regulation of PD-L1 through TLR2 on neutrophils. The binding of PD-L1 and PD-1 on T lymphocytes leads to increased apoptosis of T lymphocytes and immune dysfunction, eventually resulting in the occurrence of sepsis immunosuppression. In vivo experiments showed that the HMGB1 inhibitor glycyrrhizic acid (GA) and the TLR2 inhibitor C29 could inhibit the HMGB1/TLR2/PD-L1 pathway, and improving sepsis-induced lung injury. In summary, this study shows that HMGB1 regulates PD-L1 and PD-1 signaling pathways through TLR2, which leads to immunosuppression.

Identification of the functional PD-L1 interface region responsible for PD-1 binding and initiation of PD-1 signaling

The PD-1/PD-L1 checkpoint pathway is important for regulating immune responses and can be targeted by immunomodulatory drugs to treat a variety of immune disorders. However, the precise protein-protein interactions required for the initiation of PD-1/PD-L1 signaling are currently unknown. Previously, we designed a series of first-generation PD-1 targeting peptides based on the native interface region of programmed death ligand 1 (PD-L1) that effectively reduced PD-1/PD-L1 binding. In this work, we further characterized the previously identified lead peptide, MN1.1, to identify key PD-1 binding residues and design an optimized peptide, MN1.4. We show MN1.4 is significantly more stable than MN1.1 in serum and retains the ability to block PD-1/PD-L1 complex formation. We further characterized the immunomodulatory effects of MN1.4 treatment by measuring markers of T cell activation in a co-culture model with ovarian cancer cells and peripheral blood mononuclear cells. We found MN1.4 treatment reduced cytokine secretion and suppressed T cell responses in a similar manner as recombinant PD-L1. Therefore, the PD-L1 interface region used to design MN1.4 appeared sufficient to initiate PD-1 signaling and likely represents the minimum necessary region of PD-L1 required for PD-1 recognition. We propose a peptide agonist for PD-1, such as MN1.4, could have several applications for treating autoimmune disorders caused by PD-1 deficiencies such as type 1 diabetes, inflammatory arthritis, or autoimmune side effects arising from monoclonal antibody-based cancer immunotherapies.

Theprogrammed death ligand 1 interactome demonstrates bidirectional signaling coordinating immune suppression and cancer progression in head and neck squamous cell carcinoma.

The programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) are validated cancer targets; however, emerging mechanisms and impact of PD-L1 intracellular signaling on cancer behavior are poorly understood. We investigated the cancer cell intrinsic role of PD-L1 in multiple patient-derived models in vitro and in vivo. PD-L1 overexpression, knockdown, and PD-L1 intracellular domain (PD-L1-ICD) deletion (Δ260-290PD-L1) models were assessed for key cancer properties: clonogenicity, motility, invasion, and immune evasion. To determine how PD-L1 transduces signals intracellularly, we used the BioID2 platform to identify the PD-L1 intracellular interactome. Both human papillomavirus-positive and negative patient-derived xenografts were implanted in NOD-scid-gamma and humanized mouse models to investigate the effects of recombinant PD-1, anti-PD-L1, and anti-signal transducer and activator of transcription 3 (STAT3) in vivo. PD-L1 intracellular signaling increased clonogenicity, motility, and invasiveness in multiple head and neck squamous cell carcinoma (HNSCC) models, and PD-1 binding enhanced these effects. Protein proximity labeling revealed the PD-L1 interactome, distinct for unbound and bound PD-1, which initiated cancer cell-intrinsic signaling. PD-L1 binding partners interleukin enhancer binding factors 2 and 3 (ILF2-ILF3) transduced their effect through STAT3. Δ260-290PD-L1 disrupted signaling and reversed pro-growth properties. In humanized HNSCC in vivo models bearing T-cells, PD-1 binding triggered PD-L1 signaling, and dual PD-L1 and STAT3 inhibition were required to achieve tumor control. Upon PD-1 binding, the PD-L1 extracellular and intracellular domains exert a synchronized effect to promote immune evasion by inhibiting T-cell function while simultaneously enhancing cancer cell-invasive properties.

Abstract 3579: Assessment of target-mediated biodistribution of an 89Zr labeled PD-L1/4-1BB bispecific Mabcalin protein

Abstract Background: PRS-344/S095012 is a novel 4-1BB (CD137) and programmed death-ligand 1 (PD-L1) bispecific antibody-Anticalin® fusion protein (MabcalinTM protein) designed to cluster 4-1BB on activated T cells exclusively in the presence of PD-L1 expressing cells. We aimed to study PRS-344/S095012 in vivo biodistribution and pharmacokinetics with 89Zr-positron emission tomography (PET) at a dose with antitumoral activity in mice and evaluate the contribution of each targeting arm. Methods: PRS-344/S095012 lacks cross-reactivity to murine 4-1BB and PD-L1. To explore the PRS-344/S095012 biodistribution in a humanized 4-1BB knock-in mouse model, we synthesized the surrogate 89Zr-Atezo-J10 with the same 4-1BB building block and cross-reactivity to murine PD-L1. Humanized 4-1BB knock-in C57BL/6J (h4-1BB KI B6) and C57BL/6J (B6) mice (n=4-6 per group) were subcutaneously engrafted with murine wildtype MC38 colon adenocarcinoma cells. Tumors were grown to a minimum of ≥50 mm3 (average 163 mm3) before tracer injection. Mice received intravenously 30 µg (2.5 MBq) of 89Zr-PRS-344/S095012 or 89Zr-Atezo-J10 supplemented with PRS-344/S095012 or Atezo-J10 up to 10 mg/kg. Four mice groups were formed to distinguish between bispecific (Atezo-J10 in h4-1BB KI B6), monospecific PD-L1 (Atezo-J10 in B6), monospecific 4-1BB (PRS-344/S095012 in h4-1BB KI B6), and isotype (PRS-344/S095012 in B6) binding up to 4 days post-injection (pi). In addition, a fifth group (5 MBq 89Zr-Atezo-J10) was studied to visualize the bispecific biodistribution up to 7 days pi. At days 1, 2, 4, or 2, 4, 7 pi, mice underwent serial PET imaging to obtain mean and maximum standardized uptake (SUVmean/max) and retro-orbital blood sampling, followed by ex vivo biodistribution. Results: PET imaging showed 89Zr-Atezo-J10 specific tumor accumulation with higher tumor-to-blood ratios of respectively 2.2-, 2.6-, and 2.4-fold (p<0.01) at 4 days pi compared to monospecific binding of PD-L1, 4-1BB, and isotype. The ex vivo biodistribution demonstrated the same trend with respectively 4.2-, 5.5-, and 6.8-fold (p<0.01) increase in tumor-to-blood uptake for 89Zr-Atezo-J10 versus monospecific binding of PD-L1, 4-1BB, and isotype 89Zr-Atezo-J10 spleen uptake was comparable (ns) with monospecific binding of PD-L1 but elevated (p<0.01) compared to 4-1BB or isotype distribution. The uptake in lymph nodes (axillary, cervical, tumor-draining, and mesenteric) did not differ between the groups. Conclusion: 89Zr-Atezo-J10 specific accumulation in PD-L1 expressing tumors is due to both PD-L1 and 4-1BB binding and is higher than with PD-L1 and 4-1BB mono-targeting. This preclinical study supports the clinical evaluation of 89Zr-PRS-344/S095012’s whole-body distribution and the development of tumor-specific 4-1BB targeting bispecifics. Citation Format: Claudia A. van Winkel, Xiaoyu Fan, Danique Giesen, Glenn Gauderat, Lucia Pattarini, Thomas Jaquin, Anissa Barakat, Anne-Marie De La Bigne, Marleen Richter, Nicole Andersen, Julie Legrand, Helene Lelièvre, Elisabeth G. de Vries, Aizea Morales-Kastresana, Marjolijn N. Lub- de Hooge. Assessment of target-mediated biodistribution of an 89Zr labeled PD-L1/4-1BB bispecific Mabcalin protein. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3579.

Abstract 1878: Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2520, targeting both PD-L1 and CD47 as cancer immunotherapy

Abstract The programmed cell death protein 1 (PD-1) immune checkpoint is well known in its ability to attenuate T cell-mediated immune responses in tumors expressing its ligand, programmed death-ligand 1 (PD-L1). CD47, a cell surface molecule in the immunoglobulin superfamily, is another established target overexpressed on various malignant cells and acts as an innate inhibitory checkpoint by interrupting the phagocytosis of tumor cells and downstream activation of innate responses. It has been reported that the simultaneous blockade on both CD47 and PD-L1 checkpoints inhibited tumor growth and circulating tumor cells metastasis more potently than a single antibody inhibition or blank control in the 4T1 tumor mouse model in vivo. IMM2520 is an antibody-receptor (mAb-Trap) fusion protein consisting of recombinant human SIRPα and anti- PD-L1 antibody, thereby targeting CD47 and PD-L1 to antagonize both innate and adaptive immune checkpoint pathways. IMM2520 binds to PD-L1 on the tumor cell membrane and restores the immune response of T cells. IMM2520 also reverses the inhibitory effect of tumor cells on macrophages by blocking the binding of CD47 on the tumor cell to SIRPα on macrophages. In vitro studies have demonstrated that IMM2520 not only possesses a potent binding activity to CD47 (EC50 = 0.8 nM) and PD-L1 (EC50 = 0.09 nM) respectively, but also exhibits a strong activity in blocking PD-1/PD-L1 binding (IC50 = 0.06 - 1.0 nM). IMM2520 also showed a potent antibody-dependent cellular cytotoxicity (ADCC, EC50 = 1.54 ng/ml) against Raji-PDL1 and induced a strong antibody-dependent cellular phagocytosis (ADCP, EC50 = 0.04 nM) activity (THP-1 against Raji-PDL1). In vivo efficacy study with CT26-hPD-L1/hCD47 colon cancer model demonstrated that IMM2520 at 3 mg/kg significantly reduced tumor volume (TGITV = 90.12%, P<0.01 vs Vehicle). Interestingly, IMM2520 demonstrated a potent and significantly higher anti-tumor activities than IMM01 (a SIRPα-Fc fusion protein) and IMM2505 (also a CD47XPD-L1 mAb-Trap, but the variable region sequence of this PD-L1 antibody is identical to atezolizumab). IMM2520 at dose of 6 mg/kg completely eradicated the established tumors. In another in vivo efficacy study with MC38-hCD47/hPD-L1 colon cancer model, IMM2520 demonstrated a significant anti-tumor activity in a dose-dependent manner at doses between 2 mg/kg to 20 mg/kg. Given its potent preclinical anti-tumor activity as well as the favorable safety profile, IMM2520 may serve as a potent immunotherapy for multiple cancer types by targeting PD-L1 and CD47 on tumor cells. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Wenqi Zhu, Yanan Yang, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Fan Zhang, Frank Gan. Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2520, targeting both PD-L1 and CD47 as cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1878.

Immunotherapeutics in lung cancers: from mechanistic insight to clinical implications and synergistic perspectives.

Lung cancer is one of the highly lethal forms of cancer whose incidence has worldwide rapidly increased over the past few decades. About 80-85% of all lung cancer cases constitute non-small cell lung cancer (NSCLC), with adenocarcinoma, squamous cell carcinoma and large cell carcinoma as the main subtypes. Immune checkpoint inhibitors have led to significant advances in the treatment of a variety of solid tumors, significantly improving cancer patient survival rates. The cytotoxic drugs in combination with anti-PD-(L)1 antibodies is a new method that aims to reduce the activation of immunosuppressive and cancer cell prosurvival responses while also improving direct cancer cell death. The most commonly utilized immune checkpoint inhibitors for patients with non-small cell lung cancer are monoclonal antibodies (Atezolizumab, Cemiplimab, Ipilimumab, Pembrolizumab etc.) against PD-1, PD-L1, and CTLA-4. Among them, Atezolizumab (TECENTRIQ) and Cemiplimab (Libtayo) are engineered monoclonal anti programmed death ligand 1 (PD-L1) antibodies that inhibit binding of PD-L1 to PD-1 and B7.1. As a result, T-cell proliferation and cytokine synthesis are inhibited leading to restoring the immune homeostasis to fight cancer cells. In this review article, the path leading to the introduction of immunotherapeutic options in lung cancer treatment is described, with analyzing the benefits and shortages of the current immunotherapeutic drugs. In addition, possibilities to co-administer immunotherapeutic agents with standard cancer treatment modalities are also considered.

Programmed death ligand 1 intracellular interactions with STAT3 and focal adhesion protein Paxillin facilitate lymphatic endothelial cell remodeling

Lymphatic endothelial cells (LECs) comprise lymphatic capillaries and vessels that guide immune cells to lymph nodes (LNs) and form the subcapsular sinus and cortical and medullary lymphatic structures of the LN. During an active immune response, the lymphatics remodel to accommodate the influx of immune cells from the tissue, but factors involved in remodeling are unclear. Here, we determined that a TSS motif within the cytoplasmic domain of programmed death ligand 1 (PD-L1), expressed by LECs in the LN, participates in lymphatic remodeling. Mutation of the TSS motif to AAA does not affect surface expression of PD-L1, but instead causes defects in LN cortical and medullary lymphatic organization following immunostimulant, Poly I:C, administration invivo. Supporting this observation, invitro treatment of the LEC cell line, SVEC4-10, with cytokines TNFα and IFNα significantly impeded SVEC4-10 movement in the presence of the TSS-AAA cytoplasmic mutation. The cellular movement defects coincided with reduced F-actin polymerization, consistent with differences previously found in dendritic cells. Here, in addition to loss of actin polymerization, we define STAT3 and Paxillin as important PD-L1 binding partners. STAT3 and Paxillin were previously demonstrated to be important at focal adhesions for cellular motility. We further demonstrate the PD-L1 TSS-AAA motif mutation reduced the amount of pSTAT3 and Paxillin bound to PD-L1 both before and after exposure to TNFα and IFNα. Together, these findings highlight PD-L1 as an important component of a membrane complex that is involved in cellular motility, which leads to defects in lymphatic organization.

Colocalized targeting of TGF-β and PD-L1 by bintrafusp alfa elicits distinct antitumor responses

BackgroundBintrafusp alfa (BA) is a bifunctional fusion protein designed for colocalized, simultaneous inhibition of two immunosuppressive pathways, transforming growth factor-β (TGF-β) and programmed death-ligand 1 (PD-L1), within the tumor microenvironment...

Abstract 2477: PD-L1 PET quantifies the pharmacodynamic effects of immunotherapy

Abstract Introduction: Immune checkpoint protein targeted therapeutics have transformed cancer treatment but only a subset of patients derive benefit. To improve efficacy, a variety of combination therapies are being studied. Programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) is used as a biomarker to guide many of those therapies. Non-invasive evaluation of therapy induced changes in PD-L1 levels in the whole body could provide insights to guide those therapies. Here, we investigated the potential of [18F]DK222, a novel PD-L1 binding positron emission tomography (PET) imaging agent, to quantify PD-L1 levels and pharmacodynamics in humanized mouse modes. Methods: Non-small cell lung cancer (NSCLC), urothelial carcinoma (UC) and melanoma cell lines were screened by flow cytometry (FC) for PD-L1 expression and density. In vitro cell uptake assays, in vivo PET imaging and ex vivo biodistribution studies were used to evaluate the specificity of [18F]DK222 to bind PD-L1 (n=5-6/tumor). To quantify immunotherapy induced changes in PD-L1, A375 melanoma xenografts were inoculated in NSG mice humanized with CD34+ cells. Mice were treated with either 5 mg/kg anti- PD-1; (pembrolizumab) or 5 mg/kg of anti-PD-1 + 4 mg/kg of anti- CTLA-4 (ipilimumab) (n=5) and [18F]DK222 PET was acquired before and 7 days after start of treatment. Tumor growth was monitored. FC of tumors was performed to measure changes in TME and immune cell infiltrates. Results: In vitro uptake of [18F]DK222 in NSCLC (H244 > H226 > A549) and UC (BFTC909 > T24 > SCaBER) correlated with PD-L1 density (R2=0.96). [18F]DK222 PET and biodistribution studies in mice with NSCLC and UC xenografts revealed high and variable tumor uptake at 60 min, which was in agreement with PD-L1 levels detected by IHC. These data established sensitivity of [18F]DK222 PET to quantify variable PD-L1 levels in vivo. We next sought to quantify therapy induced changes in PD-L1 levels. We observed higher [18F]DK222 uptake in tumors of mice treated with combination therapy compared to monotherapy (1.83 vs 0.47 %ID/cc). A significant delay in tumor growth was observed with combination therapy compared to monotherapy. Two out of five mice with highest increase in [18F]DK222 uptake in combination therapy group exhibited least tumor growth. Also, final tumor volumes negatively correlated with [18F]DK222 uptake (R2=0.33) irrespective of treatment regimen. FC analysis showed higher immune infiltrates (1320 vs 230 per milligram of tumor), PD-L1+ cells (6005 vs 2739/mg) and ratio of CD8+ to CD4+ cells (1.94 vs 0.47) in tumors treated with combination therapy compared with monotherapy. These observations confirmed that immunotherapy induced changes in PD-L1 levels in tumors can be quantified using [18F]DK222 PET. Conclusion: [18F]DK222 PET can effectively quantify variable PD-L1 levels in tumors. Moreover, [18F]DK222 PET exhibits the sensitivity to quantify PD-L1 pharmacodynamics to inform on tumor response to therapy. Citation Format: Akhilesh Mishra, Dhiraj Kumar, Kuldeep Gupta, Patrick M. Forde, Edward Gabrielson, Sridhar Nimmagadda. PD-L1 PET quantifies the pharmacodynamic effects of immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2477.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-β receptor I versus II.

SUMMARYIntrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-β. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody. We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-β receptors I (TβRI) and II (TβRII). While the extracellular domain of PD-L1 (amino acids 19–238) targets TβRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TβRI mRNA from degradation by the RNA exosome complex. PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.

Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model.

Immune checkpoints such as programmed death-1 (PD-1) have been proven as antitumor targets by enhancing cytotoxic T cell activity. All immune checkpoint blockades are antibody therapeutics that have large size and high affinity, as well as known immune-related side effects and low responses. To overcome the limitation of antibody therapeutics, we have explored PD-1/PD-L1 (programmed death-ligand 1) blockades in traditional oriental medicine, which has a long history but has not yet studied PD-1/PD-L1 blockades. Sanguisorbae Radix extract (SRE) blocked PD-1 and PD-L1 binding in competitive ELISA. SRE effectively inhibited the PD-1/PD-L1 interaction, thereby improving T cell receptor (TCR) signaling and the NFAT-mediated luciferase activity of T cells. SRE treatment reduced tumor growth in the humanized PD-L1 MC38 cell allograft humanized PD-1 mouse model. Additionally, the combination of SRE and pembrolizumab (anti-PD-1 antibody) suppressed tumor growth and increased infiltrated cytotoxic T cells to a greater extent did either agent alone. This study showed that SRE alone has anticancer effects via PD-1/PD-L1 blockade and that the combination therapy of SRE and pembrolizumab has enhanced immuno-oncologic effects.

First-in-Humans Evaluation of a PD-L1-Binding Peptide PET Radiotracer in Non-Small Cell Lung Cancer Patients.

68Ga-NOTA-WL12 is a peptide-based PET imaging agent. We conducted a first-in-human study of 68Ga-NOTA-WL12 for PET to study the invivo biodistribution, metabolism, radiation dosimetry, safety, and potential for quantifying programmed death ligand-1 (PD-L1) expression levels in patients with advanced non-small cell lung cancer (NSCLC). Methods: In vitro assessment of the PD-L1 expression and cellular uptake of 68Ga-NOTA-WL12 was performed, followed by invivo evaluation of 68Ga-NOTA-WL12 uptake in mouse models with tumors. Nine patients with NSCLC with lesions expressing PD-L1 were enrolled and monitored for adverse events during the study. 68Ga-NOTA-WL12 and paired 18F-FDG PET/CT imaging were performed. Uptake (SUV, SUL [SUVlean], and kBq/mL) values of tumors and normal organs were obtained. Radiopharmaceutical biodistribution, radiation dosimetry, and the relationship of tumor uptake to PD-L1 expression were evaluated. Follow-up 18F-FDG PET/CT was performed in patients who had undergone treatment with a combination of pembrolizumab with chemotherapy. Results:68Ga-NOTA-WL12 exhibited PD-L1-specific uptake invitro and in PD-L1-positive tumors invivo. 68Ga-NOTA-WL12 PET imaging proved safe with acceptable radiation dosimetry. Physiologic tracer uptake was mainly visible in the liver, spleen, small intestine, and kidney. Tumors were clearly visible, particularly in the lungs, with a tumor-to-lung ratio of 4.45 ± 1.89 at 1 h. One hour was a suitable time point for image acquisition because no significant differences were noted in tumor-to-background ratios between 1 and 2 h. A strong, positive correlation was found between tumor uptake (SUVpeak) and PD-L1 immunohistochemistry results (r = 0.9349; P = 0.002). 68Ga-NOTA-WL12 and 18F-FDG PET studies suggest that PD-L1 PET before therapy may indicate the therapeutic efficacy of pembrolizumab plus chemotherapy combination treatment. Conclusion: Our first-in-human findings demonstrate the safety and feasibility of 68Ga-NOTA-WL12 for noninvasive, invivo detection of tumor PD-L1 expression levels, indicating potential benefits for clinical PD-L1 therapy.

A Small Molecule Antagonist of PD-1/PD-L1 Interactions Acts as an Immune Checkpoint Inhibitor for NSCLC and Melanoma Immunotherapy

Immune checkpoint inhibitors, such as monoclonal antibodies targeting programmed death 1 (PD-1) and programmed death ligand-1 (PD-L1), have achieved enormous success in the treatment of several cancers. However, monoclonal antibodies are expensive to produce, have poor tumor penetration, and may induce autoimmune side effects, all of which limit their application. Here, we demonstrate that PDI-1 (also name PD1/PD-L1 inhibitor 1), a small molecule antagonist of PD-1/PD-L1 interactions, shows potent anti-tumor activity in vitro and in vivo and acts by relieving PD-1/PD-L1-induced T cell exhaustion. We show that PDI-1 binds with high affinity to purified human and mouse PD-1 and PD-L1 proteins and is a competitive inhibitor of human PD-1/PD-L1 binding in vitro. Incubation of ex vivo activated human T cells with PDI-1 enhanced their cytotoxicity towards human lung cancer and melanoma cells, and concomitantly increased the production of granzyme B, perforin, and inflammatory cytokines. Luciferase reporter assays showed that PDI-1 directly increases TCR-mediated activation of NFAT in a PD-1/PD-L1-dependent manner. In two syngeneic mouse tumor models, the intraperitoneal administration of PDI-1 reduced the growth of tumors derived from human PD-L1-transfected mouse lung cancer and melanoma cells; increased and decreased the abundance of tumor-infiltrating CD8+ and FoxP3+ CD4+ T cells, respectively; decreased the abundance of PD-L1-expressing tumor cells, and increased the production of inflammatory cytokines. The anti-tumor effect of PDI-1 in vivo was comparable to that of the anti-PD-L1 antibody atezolizumab. These results suggest that the small molecule inhibitors of PD-1/PD-L1 may be effective as an alternative or complementary immune checkpoint inhibitor to monoclonal antibodies.

The Extrinsic and Intrinsic Roles of PD-L1 and Its Receptor PD-1: Implications for Immunotherapy Treatment.

Programmed death-ligand 1 (PD-L1) is an immune checkpoint inhibitor that binds to its receptor PD-1 expressed by T cells and other immune cells to regulate immune responses; ultimately preventing exacerbated activation and autoimmunity. Many tumors exploit this mechanism by overexpressing PD-L1 which often correlates with poor prognosis. Some tumors have also recently been shown to express PD-1. On tumors, PD-L1 binding to PD-1 on immune cells promotes immune evasion and tumor progression, primarily by inhibition of cytotoxic T lymphocyte effector function. PD-1/PD-L1-targeted therapy has revolutionized the cancer therapy landscape and has become the first-line treatment for some cancers, due to their ability to promote durable anti-tumor immune responses in select patients with advanced cancers. Despite this clinical success, some patients have shown to be unresponsive, hyperprogressive or develop resistance to PD-1/PD-L1-targeted therapy. The exact mechanisms for this are still unclear. This review will discuss the current status of PD-1/PD-L1-targeted therapy, oncogenic expression of PD-L1, the new and emerging tumor-intrinisic roles of PD-L1 and its receptor PD-1 and how they may contribute to tumor progression and immunotherapy responses as shown in different oncology models.

Gated Resonance Energy Transfer (gRET) Controlled by Programmed Death Protein Ligand 1.

The resonance energy transfer (RET) between an excited fluorescent probe molecule and a plasmonic nanoparticle (AuNP) has been investigated to evaluate the effect of protein molecules on the RET efficiency. We have found that the energy transfer to a functionalized AuNP can be modulated by a sub-monolayer film of programmed death-ligand 1 (PD-L1) protein. The interactions of PD-L1 with AuNP@Cit involve incorporation of the protein in AuNP shell and formation of a submonolayer adsorption film with voids enabling gated surface plasmon resonance energy transfer (SPRET). A model of the gated-RET system based on the protein size, estimated using Fisher–Polikarpov–Craievich density approximation, has been developed and can be utilized for other proteins, with minimum data requirement, as well. The value of the equilibrium constant KL determined for the Langmuir isotherm is high: KL = 1.27 × 108 M−1, enabling highly sensitive control of the gated-RET by PD-L1. Thus, with the gated-RET technique, one can determine PD-L1 within the dynamic range, extending from 1.2 to 50 nM. Moreover, we have found that the Gibbs free energy for PD-L1 binding to AuNP@Cit is −46.26 kJ/mol (−11.05 kcal/mol), indicating a strong adsorption with supramolecular interactions. The proposed gated-RET system, with the fluorescence intensity of the fluorophore probe molecule modulated by plasmonic quenching with AuNP and shielding of energy transfer by the adsorbed PD-L1 can be further developed for determination of PD-L1 in pharmaceutical formulations for immune checkpoint control in cancer therapy.

Abstract 3448: A highly specific 18F-labelled peptide for non-invasive quantification of PD-L1 levels in tumors

Abstract Introduction. Blockade of programmed death ligand-1 (PD-L1) and its receptor (PD-1) has emerged as a foundational treatment strategy for cancer. Immunohistochemical analysis (IHC) of PD-L1 expression is one of the biomarkers used to guide those treatments. However, total PD-L1 levels, dynamics and their relevance to treatment response are poorly understood. To facilitate non-invasive quantification of total PD-L1 levels and dynamics, here we report the synthesis of a highly specific PD-L1 binding radiolabeled peptide, [18F]DK222, and demonstrate its specificity for PD-L1 quantification in vivo in xenograft models of multiple cancer types. Experimental procedures. A 14 amino acid macrocyclic peptide (DK222) was conjugated with a bifunctional chelator (NCS-MP-NODA, Chematech) and the corresponding fluorinated radioactive and non-radioactive analogs, [18/19F]DK222, were synthesized by aluminum fluoride method. In vitro competitive inhibition assays were used to establish peptide analog affinity for PD-L1. We then tested in vitro and in vivo specificity of [18F]DK222 for PD-L1 in cell binding, positron emission (PET) imaging and ex vivo biodistribution studies. Those studies were performed in xenograft models having varying levels of PD-L1 expression and derived from breast, bladder, lung and skin cancers. In vivo PD-L1 specificity was confirmed by co-injecting a blocking dose of 50 mg/kg of parent peptide. PD-L1 expression in cell lines and tumors was validated by flow cytometry and IHC, respectively. Results. We observed half-maximal inhibitory concentrations of < 25 nM for DK222 and its analogs to inhibit PD-L1 interaction with its receptor. We generated [18F]DK222 in 25% radiochemical yield (non-decay corrected) with high purity (>95%) and with a specific activity of 256±18 mCi/micromole. In vitro studies showed that cell uptake of [18F]DK222 is reflective of PD-L1 expression detected by flow cytometry in the PD-L1high (MDAMB231, LOXIMVI, BFTC909) and PD-L1low (SUM149, MeWo, and T24) cells. PET imaging and biodistribution studies showed robust tracer uptake in PD-L1 high but not in PD-L1 low tumors. Blocking studies showed >80% reduction in the uptake of [18F]DK222 in PD-L1high tumors (P<0.0001). Imaging and biodistribution studies collectively demonstrated that [18F]DK222 binds rapidly and specifically to human PD-L1. PET imaging observations were consistent with high immunoreactivity observed in PD-L1 high tumors compared with PD-L1 low tumors. Conclusions. The pharmacokinetics of [18F]DK222 indicate that quantification of PD-L1 levels is feasible within 60 min of radiotracer administration. The developed radiotracer [18F]DK222 could potentially enable non-invasive quantification of PD-L1 levels in vivo by PET imaging. Citation Format: Dhiraj Kumar, Akhilesh Mishra, Ala Lisok, Sagar Shelake, Bryan Wharram, Ravindra Desilva, Ronnie Mease, Sridhar Nimmagadda. A highly specific 18F-labelled peptide for non-invasive quantification of PD-L1 levels in tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3448.

Dual-function chimeric antigen receptor T cells targeting c-Met and PD-1 exhibit potent anti-tumor efficacy in solid tumors.

Purpose Programmed cell death 1 (PD-1), which is upregulated under the continuous induction of the tumor microenvironment, causes chimeric antigen receptor (CAR)-T cell hypofunction via interaction with programmed death ligand 1 (PD-L1). This study aimed to construct CAR-T cells that are resistant to PD-1 inhibition to improve the effect of CAR-T cells in solid tumors. Methods We constructed a type of dual-function CAR-T cell that targets tumor-associated antigen c-Met and blocks the binding of PD-1 with PD-L1. The expression of c-Met, PD-L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR-T cells were determined using lactate dehydrogenase release assay, enzyme-linked immunosorbent assay, and flow cytometry, respectively. The levels of p-Akt, p-MAPK, caspase-3, and Bcl2 were detected by western blot. The in vivo anti-tumor effect was evaluated using tumor xenograft models. Results Dual-function CAR-T cells could mediate enhanced active signals upon encountering target antigens and had targeted cytotoxicity to target cells. However, the cytotoxicity of c-Met-CAR-PD-1+ T cells was impaired due to the interaction of PD-1 with PD-L1. By blocking the binding of PD-1 and PD-L1, the novel dual-function CAR-PD-1+ T cells could maintain cytotoxicity to PD-L1+ tumor cells. In tumor tissue, the dual-function CAR-T cells showed lower inhibitory receptor expression and lower differentiation characteristics, which resulted in potent anti-tumor effects and prolonged survival in PD-L1+ tumor xenograft models compared to single-target CAR-T cells. Conclusion These results confirm that the novel dual-function CAR-T cells exhibit stronger anti-tumor activity against solid tumors than traditional single-target CAR-T cells and present a new approach that enhance the activity of CAR-T cells in solid tumors.

Guidance and best practices for nuclear cardiology laboratories during the coronavirus disease 2019 (COVID-19) pandemic: An Information Statement from ASNC and SNMMI

1022 Purpose: The programmed death-ligand 1 (PD-L1) immunohistochemistry correlates moderately with the response to PD-L1 immunomodulatory therapy. Here we present the initial results from the studies to assess the feasibility of non-invasive PD-L1 imaging with 68Ga-WL12 (a 12-mer anti-PD-L1 peptide) radiotracer from animal experiments to the first-in-human study. Methods: PD-L1 binding peptide, NOTA-WL12, was synthesized by custom services with >95% chemical purity. , we generated 68Ga-WL12 with high specific radioactivity and radiochemical purity. We select human non-small cell lung cancer cells ( HCC827) , which with moderate expression of PD-L1. To demonstrate PD-L1-specific binding of 68Ga-WL12, both binding affinity test and cell uptakes were carried out with excess WL12. Normal Mice were used for PET/CT imaging and biodistribution study. Finally, we recruited a patient ( 80 years old female ;ethical approval No.2019KT) with non-small cell lung cancer who was positive for PD-L1(>80%) and performed PET/CT imaging with 68Ga-WL12 before and after immunomodulatory therapy. Results: To demonstrate PD-L1 specificity and cell uptake, we generated 68Ga-WL12 with radiochemical purity >99% after purification, specific activity 27.8-111.2 GBq/umol. High 68Ga-WL12 uptake was observed in PD-L1-positive HCC827 cells. TO confirming radiotracer specificity, we observed a significant reduction in bound 68Ga-WL12 in the presence of the excess unmodified peptide, indicating that 68Ga-WL12 binding to PD-L1 is specific. We performed PET imaging of the tumor model with 68Ga-WL12. The uptake value was consistent with tracer biodistribution. Finally, in this first-in-human assessment of 68Ga-WL12, we show that the imaging signal corresponds to PD-L1 expression at sites of the primary tumor and metastatic focus. The SUVpeak of different tumor lesions ranged from 2.0 to 6.0. The SUV value of PD-L1 positive tumors decreased significantly after immunomodulatory therapy. Conclusions: In this study, we demonstrated that 68Ga-WL12 exhibited a high affinity to PD-L1 in the Cell uptake experiment in vitro. 68Ga-WL12 demonstrated PD-L1 specific uptake first-in-human study. Therefore, 68Ga-WL12 PET/CT imaging may be a useful tool to assess PD-L1 expression in lesions. Future clinical studies are needed to confirm our findings in a larger patient population, to comprehensively assess different 68Ga-WL12 uptake features in combination with other clinical data to optimize therapy response prediction, and to evaluate whether 68Ga-WL12 PET could also be used as a response predictor for treatment with other monoclonal antibodies targeting the PD-1/PD-L1 axis.

Guidance and best practices for nuclear cardiology laboratories during the coronavirus disease 2019 (COVID-19) pandemic: An Information Statement from ASNC and SNMMI.

1022 Purpose: The programmed death-ligand 1 (PD-L1) immunohistochemistry correlates moderately with the response to PD-L1 immunomodulatory therapy. Here we present the initial results from the studies to assess the feasibility of non-invasive PD-L1 imaging with 68Ga-WL12 (a 12-mer anti-PD-L1 peptide) radiotracer from animal experiments to the first-in-human study. Methods: PD-L1 binding peptide, NOTA-WL12, was synthesized by custom services with >95% chemical purity. , we generated 68Ga-WL12 with high specific radioactivity and radiochemical purity. We select human non-small cell lung cancer cells ( HCC827) , which with moderate expression of PD-L1. To demonstrate PD-L1-specific binding of 68Ga-WL12, both binding affinity test and cell uptakes were carried out with excess WL12. Normal Mice were used for PET/CT imaging and biodistribution study. Finally, we recruited a patient ( 80 years old female ;ethical approval No.2019KT) with non-small cell lung cancer who was positive for PD-L1(>80%) and performed PET/CT imaging with 68Ga-WL12 before and after immunomodulatory therapy. Results: To demonstrate PD-L1 specificity and cell uptake, we generated 68Ga-WL12 with radiochemical purity >99% after purification, specific activity 27.8-111.2 GBq/umol. High 68Ga-WL12 uptake was observed in PD-L1-positive HCC827 cells. TO confirming radiotracer specificity, we observed a significant reduction in bound 68Ga-WL12 in the presence of the excess unmodified peptide, indicating that 68Ga-WL12 binding to PD-L1 is specific. We performed PET imaging of the tumor model with 68Ga-WL12. The uptake value was consistent with tracer biodistribution. Finally, in this first-in-human assessment of 68Ga-WL12, we show that the imaging signal corresponds to PD-L1 expression at sites of the primary tumor and metastatic focus. The SUVpeak of different tumor lesions ranged from 2.0 to 6.0. The SUV value of PD-L1 positive tumors decreased significantly after immunomodulatory therapy. Conclusions: In this study, we demonstrated that 68Ga-WL12 exhibited a high affinity to PD-L1 in the Cell uptake experiment in vitro. 68Ga-WL12 demonstrated PD-L1 specific uptake first-in-human study. Therefore, 68Ga-WL12 PET/CT imaging may be a useful tool to assess PD-L1 expression in lesions. Future clinical studies are needed to confirm our findings in a larger patient population, to comprehensively assess different 68Ga-WL12 uptake features in combination with other clinical data to optimize therapy response prediction, and to evaluate whether 68Ga-WL12 PET could also be used as a response predictor for treatment with other monoclonal antibodies targeting the PD-1/PD-L1 axis.

N-glycosylation controls inflammatory licensing-triggered PD-L1 upregulation in human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are characterized by their multipotency, regenerative potential, and immunoregulatory properties. Nowadays, MSCs represent a promising cell-therapeutic option for hyperinflammatory conditions such as graft-vs-host disease following allogeneic hematopoietic stem cell transplantation. A better understanding of their biology is a prerequisite for improving their treatment efficacy. Emerging evidence suggests that immunosuppressive properties are not constitutively active in MSCs. Instead, microenvironmental inflammatory stimuli such as the cytokines interferon (IFN)-γ or tumor necrosis factor (TNF)-α license MSCs to acquire a tolerance-promoting phenotype. The immunological checkpoint molecule programmed death-ligand 1 (PD-L1) is an important regulator of T-cell responses. Binding of PD-L1 to the programmed cell death protein 1 (PD-1) receptor on T-cells suppresses their activation, proliferation, and induces apoptosis. Previous studies have revealed that cell surface expression and secretion of PD-L1 are part of the MSCs' immunomodulatory armamentarium. Here, we report that inflammatory licensing leads to an enhanced PD-L1 cell surface expression and secretion, which are both accompanied by an increased posttranslational protein N-glycosylation. These post-translational modifications have been shown to be critical for key biological processes such as cell trafficking, receptor signaling, and immunohomeostasis. In fact, promoting N-glycosylation in MSCs yielded increased PD-L1 levels. We report for the first time that PD-L1 N-glycosylation plays a decisive role for its transport to the MSCs' cell surface and its subsequent secretion (in response to proinflammatory trigger). Our data offer insights into a novel regulatory mechanism with the potential to be exploited as a means to foster the immunosuppressive potency of human MSCs.