anti-PDL1 mAb Research Articles

Ketogenic diet enhances the anti-cancer effects of PD-L1 blockade in renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by a predominant metabolic reprogramming triggering energy production by anaerobic glycolysis at the expense of oxydative phosphorylation. Ketogenic diet (KD), which consists of high fat and low carbohydrate intake, could bring required energy substrates to healthy cells while depriving tumor cells of glucose. Our objective was to evaluate the effect of KD on renal cancer cell tumor metabolism and growth proliferation. Growth cell proliferation and mitochondrial metabolism of ACHN and Renca renal carcinoma cells were evaluated under ketone bodies (KB) exposure. In vivo studies were performed with mice (nude or Balb/c) receiving a xenograft of ACHN cells or Renca cells, respectively, and were then split into 2 feeding groups, fed either with standard diet or a 2:1 KD ad libitum. To test the effect of KD associated to immunotherapy, Balb/c mice were treated with anti-PDL1 mAb. Tumor growth was monitored. In vitro, KB exposure was associated with a significant reduction of ACHN and Renca cell proliferation and viability, while increasing mitochondrial metabolism. In mice, KD was associated with tumor growth reduction and PDL-1 gene expression up-regulation. In Balb/c mice adjuvant KD was associated to a better response to anti-PDL-1 mAb treatment. KB reduced the renal tumor cell growth proliferation and improved mitochondrial respiration and biogenesis. KD also slowed down tumor growth of ACHN and Renca in vivo. We observed that PDL-1 was significantly overexpressed in tumor in mice under KD. Response to anti-PDL-1 mAb was improved in mice under KD. Further studies are needed to confirm the therapeutic benefit of adjuvant KD combined with immunotherapy in patients with kidney cancer.

Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma.

Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable anti-tumor immune response in an MYCN amplified murine model of NB. Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or in combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated. Herein, we report that Syk is a marker of NB-associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm3) but not larger tumors (250 mm3). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors. Collectively, our findings demonstrate the central role of macrophage Syk in NB progression and demonstrate that Syk blockade can "reeducate" TAMs towards immunostimulatory phenotype, leading to enhanced T cell responses. These findings further support the clinical evaluation of fostamatinib alone or with radiation and ICB, as a novel therapeutic intervention in neuroblastoma.

Abstract 3246: Targeting Syk reprograms tumor-associated macrophages and enhances responses to immune checkpoint blockade and radiation therapy in high-risk neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid malignancy in children. As leading regulators of inflammation and tumor progression, tumor associated macrophages (TAMs) have gained major interest as immunotherapeutic targets in NB. Hence targeting or “re-educating” tumorigenic macrophages towards an immunostimulatory phenotype might improve responses to immunotherapy in this childhood cancer. Spleen tyrosine kinase (Syk) has recently been identified by our group as a novel immune-oncology target. We found that Syk is abundantly present in TAMs infiltrated in MYCN and non-MYCN amplified neuroblastoma tumors. Furthermore, genetic deletion or pharmacological inhibition of Syk markedly impaired neuroblastoma tumor growth. This effect was facilitated by macrophages that became immunogenic in the absence of Syk, skewing the suppressive tumor microenvironment towards immunostimulation and activating antitumor immune responses. Moreover, combining Syk inhibitor, R788 along with anti-PDL1 mAb and radiation provided a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in aggressive MYCN-and non-MYCN-driven murine neuroblastoma tumor models. Collectively, our findings demonstrate the importance of Syk inhibitor, R788, in enhancing the anti-tumor immune responses in NB and support the clinical evaluation of R788 either alone or together with radiation and immune check point inhibitors as a potential treatment strategy for NB. Citation Format: Deepak Rohila, In Hwan Park, Timothy Pham, Riley Jones, Pablo Tamayo, Alice Yu, Andrew Sharabi, Shweta Joshi. Targeting Syk reprograms tumor-associated macrophages and enhances responses to immune checkpoint blockade and radiation therapy in high-risk neuroblastoma [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 3246.

Abstract 1863: Evaluation of BDB101, a first-in-class TLR7/8 dual agonist conjugated to an anti-PD-L1 mAb, in cancer treatment

Abstract Background Responses to antiPDL1 checkpoint inhibitors (CPIs) in many tumors are governed by the PDL1 expression on tumor and immune cells in the tumor microenvironment (TME). Increasing PDL1 expression on cells may enhance the response to CPIs. Tolllike receptor (TLR) agonists have shown to induce PDL1 expression in the TME and may enhance the antitumor effects of CPIs. The effective dose of TLR agonist administered to patients is limited by systemic toxicities. To overcome this limitation, one approach is to conjugate the TLR agonist to an antibody. This allows for delivery and release of higher doses of the TLR agonist in the TME while limiting systemic exposure.BDB001 is an intravenously (IV) delivered TLR7/8 dual agonist that activates both the innate and adaptive immune system. In clinical trials, BDB001 has been well tolerated and shown promising antitumor responses in monotherapy and in combination with antiPDL1 therapy (Patel M. et al, SITC 2020 and SITC 2021). To further enhance the antitumor effect and allow for increased concentration of the TLR7/8 dual agonist in the TME, we developed BDB101, a proprietary firstinclass TLR7/8 dual agonist - antiPDL1 monoclonal antibody (mAb) conjugate using our TollLike receptor Agonist Conjugate (TLAC) platform. Here we report on the preclinical and nonhuman primate (NHP) evaluation of BDB101. Methods BDB101 utilizes a cleavable linker, allowing for the TLR7/8 dual agonist payload to be released in the TME and enabling a bystander effect. BDB101 was assessed for in vitro TLR7/8 dual agonist activity and PDL1 binding, and in vivo antitumor efficacy in immune competent mouse models. The pharmacokinetic and pharmacodynamic effects of BDB101 was evaluated in NHP. Results The conjugated TLR7/8 dual agonist did not change the PDL1 binding specificity of the antibody, and the uncleaved conjugate did not activate TLR7 or TLR8. In the C26 mouse model, a BDB101 mouse surrogate showed a significantly more robust antitumor effect compared to the TLR7/8 dual agonist or the antiPDL1 mAb alone. In NHP, a single IV infusion of BDB101 showed no significant differences in PK parameters between BDB101 and the total antiPDL1 mAb. BDB101 demonstrated remarkable in vitro stability in human plasma for up to 21 days. In NHP, BDB101 was also very stable with minimal release of free TLR7/8 dual agonist payload. Pharmacodynamic studies in NHP were consistent with PK behavior. Conclusions BDB101 exhibits robust preclinical efficacy and prolonged stability in NHP. The low rate of free TLR7/8 dual agonist payload released into the systemic circulation from the conjugated form of BDB101, should minimize the risk of systemic TLR7/8 dual agonist exposure, while maintaining the ability to deliver the payload to the TME. These results indicate that BDB101 will likely have a good in vivo therapeutic window and strongly support the clinical development of BDB101. Citation Format: Jing Yang, Robert H. Andtbacka, Walter Lau, Lixin Li. Evaluation of BDB101, a first-in-class TLR7/8 dual agonist conjugated to an anti-PD-L1 mAb, in cancer treatment [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 1863.

Activation of T Lymphocytes with Anti-PDL1-BiTE in the Presence of Adipose-Derived Mesenchymal Stem Cells (ASCs).

Due to their ability to recruit immune cells to kill tumor cells directly, bispecific T cell engager antibodies (BiTE) hold great potential in T cell redirecting therapies. BiTE is able to activate T cells through CD3 and target them to tumor-expressed antigens. However, there are many components in the tumor microenvironment (TME) such as mesenchymal stem cells (MSCs) that may interfere with BiTE function. Herein, we designed an anti-PDL1-BiTE that targets programmed death ligand 1 (PDL1) and CD3 and investigated its effect on PDL1pos cancer cells in the presence or absence of adipose-derived MSCs (ASCs). Our anti-PDL1-BiTE comprises of VL and VH chains of anti-CD3 monoclonal antibody (mAb) linked to the VL and VH chains of anti-PDL1 mAb, which simultaneously bind to the CD3ε subunit on T cells and PDL1 on tumor cells. Flow cytometry was employed to assess the strength of binding of anti-PDL1-BiTE to tumor cells and T cells. Cytotoxicity, proliferation, and activation of peripheral blood lymphocyte (PBLs) were evaluated by CFSE assay and flow cytometry after using anti-PDL1-BiTE in the presence or absence of ASCs and their conditioned media (C.M.). Anti-PDL1-BiTE had the ability to induce selective lysis of PDL1pos U251-MG cancer cells while PDL1neg cells were not affected. Also, anti-PDL1-BiTE significantly stimulated peripheral blood lymphocyte (PBL) proliferation and CD69 expression. ASCs/C.M. did not show a significant effect on the biological activity of anti-PDL1-BiTE. Overall, anti-PDL1-BiTE selectively depletes PDL1pos cells and represents a new immunotherapeutic approach. It would increase the accumulation of T cells and can improve the prognosis of PDL1pos cancers in spite of the immunomodulatory effects of ASCs and C.M.

A Paradigm of Cancer Immunotherapy Based on 2-[18F]FDG and Anti-PD-L1 mAb Combination to Enhance the Antitumor Effect.

Efforts have been devoted to select eligible candidates for PD-1/PD-L1 immune checkpoint blocker (ICB) immunotherapy. Here, we have a serendipitous finding of positron emission tomography (PET) imaging tracer 2-[18F]FDG as a potential immunomodulator. Therefore, we hypothesize that 2-[18F]FDG could induce PD-L1 expression change and create an immune-favorable microenvironment for tumor immunotherapy. We designed a series of assays to verify PD-L1 upregulation, and tested immunotherapy regimens based on 2-[18F]FDG and anti-PD-L1 mAb, as monotherapy and in combination, in fully immunocompetent mice of MC38 and CT26 models. PD-L1 expression and tumor microenvironment (TME) changes were analyzed by Western blot, transcriptomics study, and flow-cytometric analysis. PD-L1 was upregulated in a time- and dose-dependent manner after being induced by 2-[18F]FDG. The activation of NF-κB/IRF3 pathway and STAT1/3-IRF1 pathway play crucial parts in modulating PD-L1 expression after DNA damage and repair. Improved αPD-L1 mAb utilization rate and significant tumor growth delay were observed when the personalized therapeutic alliance of 2-[18F]FDG stimulation and ICB was used. In addition, combination of 2-[18F]FDG with αPD-L1 mAb could reprogram a TME from "cold" to "hot," to make low immunoactivity tumors sensitive to ICB therapy. In summary, this promising paradigm has the potential to expand the traditional tumor theranostics. 2-[18F]FDG-based ICB immunotherapy is highly significant in enhancing antitumor effect. A research of 2-[18F]FDG-based ICB immunotherapy has been proposed to enhance the antitumor effect.

A Novel Bispecific Antibody Targeting PD-L1 and VEGF With Combined Anti-Tumor Activities.

Therapeutic monoclonal antibodies (mAbs) blocking immune checkpoints have been mainly used as monotherapy. Recently, combination therapy targeting multiple immune checkpoints has recently been explored to increase anti-cancer efficacy. Particularly, a single molecule targeting more than one checkpoints has been investigated. As dual blocking of PD-1/PD-L1 and VEGF/VEGFR has demonstrated synergism in anti-tumor activities, we developed a novel bispecific antibody, termed HB0025, which is formed via fusing the domain 2 of vascular endothelial growth factor receptor 1 (VEGFR1D2) and anti-PD-L1 mAb by using mAb-Trap technology. HB0025 almost completely retains the binding affinities and the biological activities in-vitro when compared with the parent anti-PD-L1 mAb or VEGFR1D2 fusion protein. Preclinical data demonstrated that HB0025 was more effective in inhibiting cancer growth than anti PD-L1 mAb or VEGFR1D2 fusion protein. Thus, our bispecific antibody may bring about greater clinical benefits and broader indications.

Rational combination of an immune checkpoint inhibitor with CSF1R inhibitor-loaded nanoparticle enhances anticancer efficacy.

Since the advent of immune checkpoint inhibitors, rapid strides have been made in the realm of cancer immunotherapy. Of the abundance of infiltrating immune cells in the tumor microenvironment (TME), macrophages contribute a significant portion and make up to 50% of the tumor mass. In addition to this, the relative plasticity of macrophages makes it an attractive target to modulate macrophage functions to initiate an anti-tumor response. However, many challenges hinder this strategy. Macrophage colony-stimulating factor (MCSF) secreted by cancer cells binds to the colony-stimulating factor receptor present on macrophages and negatively influences macrophage functions. MCSF, along with a cocktail of immunosuppressive cytokines present in the TME, polarizes macrophages to an immunosuppressive pro-tumorigenic M2-like phenotype. M2-like macrophages dampen tumor response and are known to be associated with increased tumor progression and metastasis. Indeed, clinical interventions aimed to reprogram macrophage response from an M2-like tumor aiding phenotype to an M1-like tumor-killing phenotype using small-molecule inhibitors of the CSF1R axis have gathered much attention in the recent past. However, poor response and systemic toxicities observed in these therapies necessitate alternative therapeutic strategies. Furthermore, another key signaling pathway that has been recently implicated in aiding the CSF1R signaling in TAMs is the PDL1 signaling axis. Hence, in this study, we designed a self-assembled lipid nanoparticle system encompassing a potent small-molecule inhibitor of the CSF1R signaling axis, while the surface of the nanoparticle was tethered with anti-PDL1 mAb. The purpose of this is twofold; the nanoparticles can deliver the cargo in a targeted manner to PDL1 expressing M2-like macrophages while simultaneously blocking the receptor. The resulting nanoparticle system termed α-PDL1-CSF-LNP showed enhanced repolarization of M2 like macrophages in vitro while also upregulating the phagocytic index. Furthermore, suboptimal dose administration of α-PDL1-CSF-LNP in an aggressive melanoma mouse model resulted in superior anti-tumor efficacy with minimal toxicities. These results were validated by ex vivo mechanistic analysis showing that TAMs have successfully been repolarized to a predominantly M1-like phenotype. This, along with increased tumor infiltration of CD8+ T cells, worked in synergy to provide an effective anti-tumor strategy.

Abstract 1880: PDL1-targeted CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors

Abstract T cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve complete activation. Tumor cells lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling at the T cell/tumor cell interface could enhance anti-tumor activity. We designed PDL1 x CD28 bispecific antibodies that conditionally costimulate CD28 only in the presence of PDL1 and TCR engagement. As PD(L)1 signaling has been shown to directly inhibit CD28 costimulation, this novel bispecific modality has potential to promote CD28 costimulation while simultaneously preventing the suppression of the same signal. We designed a set of stability-optimized anti-CD28 antibodies that can be paired with anti-PDL1 antibodies to engage both PDL1 and CD28 monovalently using Xencor's XmAb® 1+1 bispecific antibody platform. In vitro T cell activation with these bispecifics was measured by T cell proliferation, cytokine production, and cytotoxicity, in co-cultures of human cancer cell lines mixed with primary human CD3-stimulated T cells. In vitro activity was validated in a CMV recall assay measuring CMV+ T cell proliferation of CMV+ PBMC co-cultured with cancer cell lines ectopically treated with pp65-derived NLV-peptide. In vivo activity was determined with hCD28 humanized mice inoculated with MC38 tumors stably expressing hPDL1-antigen. Finally, safety, tolerability, and pharmacodynamics of PDL1 x CD28 were determined in cynomolgus monkeys. PDL1 x CD28 bispecifics were generated by incorporating an anti-PDL1 mAb capable of blocking PDL1-PD1 interaction and anti-CD28 scFv covering a range of affinities. Multiple PDL1 x CD28 antibodies enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity in concert with CD3 stimulation only in the presence of PDL1. PDL1 x CD28 enhanced proliferation of CMV+ T cells recognizing cancer cells loaded with pp65-derived NLV peptide. In hCD28 mice inoculated with MC38 tumors expressing hPDL1, PDL1 x CD28 inhibited tumor growth significantly greater than an anti-PDL1 antibody alone. PDL1 x CD28 was well tolerated in cynomolgus monkeys. PDL1 x CD28 bispecific antibodies show promising anti-tumor activity and warrant further development. Citation Format: Gregory L. Moore, Veronica Zeng, Juan Diaz, Christine Bonzon, Kendra N. Avery, Ruschelle Love, Matthew Dragovich, Rumana Rashid, Michael Hackett, Irene W. Leung, Jing Qi, Charles G. Bakhit, Umesh S. Muchhal, Norman J. Barlow, John R. Desjarlais, Michael Hedvat. PDL1-targeted CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1880.

Interim Results of an Open-Label Phase IB/II Multi-Arm Study of OX40 Agonist Monoclonal Antibody (mAb), Anti-PDL1 Mab, Smoothened Inhibitor, Anti-CD33 Mab, Bcl-2 Inhibitor, and Azacitidine As Single-Agents and As Combinations for Relapsed/Refractory Acute Myeloid Leukemia

Interim Results of an Open-Label Phase IB/II Multi-Arm Study of OX40 Agonist Monoclonal Antibody (mAb), Anti-PDL1 Mab, Smoothened Inhibitor, Anti-CD33 Mab, Bcl-2 Inhibitor, and Azacitidine As Single-Agents and As Combinations for Relapsed/Refractory Acute Myeloid Leukemia

Ultrasound targeting of microbubble-bound anti PD-L1 mAb to enhance anti-tumor effect of cisplatin in cervical cancer xenografts treatment

AimsAnti-PD-L1 monoclonal antibody (mAb)-conjugated ultrasound (US) lipid-shelled microbubbles (PD-L1-MBs) were successfully synthesized to investigate whether that PD-L1-MBs could enhance anti-tumor effect in combination therapy with cisplatin (CDDP) under ultrasound mediation. Main methodsBased on affinity between biotin and streptavidin, we prepared microbubbles conjugated with anti-PD-L1 mAb by membrane hydration and mechanical oscillation. A subcutaneous tumor model was established to test the anti-tumor effect and immunological activity of this combination therapy. Bax and Bcl-2 expression were detected by RT-qPCR and Immunohistochemistry. Cells undergoing apoptosis in tissue section were determined by TUNEL. Proliferation of splenocytes was analyzed by Flow cytometry. A cytotoxic T lymphocyte assay was performed by CTL. Expression of PD-L1 and CD8 in tissue section was examined by immunologfluorescence. Expression of IFN-γ, TNF-α, CD86 and CD80 was also detected by RT-qPCR. Key findingsWe observed that the growth of the subcutaneous tumor was significantly slower in combined group than that in the group treated with either drug or microbubbles. Moreover, higher antitumor activity was observed in the combined group than that in cisplatin alone, which could be reflected by the number of apoptotic cells in tumor tissues and over expression of bax in the combined group. This combination treatment also exhibited a better immunological activity, increasing the infiltration of CD8+ T cells and the expression of several revelant cytokines. SignificanceThe ultrasound lipid-shelled PD-L1-MBs may enhance anti-tumor effects of cisplatin by blocking the PD-L1 site and improving immune function.

DC-HIL/Gpnmb Is a Negative Regulator of Tumor Response to Immune Checkpoint Inhibitors.

Immune checkpoint inhibitors (ICI) benefit only a minority of treated patients with cancer. Identification of biomarkers distinguishing responders and nonresponders will improve management of patients with cancer. Because the DC-HIL checkpoint differs from the PD1 pathway in expression and inhibitory mechanisms, we examined whether DC-HIL expression regulates ICI responsiveness. Plasma samples were collected from patients with advanced non-small cell lung carcinoma (NSCLC) (n = 76) at baseline and/or follow-up after ICI monotherapy. Blood-soluble DC-HIL (sDC-HIL) was determined and analyzed for correlation with the early tumor response. To study the mechanisms, we measured effect of anti-DC-HIL versus anti-PDL1 mAb on growth of mouse tumor cells in experimentally metastatic lung. Influence of DC-HIL to anti-PDL1 treatment was assessed by changes in tumor response after deletion of host-DC-HIL gene, injection of DC-HIL-expressing myeloid-derived suppressor cells (MDSC), or induction of sDC-HIL expression. Nonresponders expressed significantly higher levels of baseline sDC-HIL levels than responders. Among patients (n = 28) for fluctuation with time, nonresponders (14/15 cases) showed increasing or persistently elevated levels. Responders (12/13) had decreasing or persistently low levels. Among various tumors, B16 melanoma exhibited resistance to anti-PDL1 but responded to anti-DC-HIL mAb. Using B16 melanoma and LL2 lung cancer, we showed that deletion of host-derived DC-HIL expression converted the resistant tumor to one responsive to anti-PDL1 mAb. The responsive state was reversed by infusion of DC-HIL+MDSC or induction of sDC-HIL expression. sDC-HIL in the blood and probably DC-HIL receptor expressed by MDSC play an important role in regulating response to ICI in advanced NSCLC.

The Mechanism of Anti-PD-L1 Antibody Efficacy against PD-L1-Negative Tumors Identifies NK Cells Expressing PD-L1 as a Cytolytic Effector.

Blockade of PD-L1 expression on tumor cells via anti-PD-L1 monoclonal antibody (mAb) has shown great promise for successful cancer treatment by overcoming T-cell exhaustion; however, the function of PD-L1 on natural killer (NK) cells and the effects of anti-PD-L1 mAb on PD-L1+ NK cells remain unknown. Moreover, patients with PD-L1 - tumors can respond favorably to anti-PD-L1 mAb therapy for unclear reasons. Here, we show that some tumors can induce PD-L1 on NK cells via AKT signaling, resulting in enhanced NK-cell function and preventing cell exhaustion. Anti-PD-L1 mAb directly acts on PD-L1+ NK cells against PD-L1 - tumors via a p38 pathway. Combination therapy with anti-PD-L1 mAb and NK cell-activating cytokines significantly improves the therapeutic efficacy of human NK cells against PD-L1 - human leukemia when compared with monotherapy. Our discovery of a PD-1-independent mechanism of antitumor efficacy via the activation of PD-L1+ NK cells with anti-PD-L1 mAb offers new insights into NK-cell activation and provides a potential explanation as to why some patients lacking PD-L1 expression on tumor cells still respond to anti-PD-L1 mAb therapy. SIGNIFICANCE: Targeting PD-L1 expressed on PD-L1+ tumors with anti-PD-L1 mAb successfully overcomes T-cell exhaustion to control cancer, yet patients with PD-L1 - tumors can respond to anti-PD-L1 mAb. Here, we show that anti-PD-L1 mAb activates PD-L1+ NK cells to control growth of PD-L1 - tumors in vivo, and does so independent of PD-1.This article is highlighted in the In This Issue feature, p. 1325.

831TiP - A phase III, randomized, double-blind, placebo-controlled, international study of durvalumab in combination with gemcitabine plus cisplatin for patients with advanced biliary tract cancers: TOPAZ-1

BackgroundAdvanced, unresectable biliary tract cancer (BTC) represents an area of unmet medical need due to its aggressive nature, limited treatment options, and poor prognosis. BTCs express PD-L1 and high levels of soluble PD-L1 correlate with poor prognosis in BTC patients (pts) treated with chemotherapy. PD-1/PD-L1 antagonists such as durvalumab (D; an anti–PD-L1 mAb) in combination with cytotoxic chemotherapy may contribute to a more effective antitumor immune response. Early clinical data demonstrate safety and efficacy for D as a single agent, but also in combination with chemotherapy in pts with BTC. Together, these data support the evaluation of D combined with the established chemotherapy regimen of gemcitabine (G) and cisplatin (C) for treatment of pts with previously untreated, unresectable locally advanced or metastatic BTC. Trial designTOPAZ-1 (NCT03875235) is the first large, phase III, randomized, double-blind, placebo-controlled, international study to evaluate immunotherapy + chemotherapy in pts with BTC in the first-line setting. Approximately 474 pts with previously untreated, unresectable locally advanced, recurrent or metastatic BTC will be randomized 1:1 to Arm A (D + G/C for up to 8 cycles, followed by D until progressive disease [PD]) or Arm B (placebo + G/C for up to 8 cycles, followed by placebo until PD). Pts will be stratified by disease status (initially unresectable vs recurrent) and primary tumor location. Eligibility criteria include previously untreated disease if unresectable or metastatic at initial diagnosis (and pts with recurrent disease >6 months after curative surgery or completion of adjuvant therapy), WHO/ECOG PS of 0 or 1, and no history of another primary malignancy, brain metastases or spinal cord compression. Pts with ampullary cancer or prior locoregional therapy will be excluded and major surgery must have been completed >28 days prior to the study. The primary endpoint is overall survival for Arm A vs Arm B. Secondary endpoints include progression-free survival, objective response rate, and duration of response by investigator assessment using RECSIST v1.1. Clinical trial identificationNCT03875235. Editorial acknowledgementJubilee Stewart, PhD, of PAREXEL, was funded by AstraZeneca. Legal entity responsible for the studyAstraZeneca. FundingAstraZeneca. DisclosureD. Oh: Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca. L. Chen: Honoraria (self), Advisory / Consultancy: AstraZeneca; Bristol-Myers Squibb; Lilly; MSD; Ono Pharmaceutical; PharmaEngine; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis; TTY; Biopharm; Advisory / Consultancy: Five Prime Therapeutics; Syncore; Research grant / Funding (institution): Celgene; GlaxoSmithKline; Merck Serono; Novartis; Pfizer; Polaris; Syncore; Licensing / Royalties: anti-alpha-enolase (ENO-1) monoclonal antibody to HuniLife Technology, Taiwan. T. Okusaka: Honoraria (self), Research grant / Funding (institution): Eisai; Lilly; Novartis; Yakult Honsha; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Taiho Pharmaceutical; Honoraria (self), Advisory / Consultancy: Daiichi Sankyo; Dainippon Sumitomo Pharma; Advisory / Consultancy: Zeria Pharmaceutical; Honoraria (self): AbbVie; AstraZeneca Japan; Bayer Yakuhin; Celgene; Chugai Pharma; EA Pharma; Fujifilm; Nobelpharma; Ono Pharmaceutical; Pfizer; Shire; Takara Bio; Teijin Pharma; Zeria Pharmaceutical. S. Chin: Full / Part-time employment: AstraZeneca. N. Rokutanda: Full / Part-time employment: AstraZeneca. H. Uchinda: Full / Part-time employment: AstraZeneca. A. Vogel: Honoraria (self): Amgen; Bayer; Bristol-Myers Squibb; Delcath Systems; Lilly; MSD; Novartis; Roche; Sanofi, Incyte, AstraZeneca; Advisory / Consultancy: Amgen; Bayer; Bristol-Myers Squibb; Delcath Systems; Lilly; MSD; Novartis; Roche; Sanofi, Incyte, AstraZeneca; Research grant / Funding (institution): Novartis; Travel / Accommodation / Expenses: Bayer; Ipsen; Roche. J.W. Valle: Honoraria (self): Ipsen; Advisory / Consultancy: Agios; AstraZeneca; Delcath Systems; Genoscience Pharma; Incyte; Ipsen; Keocyt; Merck; Novartis; PCI Biotech; Pfizer; Pieris Pharmaceuticals; QED; Speaker Bureau / Expert testimony: Ipsen; Novartis; Research grant / Funding (institution): Novartis; Travel / Accommodation / Expenses: Celgene; Nucana. H. Kim: Full / Part-time employment: AstraZeneca. All other authors have declared no conflicts of interest.

Abstract 3238: SIRPa blockade reinvigorates myeloid cells in the tumor microenvironment and reverses T-cell exclusion

Abstract Cancer immunotherapies by immune checkpoint blockade (CKI) has shown great therapeutic efficacy by helping the immune system to recognize and attack cancer cells. However, a significant percentage of patients do not respond or develop resistance. T-cell exclusion from tumor core, mediated by myeloid cells and fibroblast colonizing the tumor micro-environment, has been described as an important factor of resistance to checkpoint inhibitors. Myeloid cells represent one of the most abundant immune cell types in many solid tumors and are often associated with a poor outcome. Interaction of SIRPalpha (SIRPá), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response, involved in the regulation of macrophages and dendritic cells functions (e.g. phagocytosis, antigen presentation). Here we evaluated the impact of selective SIRPá blockade on the function of myeloid cells from tumor micro-environment. In vivo, in an orthotopic and syngeneic hepatocellular carcinoma (HCC) mouse model, anti-SIRPá antagonist monoclonal antibody in combination with an adaptive immune checkpoint inhibitor (anti-PD-L1) dramatically enhanced overall survival and lead to complete remission in up to 60% of immunocompetent mice (p<0.001; n=11/18). A robust memory immune response developed since a second tumor challenge after drug elimination was always rejected (p<0.01). Transcriptional analysis of tumor at early stage (10 days after treatment) using Nanostring technology revealed a significant enrichment of a multiple chemokine signature in animals that received anti-SIRPá and anti-PD-L1 combination. Histological analysis of HCC tumors revealed that anti-PDL1 mAb alone increased T-cell infiltrates, but T-cell remained excluded from tumor core. In contrast, tumor nodules were strongly reduced, and T-cell significantly enriched in tumor nest in combination with the SIRPá antagonist (p<0.01). In vitro, we next observed that CD47-induced signals inhibited secretion of chemokines by immature human macrophages. This inhibition could be reversed by a selective anti-SIRPá antagonist mAb. Ex-vivo, selective anti- SIRPá antagonist mAbs significantly increased the chemokine transcriptomic expression signature of human myeloid cells purified from ovarian cancer ascites or of HCC tumor explant fragment maintained in culture for 48 hours. Finally, we found in vivo in a humanized mice model of transmigration that SIRPá blockade significantly increased human T cell migration in microenvironment locally enriched by human macrophages. In conclusion, we showed that selective SIRPá antagonist mAbs synergized with T-cell CKI by modifying the tumor microenvironment, in particular the chemokine secretion by mouse or human myeloid cells which contributes to limit T cell exclusion and favors efficient and robust anti-tumor immune responses. Citation Format: Vanessa Gauttier, Sabrina Pengam, Justine Durand, Kévin Biteau, Mélanie Néel, Sophie Conchon, Dominique Costantini, Bernard Vanhove, Nicolas Poirier. SIRPa blockade reinvigorates myeloid cells in the tumor microenvironment and reverses T-cell exclusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3238.

Blocking Monocytic Myeloid-Derived Suppressor Cell Function via Anti-DC-HIL/GPNMB Antibody Restores the In Vitro Integrity of T Cells from Cancer Patients.

Blocking the function of myeloid-derived suppressor cells (MDSC) is an attractive approach for cancer immunotherapy. Having shown DC-HIL/GPNMB to be the T-cell-inhibitory receptor mediating the suppressor function of MDSCs, we evaluated the potential of anti-DC-HIL mAb as an MDSC-targeting cancer treatment. Patients with metastatic cancer (n = 198) were analyzed by flow cytometry for DC-HIL or PDL1 expression on blood CD14+HLA-DRno/lo MDSCs. Their suppressor function was assessed by in vitro coculture with autologous T cells, and the ability of anti-DC-HIL or anti-PDL1 mAb to reverse such function was determined. Tumor expression of these receptors was examined histologically, and the antitumor activity of the mAb was evaluated by attenuated growth of colon cancers in mice. Patients with metastatic cancer had high blood levels of DC-HIL+ MDSCs compared with healthy controls. Anti-DC-HIL mAb reversed the in vitro function in ∼80% of cancer patients tested, particularly for colon cancer. Despite very low expression on blood MDSCs, anti-PDL1 mAb was as effective as anti-DC-HIL mAb in reversing MDSC function, a paradoxical phenomenon we found to be due to upregulated expression of PDL1 by T-cell-derived IFNγ in cocultures. DC-HIL is not expressed by colorectal cancer cells but by CD14+ cells infiltrating the tumor. Finally, anti-DC-HIL mAb attenuated growth of preestablished colon tumors by reducing MDSCs and increasing IFNγ-secreting T cells in the tumor microenvironment, with similar outcomes to anti-PDL1 mAb. Blocking DC-HIL function is a potentially useful treatment for at least colorectal cancer with high blood levels of DC-HIL+ MDSCs.See related commentary by Colombo, p. 453.

MA15.02 Long-Term Safety and Clinical Activity Results from a Phase Ib Study of Erlotinib Plus Atezolizumab in Advanced NSCLC

Patients with EGFR mutation–positive non-small cell lung cancer (NSCLC) achieve favorable outcomes with EGFR tyrosine kinase inhibitors (TKIs); however, the long-term efficacy of these agents is limited by development of acquired resistance. Atezolizumab (anti–PD-L1 mAb) monotherapy has shown tolerability and durable clinical activity in NSCLC. By selectively targeting PD-L1 to block its interaction with receptors PD-1 and B7.1, atezolizumab can reinvigorate anti-cancer T-cell activity. Thus, combining atezolizumab and erlotinib could result in improved anti-tumor immunity and durable anti-tumor effects. Safety and preliminary clinical activity from a Phase Ib study of erlotinib plus atezolizumab in locally advanced or metastatic NSCLC have been previously reported (Rudin, et al. WCLC 2016). Here we describe updated findings from this study (NCT02013219). EGFR TKI-naive patients with NSCLC were enrolled into a safety-evaluation stage (Stage 1) regardless of EGFR status, and an expansion stage (Stage 2) enrolled patients with EGFR-mutant NSCLC who were previously untreated or treated with 1 prior non–EGFR TKI therapy. A 7-day run-in period with erlotinib 150 mg PO QD was followed by addition of atezolizumab 1200 mg IV q3w. The primary endpoint was safety/tolerability of the combination; secondary endpoints included clinical activity per RECIST v1.1. At data cutoff (August 18, 2017), 28 patients (Stage 1, n = 8; Stage 2, n = 20) were evaluable for safety, and the median survival follow-up was 26.0 months (range, 7.8-32.9; Stage 2). The median age was 61 years (range, 47-84), and the most common EGFR mutation was exon 19 deletion (44%). Grade 3 treatment-related AEs (TRAEs) were reported in 43% of patients; no Grade 4 or 5 TRAEs occurred. The most common TRAEs were increased ALT, pyrexia, rash and diarrhea (2 patients each). Serious AEs occurred in 54% of patients; treatment-emergent AEs led to atezolizumab discontinuation in 18% and erlotinib discontinuation in 11%. Clinical activity was evaluated in Stage 2 patients. ORR was 75% (95% CI: 50.9, 91.3), with a median DOR of 16.7 months (range, 4.2-26.0+). Median PFS was 15.4 months (95% CI: 8.4, not estimable [NE]), and median OS was 32.7 months (95% CI: 32.7, NE). Atezolizumab plus erlotinib demonstrated a tolerable safety profile and compared favorably with prior reports of efficacy with erlotinib monotherapy. OS data are expected to mature and improve with longer follow-up; updated clinical and biomarker data will be presented. Further investigation of the combination is warranted to assess its full potential.

Abstract PD6-01: Prevalence of PDL1 and tumor infiltrating lymphocytes (TILs) in primary and metastatic TNBC

Abstract Background: Metastatic Triple Negative Breast Cancer (TNBC) is an aggressive breast cancer subtype with a high unmet medical need and limited therapeutic options. PDL1 on immune cells (IC) and TILs were associated with activity to the anti-PDL1 mAB atezolizumab in mTNBC (Schmid AACR 2017). PDL1 IC >=1% is the diagnostic hypothesis for TNBC atezolizumab studies. The goal of the current project was to evaluate the prevalence of PDL1 and TILs in primary and metastatic TNBC samples. Methods: FFPE tumor tissue from pathology documented TNBC tissues were centrally tested for PDL1 IHC on tumor cells (TC) and infiltrating immune cells (IC) (VENTANA SP142 assay) and quantified as percentage of tumor area. TILs were evaluated as a percentage of tumor area and PD-L1 gene (CD274) expression was quantified by Fluidigm. Results: 669 tumors were evaluable for PD-L1 by IHC and TILs. 280 samples were annotated as primary tumors and 179 as metastases with 210 denoted as unknown. PDL1 IC and TC immunostaining was correlated to PD-L1 transcript expression (r = 0.55 and r = 0.51, respectively). 84% of samples had no tumor cells expressing PDL1 and 16% had at least 1% of PDL1 in TC. 44% of the samples had no PDL1 in the immune cells, 35% expressed PDL1 IC [31% to <5%] and 21% PDL1 IC 35%. PDL1 distribution was similar between primary and metastases, with median of 1% and 0% for PDL1 IC and TC, respectively. Analyses of anatomical locations suggest that PDL1 IC is highest in lymph nodes (median IC = 3%), lowest in liver metastases (median IC = 0.5%) while breast tissue is intermediate (median IC = 1%). The correlation between PDL1 IC and TILs is r = 0.69. TILs were numerically higher in primary compared to metastases (10% vs 5%). Anatomical TIL distribution confirmed liver metastases as least infiltrated, while breast, lymph node and lung metastases with similarly higher values (5% vs 10%, respectively). Concordance analyses in intra-individual synchronous and asynchronous matched pairs were performed at a cutoff of PDL1 IC 31%. Concordance of matched specimens was 82% in 11 synchronous pairs and 75% in 20 asynchronous pairs. Conclusions: Prevalence of PDL1 IHC is similar between primary and metastatic TNBC samples. Although PDL1 IC and TILs are highly correlated, TILs are less prevalent in metastases compared to primary tissue. Anatomical location may influence prevalence of both PDL1 IC and TILs. Concordance of PDL1 in matched pairs is high, suggesting PDL1 IC may be a stable biomarker. Citation Format: Li Y, Chang C-W, Tran D, Denker M, Hegde P, Molinero L. Prevalence of PDL1 and tumor infiltrating lymphocytes (TILs) in primary and metastatic TNBC [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-01.

Phase I/III trial of atezolizumab with carboplatin and etoposide in ES-SCLC in first-line setting (IMpower133).

TPS8584 Background: The first-line standard of care for the majority of patients (pts) with extensive-stage small-cell lung cancer (ES-SCLC) is platinum-based chemotherapy with etoposide, but survival outcomes remain poor (median OS, < 1 year) despite initial response rates ranging from 50-70%. Atezolizumab (atezo), an anti–PD-L1 mAb, prevents the binding of PD-L1 with its receptors PD-1 and B7.1 and restores anticancer T-cell activity. Tolerable safety with promising durability of response has been shown with atezo in pts with ES-SCLC: confirmed ORR was 6% (n = 1/17 [partial response]; DOR of 7 mo) by RECIST v1.1 and 24% by immune-related response criteria (irRC; n = 4/17, with 2 pts on atezo for ≥ 12 mo). Preliminary data also indicate the potential synergy between atezo and platinum-based chemotherapy in NSCLC, whereby durable responses may translate into improved survival over atezo alone. IMpower133 (NCT02763579), a global, Phase I/III, randomized, multicenter, double-blinded, placebo-controlled trial will evaluate the efficacy and safety of 1L atezo + carboplatin + etoposide compared with placebo + carboplatin + etoposide in treatment-naive pts with ES-SCLC. Methods: Pts with measurable (RECIST v1.1) ES-SCLC, who have ECOG PS 0-1 and no prior systemic anticancer treatment, are eligible for the study. Exclusion criteria include untreated CNS metastases and history of autoimmune disease. The study requires submission of tumor tissue, but pts will be enrolled regardless of biomarker status. Pts will be stratified by sex, ECOG PS and presence of treated brain metastases. Eligible pts will be randomized 1:1 to receive four 21-day cycles of atezo (1200 mg IV) or placebo in combination with carboplatin (AUC 5 mg/mL/min IV, d 1) and etoposide (100 mg/m2 IV, d 1-3), followed by maintenance therapy with atezo or placebo until PD per RECIST v1.1. Pts can continue with treatment until persistent radiographic PD, symptomatic deterioration or unacceptable toxicity. Co-primary endpoints are investigator-assessed PFS per RECIST v1.1 and OS. Secondary efficacy endpoints include investigator-assessed ORR and DOR. Safety and tolerability will also be assessed. Approximately 400 pts will be enrolled. Clinical trial information: NCT02763579.

IMpower132: A phase III clinical program—1L atezolizumab plus platinum-based chemotherapy in chemo-naive advanced non-squamous NSCLC.

TPS9101 Background: Platinum-based chemotherapy (chemo) is the current standard of care for patients (pts) with newly diagnosed advanced non-squamous NSCLC. The combination of platinum-based chemo and pemetrexed (pem) provides comparable benefit to pts as other standard platinum doublets commonly used and has a favorable toxicity profile. However, the survival benefit conferred by this combination leaves considerable room for improvement. The anti–PD-L1 mAb atezolizumab (atezo) inhibits the interaction of PD-L1 with its receptors PD-1 and B7.1, thereby restoring tumor-specific T-cell immunity. In the OAK trial, pts with 2L/3L advanced NSCLC had improved mOS in the atezo arm (13.8 mo) vs the docetaxel arm (9.6 mo), with a survival benefit observed across PD-L1 expression levels. The potential for chemo to further augment responses to atezo, with tolerable safety, has also been demonstrated. A global, Phase III, randomized, open-label trial, IMpower132 (NCT02657434), is being conducted to evaluate 1L atezo + platinum-based chemo + pem in chemo-naive pts with stage IV NSCLC. Methods: Eligibility criteria include stage IV NSCLC, measurable disease (RECIST v1.1), no prior chemo and ECOG PS 0-1. Exclusion criteria include tumors known to harbor EGFR or ALK driver mutations, untreated CNS metastases, autoimmune disease and prior exposure to immunotherapy. Pts will be enrolled regardless of PD-L1 expression status and randomized to the treatment arms. Pts will be stratified by sex, ECOG PS, type of chemo (carboplatin [carbo] vs cisplatin [cis]) and smoking status. Pts will receive four or six 21-day cycles of either atezo + carbo/cis + pem or carbo/cis + pem. Following the induction phase, pts will receive maintenance atezo + pem or pem alone, depending on their allocated treatment regimen. Pts receiving atezo may continue until loss of clinical benefit.Co-primary endpoints are investigator-assessed PFS and OS. Secondary endpoints include IRF-assessed PFS, investigator-assessed ORR and safety. Predictive biomarkers associated with efficacy will be evaluated. Approximately 568 pts will be enrolled. Clinical trial information: NCT02657434.