PD-L1 Blocking Research Articles

Exploring the potential of combining IL-2-activated NK cells with an anti-PDL1 monoclonal antibody to target multiple myeloma-associated macrophages

Multiple myeloma (MM) is an incurable disease, characterized by malignant plasma cells in the bone marrow. MM growth is largely dependent on the tumor microenvironment (TME), consisting of complex cellular networks that shape a tumor-permissive environment. Within the TME, tumor-associated cells (TAC) comprise heterogeneous cell populations that collectively support immunosuppression. Reshaping the TME toward an immunostimulatory environment may enhance effectiveness of immunotherapies. Here, we investigated interactions between donor-derived natural killer (NK) cells and TAC, like tumor-associated macrophages (TAM) and M1 macrophages, and assessed whether anti-tumor effector functions of NK cells could be enhanced by an ADCC-triggering antibody targeting macrophages. Monocytes were polarized in vitro toward either M1 or TAM before co-culture with high-dose IL-2-activated NK cells. NK cell responses were assessed by measuring degranulation (CD107a) and IFN-γ production. We found that NK cells degranulated and produced IFN-γ upon interaction with both macrophage types. NK cell responses against PD-L1+ M1 macrophages could be further enhanced by Avelumab, an anti-PD-L1- and ADCC-inducing antibody. Additionally, NK cell responses were influenced by HLA class I, shown by stronger degranulation in NK cell subsets for which the corresponding HLA ligand was absent on the macrophage target cells (KIR-ligand mismatch) compared to degranulation in the presence of the HLA ligand (KIR-ligand match). Our results suggest that NK cells could, next to killing tumor cells, get activated upon interaction with TAC, like M1 macrophages and TAMs, and that NK cells combined with PD-L1 blocking antibodies with ADCC potential could, through IFN-γ secretion, promote a more immune-favorable TME.

PD-1/PD-L1 Immunotherapy: Combating Drug Resistance in Breast Cancer

In recent years, immune-checkpoint blocking therapy targeting the PD-1/PD-L1 axis has advanced tumor immunotherapy to a new level, with positive outcomes in a variety of malignant tumors. Tumors can avoid an antigen-specific T cell immune response by means of PD-1/PD-L1 signaling, which adversely controls T cell-mediated immune response. In clinical practice, it was shown that while some patients did have long-term success with immunotherapy, the majority eventually had resistance to drugs and recurrence. Hence, one of the main challenges that severely restricts the long-lasting benefits and widespread use of PD-1/PD-L1 blocking treatment is both primary and acquired resistance. Therefore, it is high time to understand the mechanisms of resistance for improving anti-PD-1/PD-L1 efficacy. In this review, we describe major signaling pathways that regulate PD-1/PD-L1 axis in cancers as well as the role of PD-1/PD-L1 in breast cancer development and progression. In addition, we further discuss the involvement of PD-1/PD-L1 axis in multi-drug resistance in cancers, which affected breast cancer and other solid tumor response rates and durability to treatment strategies.

Review and prospect of immune checkpoint blockade therapy represented by PD-1/PD-L1 in the treatment of clear cell renal cell carcinoma.

As a common tumor of the urinary system, the morbidity and mortality related to renal carcinoma, are increasing annually. Clear cell renal cell carcinoma (CCRCC) is the most common subtype of renal cell carcinoma, accounting for approximately 75% of the total number of patients with renal cell carcinoma. Currently, the clinical treatment of ccRCC involves targeted therapy, immunotherapy, and a combination of the two. In immunotherapy, PD-1/PD-L1 blocking of activated T cells to kill cancer cells is the most common treatment. However, as treatment progresses, some patients gradually develop resistance to immunotherapy. Meanwhile, other patients experience great side effects after immunotherapy, resulting in a survival status far lower than the expected survival rate. Based on these clinical problems, many researchers have been working on the improvement of tumor immunotherapy in recent years and have accumulated numerous research results. We hope to find a more suitable direction for future immunotherapy for ccRCC by combining these results and the latest research progress.

Histone Deacetylase Inhibitor Panobinostat Benefits the Therapeutic Efficacy of Oncolytic Herpes Simplex Virus Combined with PD-1/PD-L1 Blocking in Glioma and Squamous Cell Carcinoma Models.

Combination therapy has been widely explored for oncolytic virus (OV), as it can be met with tumor resistance. The HDAC inhibitor (HDACi) panobinostat is a potent pan-deacetylase inhibitor which blocks multiple cancer-related pathways and reverses epigenetic events in cancer progression. In this study, oncolytic activity in vitro and antitumor therapeutic efficacy in vivo when combined with oHSV and panobinostat were investigated. (1) Treatment with panobinostat enhanced oHSV propagation and cytotoxicity in human glioma A172 and squamous cell carcinoma SCC9 cells. (2) Combined treatment with oHSV and panobinostat enhanced virus replication mediated by the transcriptional downregulation of IFN-β- and IFN-responsive antiviral genes in human glioma A172 and squamous cell carcinoma SCC9 cells. (3) Panobinostat treatment induced upregulation of PD-L1 expression in both glioma and squamous cell carcinoma cells. (4) A significantly enhanced therapeutic efficacy was shown in vivo for the murine glioma CT-2A and squamous cell carcinoma SCC7 models when treated with a combination of oHSV, including PD-1/PD-L1 blockade and HDAC inhibition. Consequently, these data provide some new clues for the clinical development of combination therapy with OVs, epigenetic modifiers, and checkpoint blockades for glioma and squamous cell carcinoma.

Trial in Progress: An Open-Label Phase II Study of Relatlimab with Nivolumab in Combination with 5-Azacytidine for the Treatment of Patients with Relapsed/Refractory and Elderly Patients with Newly Diagnosed Acute Myeloid Leukemia (AARON)

The approval of venetoclax in combination with hypomethylating agents (HMA) for newly diagnosed acute myeloid leukemia (AML) has significantly improved the outcomes of patients that are not fit for intensive chemotherapy regimens. However, a considerable proportion of patients are primary refractory or relapse, highlighting the need for novel combinatorial treatment options in this patient population. Preclinical models have shown that blocking of the PD-1/PD-L1 pathway enhances antileukemic responses. However, in AML, early clinical trials evaluating the single-agent efficacy of nivolumab and other PD-1 inhibitors have demonstrated only limited activity. Based on preclinical findings that hypomethylating agents (HMA) like 5-Azacytidin (AZA) can induce an upregulation of PD-1 expression and the IFNγ signaling pathway, combinatorial treatment approaches have been studied. In relapsed/refractory (R/R) AML, the combination of AZA + Nivolumab demonstrated higher response rates and improved median overall survival compared to historical outcomes with HMA-based salvage clinical trials (Daver N et al, Cancer Discovery 2019). In our own previous work, we uncovered that blocking the inhibitory checkpoint molecule LAG-3 significantly increases the generation of anti-leukemic T-cell responses, in particular in combination with PD-1/PD-L1 blocking (Lichtenegger FS et al, Front Immunol 2018). Clinical trials are evaluating relatlimab, a monoclonal IgG4 antibody blocking LAG-3 and its interaction with HLA-class II molecules, in various indications, and it has recently been approved in combination with nivolumab for the treatment of patients with untreated advanced malignant melanoma by the FDA. Here, we describe a trial designed to assess the safety and efficacy of the combination of 5-azacytidine, nivolumab, and relatlimab in patients with r/r AML (cohort 1). The AARON trial (NCT04913922) is a phase 2, single arm, open-label trial. Additionally, we plan to amend the protocol to enroll patients aged ≥65 years with previously untreated AML (excluding patients with NPM1 and IDH1/2 mutations) who are unfit for or decline standard induction therapy (cohort 2). For this cohort, the trial will evaluate the combination of relatlimab, nivolumab and 5-azacytidine in combination with venetoclax. Importantly, in preclinical data venetoclax has been shown to promote T cell-mediated antileukemic activity (Lee JB et al, Blood 2021), making it a promising combination partner. The study will enroll a total of 30 adult patients with histologically or cytologically documented AML (excluding APL, and excluding patients with NPM1 and IDH1/2 mutations in the first-line cohort). A lead-in phase, recruited independently for both patient cohorts, will establish the safety of the study treatment in 6-12 patients per cohort. Patients must have adequate organ function and an ECOG performance score of ≤2. Patients with previous allogeneic stem cell transplantation can be enrolled in cohort 1 (from day +100 after allo-SCT) in the absence of higher-grade GvHD. Pre-treatment with HMAs is allowed for patients in cohort 1. Patients with a history of or active autoimmune disease are excluded. 5-azacytidine is applied as in routine care (75 mg/m2 BSA, days 1-7 of a 4-week cycle). Nivolumab (480mg flat dose) and relatlimab (160mg flat dose) are infused on day 1 of each cycle. For cohort 2, we plan to dose venetoclax at 70mg daily (with mandatory posaconazole co-medication from d1 to 28, or shorter in case of significant hematotoxicity). If dose-limiting toxicities occur in ≥2/6 patients in the lead-in phase, the dose of relatlimab will be reduced to 80mg (flat dose), and the safety of the treatment will be re-assessed with a new 6-patient lead-in phase. Response will be assessed after completion of cycle 1, then every 1-3 cycles. The primary endpoint of the lead-in phase is the establishment of a recommended phase 2 dose, and overall response rate. The latter will be assessed in both patients from the lead-in and the expansion phase. Secondary endpoints, assessed in all enrolled patients, include the safety, duration of response and survival. A comprehensive accompanying immunomonitoring program will study immunological changes in the peripheral blood and bone marrow. Funding: Bristol-Myers Squibb. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A mouse pancreatic organoid model to compare PD-L1 blocking antibodies

ABSTRACT Immune checkpoint inhibitors (ICIs) have changed the therapeutic landscape for cancer patients, but diabetes, a rare, severe immune-related endocrinopathy, is linked to ICI therapy. It is unclear whether glycosylation of ICIs may play a role in the development of this adverse event and how the physiological effects of different ICIs on pancreatic cells should be evaluated. We used a mouse pancreatic organoid model to compare three PD-L1 blocking antibodies in the presence or absence of IFNγ using a metabolic bioanalyzer. Modulation of ICI glycosylation altered its metabolic effects on mouse pancreatic organoids, suggesting that this model could be used to monitor and compare ICIs and to study the mechanisms underlying the development of IC-mediated diabetes.

Mesothelin CAR‐T cells secreting PD‐L1 blocking scFv for pancreatic cancer treatment

PD-1/PD-L1 pathway caused immunosuppression accounts, at least partly, for the poor therapeutic effect of Chimeric Antigen Receptor T (CAR-T) on solid tumors. In this study, we designed and prepared CAR-T cells that could secrete PD-L1 blocking antibody and target Mesothelin antigen (Sec-MesoCAR-T), to remove the immunosuppressive effect of tumor on CAR-T cells, thereby increasing the therapeutic effect of CAR-T cells on pancreatic cancer. The CAR-T cells that could not secret PD-L1 blocking antibodies (MesoCAR-T) were used as a control. Sec-MesoCAR-T cells showed an enhanced inhibitory effect on BxPC-3 tumor than MesoCAR-T cells in vitro and in vivo. Besides, Sec-MesoCAR-T cells secreted higher level of cytokines including IL-2, IL-6 and IFN-γ in vitro than MesoCAR-T cells. Following injection, there were significantly more CAR-T cells in the peripheral blood of Sec-MesoCAR-T group than that of MesoCAR-T group. This work demonstrated that the PD-L1 antibody secreted by Sec-MesoCAR-T cells relieved the immunosuppressive effect of pancreatic cancer on CAR-T cells and improved the anti-tumor activity of CAR-T cells, which has a good guiding significance for the clinical application of CAR-T cells in treating solid tumors.

Development of the Novel Bifunctional Fusion Protein BR102 That Simultaneously Targets PD-L1 and TGF-β for Anticancer Immunotherapy.

Simple SummaryImmune checkpoint inhibitors (ICIs), such as anti-PD-1/PD-L1 antibodies, have revolutionized the therapy landscape of cancer immunotherapy. However, poor clinical response to ICIs and drug resistance are the main challenges for ICIs immunotherapy. TGF-β produced in the TME was found to confer resistance to PD-1/PD-L1-targeted immunotherapy. The independent and complementary immunosuppressive role of PD-L1 and TGF-β in cancer progression provides a rationale for simultaneously targeting TGF-β and PD-L1 to improve anti-PD-L1 therapy. Consequently, we develop and characterize a novel anti-PD-L1/TGF-β bifunctional fusion protein termed BR102. The data suggest that BR102 could simultaneously disrupt TGF-β- and PD-L1-mediated signals and display high antitumor efficacy and safety. The data support further clinical advancement of BR102 as a promising approach to cancer immunotherapy.Immune checkpoint inhibitors (ICIs) are remarkable breakthroughs in treating various types of cancer, but many patients still do not derive long-term clinical benefits. Increasing evidence shows that TGF-β can promote cancer progression and confer resistance to ICI therapies. Consequently, dual blocking of TGF-β and immune checkpoint may provide an effective approach to enhance the effectiveness of ICI therapies. Here, we reported the development and preclinical characterization of a novel bifunctional anti-PD-L1/TGF-β fusion protein, BR102. BR102 comprises an anti-PD-L1 antibody fused to the extracellular domain (ECD) of human TGF-βRII. BR102 is capable of simultaneously binding to TGF-β and PD-L1. Incorporating TGF-βRII into BR102 does not alter the PD-L1 blocking activity of BR102. In vitro characterization further demonstrated that BR102 could disrupt TGF-β-induced signaling. Moreover, BR102 significantly inhibits tumor growth in vivo and exerts a superior antitumor effect compared to anti-PD-L1. Administration of BR102 to cynomolgus monkeys is well-tolerated, with only minimal to moderate and reversing red cell changes noted. The data demonstrated the efficacy and safety of the novel anti-PD-L1/TGF-β fusion protein and supported the further clinical development of BR102 for anticancer therapy.

Ginsenoside Rg3 nanoparticles with permeation enhancing based chitosan derivatives were encapsulated with doxorubicin by thermosensitive hydrogel and anti-cancer evaluation of peritumoral hydrogel injection combined with PD-L1 antibody.

Combination of chemotherapy and immune checkpoint inhibitor therapy has greatly improved the anticancer effect on multiple malignancies. However, the efficiency on triple-negative breast cancer (TNBC) is limited, since most patients bear "cold" tumors with low tumor immunogenicity. Doxorubicin (DOX), one of the most effective chemotherapy agents, can induce immunogenic cell death (ICD) and thus initiating immune response. In this study, to maximize the ICD effect induced by DOX, chitosan and cell-penetrating peptide (R6F3)-modified nanoparticles (PNPs) loaded with ginsenoside Rg3 (Rg3) were fabricated using the self-assembly technique, followed by co-encapsulation with DOX based on thermo-sensitive hydrogel. Orthotopic tumor model and contralateral tumor model were established to observe the antitumor efficacy of the thermo-sensitive hydrogel combined with anti-PD-L1 immunotherapy, besides, the biocompatibility was also evaluated by histopathological. Rg3-PNPs strengthened the immunogenic cell death (ICD) effect induced by DOX. Moreover, the hydrogel co-loading Rg3-PNPs and DOX provoked stronger immune response in originally nonimmunogenic 4T1 tumors than DOX monotherapy. Following combination with PD-L1 blocking, substantial antitumor effect was achieved due to the recruitment of memory T cells and the decline of adaptive PD-L1 enrichment. The hydrogel encapsulating DOX and highly permeable Rg3-PNPs provided an efficient strategy for remodeling immunosuppressive tumor microenvironment and converting immune "cold" 4T1 into "hot" tumors.

PD-L1 antibody enhanced β-glucan antitumor effects via blockade of the immune checkpoints in a melanoma model.

In the tumor microenvironment (TME), one of the major functions of tumor-recruited CD11b+ cells are the suppression of the T-cell-mediated anti-tumor immune response. β-glucan could convert the phenotype of tumor-recruited CD11b+ cells from the suppressive to the promotive, and enhanced their anti-tumor effects. However, β-glucan could enhance the PD-1/PD-L1 expression on CD11b+ cells, while PD-1 could inhibit macrophage phagocytosis and PD-L1 could induce a co-inhibitory signal in T-cells and lead to T-cell apoptosis and anergy. These protumor effects may be reversed by PD-1/PD-L1 block therapy. In the present study, we focused on the efficacy of β-glucan anti-tumor therapy combined with anti-PD-L1 mAb treatment, and the mechanism of their synergistic effects could be fully verified. We verified the effect of β-glucan (i.e., inflammatory cytokine secretion of TNF-α, IL-12, IL-6, IL-1β and the expression of immune checkpoint PD-1/PD-L1) in naïve mouse peritoneal exudate CD11b+ cells. In our mouse melanoma model, treatment with a PD-L1 blocking antibody with β-glucan synergized tumor regression. After treatment with β-glucan and anti-PD-L1 mAb antibody, tumor infiltrating leukocyte (TILs) not only showed a competent T-cell function (CD107a, perforin, IL-2, IFN-γ and Ki67) and CTL population, but also showed enhanced tumor-recruited CD11b+ cell activity (IL-12, IL-6, IL-1β and PD-1). This effect was also verified in the peritoneal exudate CD11b+ cells of tumor-bearing mice. PD-1/PD-L1 blockade therapy enhanced the β-glucan antitumor effects via the blockade of tumor-recruited CD11b+ cell immune checkpoints in the melanoma model.

BioIns-O-05 - Dual blocking of CD47 and PD-L1 increases innate and adaptive immune responses in CTCL

BioIns-O-05 - Dual blocking of CD47 and PD-L1 increases innate and adaptive immune responses in CTCL

Computational design of PD-L1 small molecule inhibitors for cancer therapy.

Drug repurposing opens new avenues in cancer therapy. Drug repurposing, or finding new uses for existing drugs, can substantially reduce drug discovery time and costs. Cheminformatics, genetics, and systems biology advances enable repositioning drugs. Clinical usage of PD-1/PD-L1 blocking has been approved because of its efficacy in improving prognosis in select groups. The PD-1/PD-L1 axis was considered to represent a mechanism for tumour evasion of host tumour antigen-specific T-cell immunity in early preclinical research. The expression of PD-L1 in cancer cells causes T lymphocytes to become exhausted by transmitting a co-inhibitory signal. A better understanding of how PD-L1 is regulated in cancer cells could lead to new therapeutic options. In this view, the study was aimed to repurpose the existing FDA-approved drugs as a potential PD-L1 inhibitor through e-Pharmacophore modelling, molecular docking and dynamic simulation. e-Pharmacophore screening retrieved 324 FDA-approved medications with the fitness score ≥ 1. The top 10-docked FDA candidates were compared with IN-35 (Clinical trial candidate) for its interaction pattern with critical amino acid residues. Mirabegron and Indacaterol exhibited a greater affinity for PD-L1 with docking scores of - 9.213kcalmol-1 and - 8.023kcalmol-1, respectively. Mirabegron retain interactions at all three major hotspots in the PD-L1 dimer interface similar to IN-35. MM-GBSA analyses indicated that Mirabegron uses less energy to create a more stable complex and retains all of the inhibitor's positive interactions found in clinical trial ligand IN-35. Molecular dynamics simulation analysis of the Mirabegron complex showed a similar pattern of deviation in correlation with IN-35, and it retains the interaction with the active key amino acids throughout the simulation time. Our present study has shown Mirabegron as a powerful inhibitor of PD-L1 expression in cancer cells using a drug-repurposing screen.

Bifidobacterium infantis Promotes Foxp3 Expression in Colon Cells via PD-L1-Mediated Inhibition of the PI3K-Akt-mTOR Signaling Pathway.

AimOur objective was to investigate whether Bifidobacterium infantis inhibits PI3K-Akt-mTOR signaling and upregulates Foxp3 expression through PD-L1 and to explore the possible mechanism of action of B. infantis in cellular immunosuppression. MethodThe effects of B. infantis supernatant on PD-L1, PD-1, Foxp3, and the PI3K-Akt-mTOR signaling pathway were observed by culturing HCT-116 cells. Simultaneously, the effects of blocking PD-L1 on PD-1, on Foxp3 protein and mRNA, and on the PI3K-Akt-mTOR signaling pathway protein were observed.ResultsB. infantis supernatant was able to upregulate the protein and mRNA expression of PD-L1 and Foxp3 and downregulate the phosphorylated protein expression of PI3K, Akt, and mTOR (P < 0.05); however, for PI3K, Akt, and mTOR, there was no change in the total protein expression. After the blocking of PD-L1, the stimulatory effect of B. infantis supernatant on Foxp3 and the inhibitory effect on the phosphorylated protein expression of PI3K, Akt, and mTOR were weakened.ConclusionB. infantis may inhibit the PI3K-Akt-mTOR signaling pathway and promote the expression of Foxp3 through PD-L1, which may be a target via which B. infantis exerts its immunosuppressive effect.

Abstract LB220: The anticalin-antibody bispecific PRS-352/S095025 strongly stimulates human CD4+ T cells in a PD-L1-dependent manner

Abstract Background: OX40, a member of the tumor necrosis factor receptor superfamily, is a costimulatory molecule promoting survival and cytokine release by effector T cells and inhibiting human regulatory T-cell activity. OX40 activation has been explored as a potential treatment for solid malignancies, using mainly agonistic anti-OX40 antibodies. These antibodies rely on Fc gamma receptor (FcγR)-mediated crosslinking to activate the OX40 pathway and have shown so far only limited antitumoral efficacy in clinical settings. One underlying hypothesis is that FcγR crosslinking of these antibodies may not allow optimal activation of the OX40 pathway to fully exploit the potential of this target for immunotherapy of cancer. To overcome this limitation, we designed PRS-352/S095025, a strong PD-L1-dependent OX40 agonistic bispecific molecule, that allows combination of OX40 costimulation and PD-L1 blockade. Methods: We generated an Anticalin protein that binds with high affinity to human OX40. Anticalin® proteins are approximately 18-20 kDa protein therapeutics derived from human lipocalins that can be engineered to bind with high affinity and specificity to different targets. The PRS-352/S095025 bispecific fusion protein was obtained by genetic fusion of OX40-targeting Anticalin protein to a PD-L1-targeting monoclonal antibody with a modified IgG4 backbone, allowing an optimal activation of OX40 in the presence of PD-L1, but not FcγRs. Results: We showed that PRS-352/S095025 retains binding to PD-L1 like the anti PD-L1 antibody backbone alone and binds with high affinity to both human and cynomolgus OX40. We showed that PRS-352/S095025 inhibited the PD-1/PD-L1 pathway, as the parental PD-L1 targeting antibody and an approved anti PD-L1 antibody. In addition to its PD-L1 blocking properties, we showed that PRS-352/S095025 activates OX40, and that this activity is dependent on the expression of PD-L1 and not on FcγR, consistent with the desired mechanism of action. Furthermore, we demonstrated that PRS-352/S095025 activity is superior to both PD-L1 benchmark antibodies and to the combination of clinically relevant OX40 and PD-L1 benchmark antibodies. We could also show that PRS352/S095025 strongly stimulated human CD4 T cells. In vivo, we showed that PRS-352/S095025 has a PK profile similar to that of the PD-L1 antibody alone. Conclusions: We provide here the preclinical characterization of the fusion protein PRS-352/S095025, a bispecific molecule composed of a PD-L1 blocking moiety and an Anticalin protein agonizing OX40. In vitro, this molecule showed the desired MoA, PD-L1 blocking and potent OX40 agonism driven by binding to PD-L1, with superior activity to a clinical stage OX40 agonist. Citation Format: Lucia Pattarini, Marina Pavlidou, Aizea Kastresana Morales, Janet Peper-Gabriel, Matthieu Riviere, Didier Demarles, Alix Scholer-Dahirel, Veronique Blanc, Shane Olwill. The anticalin-antibody bispecific PRS-352/S095025 strongly stimulates human CD4+ T cells in a PD-L1-dependent manner [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 LB220.

Abstract 4169: A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells for cancer immunotherapy

Abstract Background: Interleukin-10 (IL-10) has demonstrated utility in aiding CD8 T cell-mediated tumor control in mouse models but phase 2 clinical trials with pegylated IL-10 have so far not been entirely successful. More recent studies revealed that another half-life-extended form of IL-10 (IL10-Fc) can rejuvenate terminally-exhausted CD8+ T cells to boost anti-tumor activity. We investigated whether combination of a PDL-1-blocking antibody with a C-terminal IL-10 fusion may enhance anti-tumor immunity, and possibly achieve synergistic effects. Methods: Recombinant proteins were produced using an expiCHO system (Thermo Fisher). PDL-1 blocking activity was evaluated by ELISA, PD-1/PDL-1 signaling (Eurofins) and mixed lymphocyte reaction assays. IL-10 activity was evaluated by MC/9 cell proliferation and CD8 T cell activation and effector assays. Mouse CT26 colon cancer and EMT6 breast cancer studies employed the services of Oneness Biotech and Crown Bioscience, respectively. Results: To develop a bifunctional anti-PDL-1 antibody/IL-10 fusion protein, anti-human PDL-1 antibodies were first developed by screening an in-house human variable domain phage display library. Three different antibodies were then C-terminally-fused with human IL-10 polypeptide. All three showed high binding affinity to PDL-1 and blocked PD-1 signaling through PD-1/PDL-1 interaction in a dose-dependent manner. In CD4 T cell/dendritic cell co-cultures, the lead anti-PDL1/IL10 protein enhanced IL-2 and IFNγ production, and it enhanced IFNγ and granzyme B production from activated CD8 T cells as well as proliferation of MC/9 cells in an IL-10-dependent manner. Also, a mouse ‘surrogate’ version (BF11, using avelumab anti-PDL-1) showed superior efficacy compared to either IL10-Fc or anti-PDL1/TGFbR in a mouse CT26 colon tumor model, and correlated with increased serum levels of CXCL9 and IL-18. BF11 also allowed greater EMT6 tumor control than anti-PDL-1 alone or anti-PDL1/TGFbR fusion protein. Such BF11-treated mice also showed long-lasting immunity against a secondary EMT6 tumor challenge. This ability of BF11 to enhance tumor control was in part dependent on increased activity of tumor-resident cells as co-treatment with FTY720, which inhibits lymphocyte egress from secondary lymphoid organs, did not appear to reduce anti-tumor activity of BF11 in some, albeit not all, EMT6-tumor-bearing mice. Conclusions: Anti-PDL1/IL10 fusion protein not only targets PDL-1 for checkpoint blockade but also activates the IL-10 pathway to boost T cell response, presumably including revitalization of exhausted T cells as recently reported. Effective anti-tumor activities of a mouse surrogate fusion protein, BF11, are demonstrated in CT26 and EMT6 tumor models. Taken together, our findings support this dual mechanism-strategy to potentiate anti-tumor immunity. Citation Format: Huey-Wen Hsiao, Chih-Lun Hsiao, Pandelakis Koni, Hung-Kai Chen. A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells for cancer 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 4169.

LB101, a conditionally tetravalent PD-L1xCD47 bispecific monoclonal antibody (mAb), combines tumor microenvironment (TME) targeted delivery (PD-L1) and a single biological high potency effector (CD47).

2562 Background: Anti-PD-L1 antibodies are well-established as potent immune effectors in solid tumors expressing PD-L1 and demonstrate binding to these tumor cells within the TME. By contrast, due to the ubiquitous expression of CD47 on normal cells and tissues, anti-CD47 therapeutic agents with an active antibody Fc can lead to serious dose-limiting toxicities and profound biodistribution problems. To overcome these issues, we have generated LB101, a next-generation conditionally activated PD-L1xCD47 bispecific mAb with two anti-CD47 domains blocked by two further anti-PD-L1 domains, linked by two proprietary IgG-derived hinges, which inhibit CD47 interactions in the periphery, but are degraded in the PD-L1+ TME, thereby activating CD47 blockade to induce antibody-dependent cellular phagocytosis (ADCP). Methods: An IgG1-based, conditionally activated bispecific molecule was examined in binding, phagocytosis, PD-L1 blocking and flow cytometry assays. Efficacy analyses were performed in an hPD-L1+ syngeneic model in hPD-L1+/hPD1+ C57BL/6 mice, and preliminary safety and exposure analyses were completed in monkey. Results: In its intact locked form, LB101 exhibited anti-PD-L1 assay potency similar to atezolizumab. CD47 binding was only observed for LB101 after unlocking by hinge linker cleavage, leading to strongly enhanced ADCP by human CD14+ cells. In monkey, plasma stability of the locked construct was established with a PK profile mimicking that of a typical PD-L1 mAb. In the MC38 mouse model, systemically administered LB101 monotherapy delivered at equimolar dose to atezolizumab, led to no anemia, weight-loss, or overt toxicity. Monotherapy LB101 exhibited greatly improved efficacy and durability of response (14/16 tumors eradicated) over isotype control IgG (0/16) and atezolizumab (1/16). The CD47 naked parent IgG1 mAb was not tolerated in mouse, with 100% lethality above 2mg/kg. In rechallenge experiments, all naïve mice established tumors rapidly while none of the mice from groups with prior LB101-induced regressions exhibited tumor growth. Conclusions: Single agent LB101 immunotherapy delivered systemically in a difficult to treat syngeneic mouse model resulted in PD-L1 directed, local tumor-specific CD47 effector engagement leading to significant tumor regressions as compared to atezolizumab without overt toxicity. Future developments will focus on developing LB101 as a single agent therapeutic and exploring the potential of LockBody technology in improving the therapeutic index of other anti-cancer biological effectors through conditional activation within the TME.

Hsa_circ_0046523 Mediates an Immunosuppressive Tumor Microenvironment by Regulating MiR-148a-3p/PD-L1 Axis in Pancreatic Cancer.

BackgroundCircular RNAs (circRNAs) are a novel type of non-coding RNA, play an important role in the progression of tumors. However, the function and mechanism of circRNAs in regulating immune microenvironment of pancreatic cancer (PC) remain largely unclear.MethodsThe effects of hsa_circ_0046523 expression on proliferation, migration and invasion of PC cells were analyzed by CCK8 and Transwell assays. Flow cytometry was used to detect the proportion of CD4+ T cells, CD8+ T cells and Tregs in peripheral blood mononuclear cells (PBMCs) after co-culture, and the apoptosis, depletion and function of CD8+ T cells. The expression levels of immunoregulatory cytokines were detected by enzyme linked immunosorbent assay (ELISA). The dual-luciferase reporter was performed to determine the interaction between hsa_circ_0046523, miR-148a-3p, and PD-L1. Rescue experiments and PD-L1 blocking experiments were employed to investigate whether hsa_circ_0046523 exerts its biological function by miR-148a-3p/PD-L1 in PC. Furthermore, an immunocompetent murine PC model was established to confirm these findings.ResultsHsa_circ_0046523 expression was remarkably upregulated in PC tissues and cell lines. Moreover, high expression of hsa_circ_0046523 was correlated with advanced pathological stage and poorer prognosis. Hsa_circ_0046523 overexpression promoted the proliferation, migration and invasion of PC cells in vitro. Co-culture experiments confirmed that forced expression of hsa_circ_0046523 could decrease the proportion of CD4+ and CD8+ T cells, as well as increase the proportion of Tregs among peripheral blood mononuclear cells (PBMCs). Meanwhile, hsa_circ_0046523 overexpression promoted the apoptosis and exhaustion of CD8+ T cells, inhibited CD8+ T cell function, increased the secretion of immunosuppressive cytokines IL-10 and TGF-β, and decreased the secretion of immune effector cytokines IFN-γ and IL-2 among PBMCs. Mechanistically, hsa_circ_0046523 exerted its biological function by binding to miR-148a-3p to upregulate PD-L1 expression in PC. Moreover, these immune modulating functions of miR-148a-3p/PD-L1 axis were also confirmed in an immunocompetent murine PC model.ConclusionsOur study suggests that hsa_circ_0046523/miR-148a-3p/PD-L1 regulatory axis mediates PC immunosuppressive microenvironment and these molecules are expected to be new targets for remodeling tumor immune microenvironment of PC.

Comprehensive molecular characterization of muscle-invasive bladder cancer (MIBC) treated with durvalumab plus olaparib in the neoadjuvant setting: Neodurvarib trial.

546 Background: Immune checkpoint inhibitors have been incorporated to early-stage bladder carcinoma treatment recently. Durvalumab is a PD-L1 blocking antibody active in advance urothelial tumors and under evaluation in other settings of the disease. PARP inhibitors have shown activity in a variety of tumors with Homologous Recombination Deficiencies (HRD). The combination of Durvalumab plus Olaparib could present a synergistic effect, but its efficacy and potential biomarkers are under exploration. NEODURVARIB is a phase II clinical trial assessing the combination of Durvalumab plus Olaparib in MIBC (NCT03534492; SOGUG-2017-AIEC(VEJ)-2). Clinical activity and safety have been previously communicated by our group. Here we present the basal molecular profiles and their evolution under treatment with this combination. Methods: cT2-T4a MIBC aimed for cystectomy were treated during 6-8 weeks precystectomy. Pre- and post-treatment tumor and blood samples from 26 patients were collected. Pattern of immune infiltration was determined by IHQ. Genomics (mutational pattern, HRD and Tumor Mutation Burden [TMB]) and transcriptomics (differentially expressed loci, functional enrichment, molecular clustering and MIBC molecular subtyping) analysis were performed. Circulating immune populations were assessed using flow cytometry. Results: In basal (TURBT) samples, the frequency of mutations in genes commonly altered in MIBC ( TP53, MLL2, ARID1A, FGFR3, among others), HRD and TMB were similar to previous reports in MIBC and did not differ between responders and non-responders. Additionally, mutational patterns remained stable between baseline (TURBT) and post-treatment (cystectomy) samples. Regarding transcriptomics, GSEA showed enrichment of Epithelial Mesenchymal Transition (EMT), TGFβ and inflammatory/infection related classes in resistant tumors. Interestingly, differentially expressed genes in responders vs. non-responders were significantly regulated by epigenetic factors (EZH2/Suz12/PRC2 network). Transcriptomic-based estimations of the stromal/immune infiltration and MIBC molecular subtyping also showed a switch of the tumor microenvironment due to the treatment (luminal to basal/squamous transitions), reinforced by significant changes in the expression of immune markers (higher PDL1 and FAP scores in cystectomies). Lastly, circulating senescent T-cells were correlated with pathological complete response. Conclusions: Genetic alterations remained unchanged in bladder cancers treated with Durvalumab plus Olaparib. However, an enrichment of EMT signatures and a switch towards basal/squamous phenotypes were observed in resistant tumors. These findings underscore the relevance of modifications in gene expression as potential mechanisms of resistance to this combination. Clinical trial information: NCT03534492.

Stimuli-responsive nano vehicle enhances cancer immunotherapy by coordinating mitochondria-targeted immunogenic cell death and PD-L1 blockade

Induction of immunogenic cell death promotes antitumor immunity against cancer. However, majority of clinically-approved drugs are unable to elicit sufficient ICD. Here, our study revealed that mitochondria-targeted delivery of doxorubicin (DOX) massively amplified ICD via substantial generation of reactive oxygen species (ROS) after mitochondrial damage. The underlying mechanism behind increased ICD was further demonstrated to be ascribed to two pathways: (1) ROS elevated endoplasmic reticulum (ER) stress, leading to surface exposure of calreticulin; (2) ROS promoted release of various mitochondria-associated damage molecules including mitochondrial transcription factor A. Nevertheless, adaptive upregulation of PD-L1 was found after such ICD-inducing treatment. To overcome such immunosuppressive feedback, we developed a tumor stimuli-responsive nano vehicle to simultaneously exert mitochondrial targeted ICD induction and PD-L1 blockade. The nano vehicle was self-assembled from ICD-inducing copolymer and PD-L1 blocking copolymer, and possessed long-circulating property which contributed to better tumor accumulation and mitochondrial targeting. As a result, the nano vehicle remarkably activated antitumor immune responses and exhibited robust antitumor efficacy in both immunogenic and non-immunogenic tumor mouse models.

991. Blockade of the PD-1/PD-L1 Immune Checkpoint Pathway Improves Mortality, Infection Severity, and Fungal Clearance in an Immunosuppressed Murine Model of Invasive Pulmonary Mucormycosis

Abstract Background Emerging experimental evidence suggests that immune checkpoint inhibitors (ICIs) enhance antifungal immunity. In addition, there is anecdotal evidence of potential benefit of adjunct PD-1 pathway blockade in patients with intractable mucormycosis. However, proof-of-concept data in animal models are lacking. Therefore, we compared the efficacy of PD-1 and PD-L1 inhibition in an immunosuppressed murine model of invasive pulmonary mucormycosis (IPM). Methods Female 8-9-week-old BALB/c mice were immunosuppressed with cyclophosphamide (150 mg/kg on days -4 and -1, 100 mg/kg on day +3) and cortisone acetate (300 mg/kg on day -1) and infected intranasally with 50,000 Rhizopus arrhizus spores (clinical isolate Ra-749, day 0). On days 0, +2, +4, and +6, mice received intraperitoneal injections of 250 µg/kg PD-1 or PD-L1 blocking antibodies versus (vs.) 250 µg/kg of the corresponding isotype antibodies (all antibodies from Leinco Technologies). Survival was monitored for 7 days post-infection. Infection severity was scored using the murine sepsis score (MSS, 0 = healthy to 3 = moribund). Fungal burden in lung tissue was determined by an 18S quantitative PCR assay on day +7 or upon death. 20 mice per treatment were assessed in 2 independent experiments. Results Control mice with IPM receiving either of the unspecific isotype antibodies developed severe infection (median MSS on day 7, 2.5-3.0) and had a high 7-day mortality (50-55%). Compared to the corresponding isotype control, PD-L1 inhibition provided a strong therapeutic benefit, significantly improving morbidity (median MSS = 1.0 vs. 2.5, p = 0.002), 7-day mortality (15% vs. 50%, p = 0.02), and fungal burden (3.6k vs. 27.2k spore equivalents/lung, p &amp;lt; 0.001). In contrast, blockade of-PD-1 modestly yet non-significantly reduced infection severity (median MSS = 2.1 vs. 3.0, p = 0.48), 7-day mortality (35% vs, 55%, p = 0.12), and fungal burden (5.6k vs. 40.7k spore equivalents/lung, p = 0.09) compared to isotype control. Conclusion Even without concomitant antifungals, blockade of PD-L1 and to a lesser extent of PD-1 improved mortality, infection severity, and fungal clearance in immunosuppressed mice with IPM. Immune phenotyping studies are in progress to better understand the protective antifungal activity of ICIs in IPM. Disclosures Dimitrios P. Kontoyiannis, MD, Astellas (Consultant)Cidara Therapeutics (Advisor or Review Panel member)Gilead Sciences (Consultant, Grant/Research Support, Other Financial or Material Support, Honoraria)