PD-1 Interaction Research Articles

Generation of NK cells with chimeric-switch receptors to overcome PD1-mediated inhibition in cancer immunotherapy

Multiple myeloma (MM) is an incurable hematological cancer, in which immune checkpoint inhibition (ICI) with monoclonal antibodies (mAbs) has failed due to uncontrollable immune responses in combination therapies and lack of efficacy in monotherapies. Although NK cell-specific checkpoint targets such as NKG2A and KIRs are currently being evaluated in clinical trials, the clinical impact of NK cells on the PD1 cascade is less well understood compared to T cells. Furthermore, while NK cells have effector activity within the TME, under continuous ligand exposure, NK cell dysfunctionality may occur due to interaction of PD1 and its ligand PD-L1. Due to above-mentioned factors, we designed novel NK cell specific PD1-based chimeric switch receptors (PD1-CSR) by employing signaling domains of DAP10, DAP12 and CD3ζ to revert NK cell inhibition and retarget ICI. PD1-CSR modified NK cells showed increased degranulation, cytokine secretion and cytotoxicity upon recognition of PD-L1+ target cells. Additionally, PD1-CSR+ NK cells infiltrated and killed tumor spheroids. While primary NK cells (pNK), expressing native PD1, showed decreased degranulation and cytokine production against PD-L1+ target cells by twofold, PD1-CSR+ pNK cells demonstrated increased activity upon PD-L1+ target cell recognition and enhanced antibody-dependent cellular cytotoxicity. PD1-CSR+ pNK cells from patients with MM increased degranulation and cytokine expression against autologous CD138+PD-L1+ malignant plasma cells. Taken together, the present results demonstrate that PD1-CSR+ NK cells enhance and sustain potent anti-tumor activity in a PD-L1+ microenvironment and thus represent a promising strategy to advance adoptive NK cell-based immunotherapies toward PD-L1+ cancers.

OR19-5 Dissecting Thyroidal Pathways Activated by PD-L1 Blockade Identifies New Mechanisms for Thyroiditis Triggered by Immune Checkpoint Inhibitors

Abstract Programmed death-ligand 1 (PD-L1) is a strong immunosuppressor that inhibits T-cell activation by binding to its receptor (PD-1) on T-cells. Blocking the PD-L1/PD-1 interaction by monoclonal antibodies (mAbs) is an effective therapy for several cancers via activation of T-cells. Destructive thyroiditis and hypothyroidism often develop during anti-PD-L1 (αPD-L1) therapy but the pathogenic mechanisms are not known. We evaluated in vivo, the role of PD-L1 and its interactions with inflammatory triggers in the development of autoimmune thyroiditis (AIT). NOD.H-2h4 mice were treated with either αPD-L1 or isotype control (CTR) for two weeks and AIT evaluated after six weeks. αPD-L1 treated mice showed significantly higher Abs (p = 6×10e-4) and T-cell responses (p = 0.02) to thyroglobulin (Tg) compared with CTR mice. Serum fT4 levels were lower (p = 0.01) and TSH higher (p = 0.05) in αPD-L1 treated vs. CTR mice. Seven of the eight αPD-L1 treated mice (and none of the CTR mice) developed thyroid lymphocytic infiltration characterized by the presence of the CD4+, CD8+, FoxP3+ T- cells and B220+ B- cells. To gain insight into the pathways regulated by PD-L1 in the thyroid we assessed gene expression in the thyroid of αPD-L1 treated mice using RNA-seq. PD-L1 blockade was associated with upregulation of pathways of lymphocyte activation (p = 1.3×10e-5), lymphocyte proliferation (p = 2×10e-5), chemotaxis (p = 3.8×10e-12), and inflammatory response (p = 7.9×10e-7). Specifically, blocking PD-L1 was associated with upregulation of cytokine/chemokine genes that have a critical role in Th1 differentiation: IFNγ, IL-12, IL-15, CCL1 to CCL5, CD300, CXCL10, CXCL11. Ingenuity Pathway Analysis identified IFNγ and TNF as the top regulators of these activated gene networks. These results indicate that blockade of PD-L1 in thyroid promoted the Th1 mediator, IFNγ, inducing activation and proliferation of thyroidal CD4+ and CD8+ T cells, triggering thyroid tissue damage and AIT. Since PD-L1 mediates immune responses to inflammatory stressors (e.g. viral infections), we evaluated PD-L1 interaction with a known inflammatory trigger of AIT, interferon alpha (IFNα). Treatment with αPD-L1 + IFNα accelerated AIT in NOD.H-2h4 mice that developed high Tg Ab and T-cell responses and thyroidal lymphocytic infiltration only two weeks after treatment. Moreover, thyroiditis was more severe in mice receiving αPD-L1 + IFNα than αPD-L1 alone, manifesting by higher T-cell responses (p = 5×10e-3), and significantly more profound hypothyroidism. These results suggest that PDL-1 has a protective role in the thyroid against inflammatory stressors, and that its blockade exposes the thyroid to inflammatory damage. Therefore, in patients undergoing αPD-L1 therapy, inflammatory conditions (e.g. infections) can be a "second hit" that accelerates the onset of thyroiditis and augments thyroidal inflammation. Presentation: Monday, June 13, 2022 12:00 a.m. - 12:15 a.m.

Docosahexaenoic acid reverses PD-L1-mediated immune suppression by accelerating its ubiquitin-proteasome degradation

PD-L1 interacts with its receptor PD-1 on T cells to negatively regulate T cell function, leading to cancer cell immune escape from the immune surveillance. Therefore, targeting PD-L1 is considered to be an attractive approach for cancer immunotherapy. In this study, we demonstrated for the first time that ω-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) reduced the expression of PD-L1 in cancer cells both in vitro and in vivo. Promotion of PD-L1 ubiquitin-proteasome degradation by DHA resulted in a decrease of PD-L1 expression, leading to reduction of PD-L1 and PD-1 interaction, and reversing PD-L1-mediated immune suppression, which in turn contributed to the inhibitory effect on tumor growth. Furtherly, DHA significantly reduced fatty acid synthase (FASN) expression in cancer cells, which inhibited the palmitoyltransferases DHHC5, promoting the CSN5-dependent PD-L1 degradation. Our present finding uncovered a novel mechanism involved in the anti-cancer activity of DHA, and implicated that DHA holds promising potential to be developed as a novel immune-enhancer for cancer treatment and prevention.

197 (PB077) - Small molecule PD-L1 inhibitor modulates expression of PD-L1 on the cell surface – a potential mechanism of blocking interaction with PD-1

Background: Blocking the PD1-PD-L1 interaction has been shown to stimulate anti-tumour responses. Although antibody-based drugs targeting either PD-1 or PD-L1 are approved, small molecule inhibitors of PD-L1 are currently in development. A subset of these small molecule inhibitors induce the loss of cell surface PD-L1 in a time- and concentration-dependent manner. INCB090244 is a small molecule that has previously been shown to bind to PD-L1 and disrupt the PD-L1/PD-1 interaction.

The role of PD-1/PD-L1 and application of immune-checkpoint inhibitors in human cancers.

Programmed cell death protein-1 (PD-1) is a checkpoint receptor expressed on the surface of various immune cells. PD-L1, the natural receptor for PD-1, is mainly expressed in tumor cells. Studies have indicated that PD-1 and PD-L1 are closely associated with the progression of human cancers and are promising biomarkers for cancer therapy. Moreover, the interaction of PD-1 and PD-L1 is one of the important mechanism by which human tumors generate immune escape. This article provides a review on the role of PD-L1/PD-1, mechanisms of immune response and resistance, as well as immune-related adverse events in the treatment of anti-PD-1/PD-L1 immunotherapy in human cancers. Moreover, we summarized a large number of clinical trials to successfully reveal that PD-1/PD-L1 Immune-checkpoint inhibitors have manifested promising therapeutic effects, which have been evaluated from different perspectives, including overall survival, objective effective rate and medium progression-free survival. Finally, we pointed out the current problems faced by PD-1/PD-L1 Immune-checkpoint inhibitors and its future prospects. Although PD-1/PD-L1 immune checkpoint inhibitors have been widely used in the treatment of human cancers, tough challenges still remain. Combination therapy and predictive models based on integrated biomarker determination theory may be the future directions for the application of PD-1/PD-L1 Immune-checkpoint inhibitors in treating human cancers.

Further investigation of blockade effects and binding affinities of selected natural compounds to immune checkpoint PD-1/PD-L1.

The breakthrough in the discovery of immune checkpoint PD-1/PD-L1 inhibitors, such as the series of Bristol Myers Squibb synthetic compounds, boosted the research of small molecules with blockade effects on the interaction of PD-1/PD-L1. However, the search for natural products derived PD-1/PD-L1 inhibitors can be impeded by the false positive and/or negative results from the screening assays. Herein, we combined a PD-1/PD-L1 blockade assay (pair ELISA) and a PD-L1/PD-L1 binding assay (surface plasmon resonance; SPR) to evaluate a panel of natural compounds previously reported to show anti-PD-1/PD-L1 activity. The test compounds included kaempferol, cosmosiin, tannic acid, pentagalloyl glucose, ellagic acid, resveratrol, urolithin A, and rifubutin. Based on the analyses of their responses to the combined screening assays, these compounds were categorized into four groups: I) PD-1/PD-L1 inhibitors that can bind to PD-1 and PD-L1; II) PD-1/PD-L1 inhibitors selectively bind to PD-L1 protein; III) PD-1/PD-L1 inhibitors without binding capacity, and IV) PD-1/PD-L1 binders without blockade effect. Discrimination of positive responders in the PD-1/PD-L1 blockade and binding assays can provide useful insights to avoid false outcomes. Examples demonstrated in this study suggest that it is crucial to adopt proper evaluation methods (including using multiple-facet functional assays and target binding techniques) for the search for natural products derived PD-1/PD-L1 inhibitors.

1034P A phase II study of KN046 (a bispecific anti-PD-L1/CTLA-4) in patients with metastatic non-small cell lung cancer (NSCLC) who failed prior EFGR-TKIs

KN046 is a novel bispecific antibody that blocks both PD-L1 interaction with PD1 and CTLA-4 interaction with CD80/CD86. In the phase II study, promising anti-tumor activity was seen with KN046 in subjects with non-small cell lung cancer (NSCLC). Here, we reported the efficacy and safety results of KN046 combined with chemotherapy in treatment of advanced NSCLC with EGFR sensitivity mutation who failed prior tyrosine kinase inhibitors (TKIs) from Cohort D in this study.

1210P The preliminary efficacy and safety of KN026 combined with KN046 treatment in HER2-positive locally advanced unresectable or metastatic gastric/gastroesophageal junction cancer without prior systemic treatment in a phase II study

KN026 is a novel bispecific antibody that simultaneously binds to two distinct HER2 epitopes. KN046 is a novel bispecific antibody that blocks both PD-L1 interaction with PD-1 and CTLA-4 interaction with CD80/CD86. An IIT study (KN046-IST-02) had proved efficacy and safety of KN026 combined with KN046 for late line HER2 positive gastrointestinal cancer.

1022P A phase II study of KN046 (a bispecific anti-PD-L1/CTLA-4) in patients with metastatic non-small cell lung cancer (NSCLC) who failed first line treatment

KN046 is a novel bispecific antibody that blocks both PD-L1 interaction with PD1 and CTLA-4 interaction with CD80/CD86. In the phase II study, promising anti-tumor activity was seen with KN046 in subjects with non-small cell lung cancer (NSCLC). Here, we reported the efficacy and safety results of KN046 in second line treatment of advanced NSCLC from Cohort A and B in this study. This is an open-label, multi-center, multiple cohorts, single arm study to evaluate the efficacy, safety and tolerability of KN046 in NSCLC. Subjects with NSCLC who had failed from first line platinum-based doublet chemotherapy without PD-(L)1 immune checkpoint blockade were enrolled. Subjects with EGFR mutation and/or ALK translocation will be excluded. All subjects enrolled will receive KN046 3 mg/kg (Cohort A) and 5 mg/kg (Cohort B) Q2W IV administration, respectively. Primary endpoint was objective response rate (ORR) per RECIST version 1.1 by BICR. Between May 16, 2019 and April 30, 2020, 64 subjects with metastatic NSCLC who failed first line treatment were enrolled. At the data cutoff of August 31, 2021, the median follow-up was 21.6 months (95% CI, 20.3, 23.2). Among all 64 subjects, the ORR was 14.1% (9/64, 95% CI, 6.64, 25.02%), median progression-free survival (mPFS) was 3.7 months (95% CI, 2.9, 5.5) and median overall survival (mOS) was 18.4 months (95% CI, 12.9, 21.9). Furthermore, the ORR was 17.1% (7/41, 95% CI, 7.15, 32.06%), mPFS was 3.7 months (95% CI, 2.76, 5.45) and mOS was 19.8 months (95% CI, 13.04, 23.36) in non-squamous NSCLC (n=41). And in squamous NSCLC (n=20), the ORR was 10.0% (2/20, 95% CI, 1.23, 31.70%), mPFS was 7.4 months (95% CI, 1.81, 14.39) and mOS was 12.9months (95% CI, 8.97, -). 27(42.2%) out of the 64 subjects had experienced treatment-related adverse event (TRAE) at grade 3 or higher levels. The most common TRAEs of grade 3 or higher were infusion reaction (7/64 [10.9%]), hepatic dysfunction (3/64 [4.7%]), pneumonia (2/64 [3.1%]), etc. The bispecific antibody, KN046 was well tolerated and effective as second line treatment of advanced NSCLC. KN046 showed promising OS benefit in both squamous and non-squamous NSCLC.

Quantification-Promoted Discovery of Glycosylated Exosomal PD-L1 as a Potential Tumor Biomarker.

Exosomal programmed cell death ligand 1 (exoPD-L1) has emerged as a promising biomarker for cancer diagnosis and immunotherapy outcome prediction. However, the existing quantitation methods are incapable of addressing the heterogeneity of exoPD-L1 glycosylation, which has been demonstrated to be the institutional basis for PD-L1/PD-1 interaction and the crucial participant in inhibiting the activity of CD8+ T cells. Herein, an aptamer- and lectin-induced proximity ligation assay combined with quantitative real-time polymerase chain reaction for precise quantitation of glycosylated exoPD-L1 is developed. Leveraging the metabolism-free lectin labeling of glycosylation, the glycosylation-independent aptamer tagging of PD-L1, and excellent selectivity of dual-recognition, this method enables glycosylated exoPD-L1 quantitation with high sensitivity and selectivity in a wash-free manner. As a result, this method is able to distinguish the levels of glycosylated exoPD-L1 between healthy donors and cancer patients with sensitivity and specificity of 100%. Compared with the total circulating exoPD-L1 level, glycosylated exoPD-L1 is for the first time identified to be a more reliable biomarker for tumor diagnosis. Overall, this strategy holds a great potential for revealing the significance of exoPD-L1 glycosylation and converting glycosylated exoPD-L1 into a reliable clinical indicator.

Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells.

Despite recent therapeutic advances, pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with limited therapeutic options. Immune checkpoint inhibitors (ICIs) have demonstrated promising results in many cancers, but thus far have yielded little clinical benefit in PDAC. Based on recent combined targeting of programmed cell death protein-1 (PD-1) and C-X-C chemokine receptor 4 (CXCR4) in patient-derived xenografts (PDXs) and a pilot clinical trial, we sought to elucidate potential interactions between PD-1 and CXCR4. We observed concomitant expression and direct interaction of PD-1 and CXCR4 in PDAC cells. This interaction was disrupted upon CXCR4 antagonism with AMD3100 and led to increased cell surface expression of PD-1. Importantly, CXCR4-mediated PDAC cell migration was also blocked by PD-1 inhibition. Our work provides a possible mechanism by which prior studies have demonstrated that combined CXCR4 and PD-1 inhibition leads to decreased tumor growth. This is the first report investigating PD-1 and CXCR4 interactions in PDAC cells and our results can serve as the basis for further investigation of combined therapeutic targeting of CXCR4 and PD-1.

Differential Engraftment of Parental A20 PD-L1 WT and PD-L1 KO Leukemia Cells in Semiallogeneic Recipients in the Context of PD-L1/PD-1 Interaction and NK Cell-Mediated Hybrid Resistance.

The contribution of natural killer (NK) cells to tumor rejection in the context of programmed death-ligand 1/programmed death 1 (PD-L1/PD-1) blockade is a matter of intense debate. To elucidate the role of PD-L1 expression on tumor cells and the functional consequences of engaging PD-1 receptor on cytotoxic cells, PD-L1 expression was genetically inactivated and WT or PD-L1-deficient parental tumor cells were adoptively transferred intravenously into F1 recipients. The engraftment of PD-L1-deficient A20 tumor cells in the spleen and liver of F1 recipients was impaired compared with A20 PD-L1 WT tumor counterparts. To elucidate the mechanism responsible for this differential tumor engraftment and determine the relevance of the role of the PD-L1/PD-1 pathway in the interplay of tumor cells/NK cells, a short-term competitive tumor implantation assay in the peritoneal cavity of semiallogeneic F1 recipients was designed. The results presented herein showed that NK cells killed target tumor cells with similar efficiency regardless of PD-L1 expression, whereas PD-L1 expression on A20 tumor cells conferred significant tumor protection against rejection by CD8 T cells confirming the role of the co-inhibitory receptor PD-1 in the modulation of their cytotoxic activity. In summary, PD-L1 expression on A20 leukemia tumor cells modulates CD8 T-cell-mediated responses to tumor-specific antigens but does not contribute to inhibit NK cell-mediated hybrid resistance, which correlates with the inability to detect PD-1 expression on NK cells neither under steady-state conditions nor under inflammatory conditions.

Combination of two novel blocking antibodies, anti-PD-1 antibody ezabenlimab (BI 754091) and anti-LAG-3 antibody BI 754111, leads to increased immune cell responses

ABSTRACT Upregulation of inhibitory receptors, such as lymphocyte activation gene-3 (LAG-3), may limit the antitumor activity of therapeutic antibodies targeting the programmed cell death protein-1 (PD-1) pathway. We describe the binding properties of ezabenlimab, an anti-human PD-1 antibody, and BI 754111, an anti-human LAG-3 antibody, and assess their activity alone and in combination. Ezabenlimab bound with high affinity to human PD-1 (KD = 6 nM) and blocked the interaction of PD-1 with PD-L1 and PD-L2. Ezabenlimab dose-dependently increased interferon-γ secretion in human T cells expressing PD-1 in co-culture with PD-L1-expressing dendritic cells. Administration of ezabenlimab to human PD-1 knock-in mice dose-dependently inhibited growth of MC38 tumors. To reduce immunogenicity, ezabenlimab was reformatted from a human IgG4 to a chimeric variant with a mouse IgG1 backbone (BI 905725) for further in vivo studies. Combining BI 905725 with anti-mouse LAG-3 antibodies improved antitumor activity versus BI 905725 monotherapy in the MC38 tumor model. We generated BI 754111, which bound with high affinity to human LAG-3 and prevented LAG-3 interaction with its ligand, major histocompatibility complex class II. In an in vitro model of antigen-experienced memory T cells expressing PD-1 and LAG-3, interferon-γ secretion increased by an average 1.8-fold versus isotype control (p = 0.027) with BI 754111 monotherapy, 6.9-fold (p < 0.0001) with ezabenlimab monotherapy and 13.2-fold (p < 0.0001) with BI 754111 plus ezabenlimab. Overall, ezabenlimab and BI 754111 bound to their respective targets with high affinity and prevented ligand binding. Combining ezabenlimab with BI 754111 enhanced in vitro activity versus monotherapy, supporting clinical investigation of this combination (NCT03156114; NCT03433898).

Abstract 5444: BPI-371153, an orally bioavailable small molecule PD-L1 inhibitor

Abstract PD-1/PD-L1 antibodies have entered mainstream of cancer treatment, but they came with limitations such as weak tumor penetration, immunogenicity, immune-related risks with a long half-life, and high cost. Small molecule inhibitors of PD-L1 are expected to overcome these limitations. We hereby present BPI-371153, an orally bioavailable small molecule that disrupts PD-1/PD-L1 interaction similar to PD-1/PD-L1 antibodies. BPI-371153 demonstrates high binding affinity to human PD-L1, induces PD-L1 dimeralization and disrupts PD-L1:PD-1 interaction (IC50 &amp;lt;1 nM by HTRF assay). BPI-371153 induces PD-L1 internalization with an IC50 value of 4.2 nM as detected by flow cytometry, activates nuclear factor of activated T cell (NFAT) signaling in cell-based reporter assay, and effectively stimulates IFN-γ release in human peripheral blood monocyte-mediated tumor cell killing assay. In a syngeneic mouse model with murine MC38 colon tumor cells expressing human PD-L1 (MC38-hPD-L1 model), BPI-371153 suppressed tumor growth to an extent comparable to an anti-PD-L1 antibody. In immunocompromised mice grafted with the same cell line, no growth inhibition was observed, confirming that the pharmacologic effect of BPI-371153 indeed came from the immune system. In addition, this compound exhibits favorable ADME properties, with high oral exposure across multiple pre-clinical species. Phase I clinical trial of BPI-371153 is planned for early 2022. Citation Format: Yiqian Wang, Xiangyan Jing, Hong Chen, Huijuan Zhang, Tianyi Ma, Yao Zhang, Chunhui Zhang, Guangzhi Zhang, Xiangyong Liu, Dan Yan, Jing Guo, Hong Lan, Jiabing Wang, Lieming Ding. BPI-371153, an orally bioavailable small molecule PD-L1 inhibitor [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 5444.

Abstract 576: Shark VNAR single domain-based CAR T cells targeting PD-L1 for cancer therapy

Abstract Background: Chimeric antigen receptor (CAR)-T therapy shows great potency against hematological malignancies, whereas it is difficult to transfer CAR T into solid tumors due to lack of appropriate antigenic targets and immunosuppressive tumor microenvironment. Checkpoint molecule PD-L1 is widely overexpressed on multiple tumor types, and PD-1/PD-L1 interaction is a primary mediator of immunosuppression in the TME. Here, we generated anti-PD-L1-specific shark single domain VNAR-based CAR T cell strategy and explored its anti-tumor efficacy in xenograft mouse models. Methods: We isolated anti-PD-L1 single domain antibodies from semi-synthetic shark VNAR phage display libraries. The binding of isolated VNARs was validated by ELISA, flow cytometry, and Octet. A peptide library based on human PD-L1 was synthesized to predict the epitope of select VNARs. Anti-tumor efficacy of CAR T cells was determined via cell luciferase-based cell assay as well as xenograft mouse models. Two tumor models, MDA-MB-231 (triple-negative breast cancer) and Hep3B (hepatocellular carcinoma or HCC) were used in the present study. Glypican-3 (GPC3) is a tumor-specific target in the Hep3B model. Results: We identified three anti-PD-L1 phage binders, B2, A11, and F5 from our shark phage libraries. All three binders showed cross-activity to human, mouse, and canine antigens, whereas only B2 functionally blocked the interaction of human PD-1 to PD-L1. Moreover, CAR (B2) T cells specifically lysed both MDA-MB-231 and Hep3B tumor cells by targeting constitutive and inducible expression of PD-L1. Importantly, the combination of anti-GPC3 CAR (hYP7) T cells with CAR (B2) T cells regress Hep3B tumors synergistically in mice. Conclusions: PD-L1-targeted shark VNAR single domain-based CAR T cell therapy is a novel strategy to treat breast cancer and liver cancer. This work provides a rationale for a potential use of anti-PD-L1 CAR T cells as a monotherapy or combination with a tumor-specific therapy in clinical studies for solid tumors. Citation Format: Dan Li, Hejiao English, Jessica Hong, Tianyuzhou Liang, Glenn Merlino, Chi-Ping Day, Mitchell Ho. Shark VNAR single domain-based CAR T cells targeting PD-L1 for cancer therapy [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 576.

Abstract CT542: Preliminary safety and efficacy results of KN046 in combination with KN026 in patients with locally advanced unresectable or metastatic HER2-positive solid cancer

Abstract Except breast cancer and gastric cancer, HER2 gene amplification or overexpression is also expressed in other solid tumors, including but not limited to colorectal cancer (CRC), non-small cell lung cancer (NSCLC), gallbladder cancer, renal pelvis cancer and pancreatic cancer. The reports of immunotherapy combined with HER2-targeted therapy are limited. KN046 is a novel bispecific antibody that blocks both PD-L1 interaction with PD-1 and CTLA4 interaction with CD80/CD86. KN026 is a novel bispecific antibody that simultaneously binds to two distinct HER2 epitopes. Here the preliminary safety and efficacy results of KN046 in combination KN026 were reported in patients with locally advanced unresectable or metastatic other solid tumors who received ≥ 1 line prior systemic therapy. Methods: HER2-positive locally advanced unresectable or metastatic other solid tumors with progression after ≥ 1 line of prior systemic therapy were recruited, including 14 CRC patients, 4 NSCLC patients, 4 gallbladder cancer patients, 1 renal pelvis cancer patient and 1 pancreatic cancer patient. These patients were by KN046 (iv. 5 mg/kg Q3W) plus KN026 (iv. 30 mg/kg Q3W, loading on C1D1, D8) until progression, unacceptable toxicity, or patient withdrawal. Efficacy was assessed according to RECIST 1.1 Q6W. The primary endpoint was objective response rate (ORR). Results: As of the August 10th, 2021, 24 non-breast or non-gastric cancer patients with the median age of 56 years (range: 37-66) were enrolled. 20 and 24 patients were evaluable for overall response and safety, respectively. The ORR was 55.0% (11 of 20, 95% CI: 31.5-76.9). And the disease control rate (DCR) was 85.0% (17 of 20, 95% CI 62.1-96.8). The 6-month progression-free survival (PFS) rate was 84.1%. 11 CRC patients were evaluable for overall response. The ORR and DCR in CRC was 45.5% (5 of 11, 95% CI: 16.7-76.6) and 90.9% (10 of 11, 95% CI 58.7-99.8), respectively. Twenty of total 24(87.9%) patients suffered from treatment-related adverse events (TRAEs) of any grade. Total 4 of 24 (16.7%) patients had experienced ≥grade 3 TRAEs, including 4 cases related to KN046 and 3 cases related to KN026. The most common (≥10%) TRAEs were infusion related reaction (29.2%), diarrhea (19.4%), alanine aminotransferase increased (16.7%), aspartate aminotransferase increased (16.7%), vomiting(12.5%) and decreased appetite (12.5%). No treatment-related deaths were observed. Conclusion: This chemotherapy-free regimen of KN046 in combination with KN026 has shown promising clinical efficacy and manageable toxicity in HER2-positive non-breast and non-gastric solid tumors with ≥ 1 line prior systemic therapy. The trial is currently ongoing. ClinicalTrials.gov Number, NCT04521179 Citation Format: Jifang Gong, Lei Chen, Meili Sun, Yanming Zhang, Jieer Ying, Xiangcai Wang, Mingli Ni, Zhixiang Zhuang, Baohong Guo, Long Xiao, Summer Xia, Lin Shen. Preliminary safety and efficacy results of KN046 in combination with KN026 in patients with locally advanced unresectable or metastatic HER2-positive solid cancer [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 CT542.

Abstract 1875: Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy

Abstract Programmed death 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T-cell immune function. Blocking the interaction of PD-1 with its ligand PD-L1 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 blockade has led to a paradigm shift in cancer drug discovery, due to its durable effect against a wide variety of cancers. However, despite its tremendous clinical success in cancer treatment, a significant fraction of patients and tumor types remain unresponsive to this therapy. This has led to the development of combination therapies combining PD-1 checkpoint inhibitors with a broad range of clinically active treatments including those targeting other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays as existing methods rely heavily on primary immune cells and are highly variable. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibody targeting human PD-1/PD-L1, or bispecific molecules target PD-1/PD-L1 and a co-stimulatory receptor (e.g., 4-1BB, OX40, ICOS) or an immune inhibitory receptor (e.g., CTLA-4, LAG-3, TIGIT). A mouse PD-1/PD-L1 assay and a human PD-1/PD-L2 assay are also developed and can be used in parallel with human PD-1/PD-L1 assay to address preclinical study and assay specificity. Each of these assays consist of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), PD-1 and the second IC receptor, and an artificial antigen presenting cells (aAPCs). Particularly, the combination bioassays are able to measure the synergetic effect of PD-1 blockade with a second IC inhibitor receptor blockade or a costimulatory receptor activation. These reporter-based assays reflect the mechanisms of action for the therapeutic drugs of interest and are sensitive and quantitative. They are prequalified according to ICH guideline and demonstrated the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assay for product release and stability studies during immunotherapy drug development and manufacture. Citation Format: Jamison Grailer, Julia Gilden, Jun Wang, Denise Garvin, Pete Stecha, Jim Hartnett, Steven Edenson, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy [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 1875.

Abstract LB530: A potent and selective small molecule antagonist of PD-L1 ABSK043 demonstrates significant anti-tumor efficacy in combination with other agents in preclinical models

Abstract Immunotherapy has revolutionized cancer treatment in the last decade. Several monoclonal PD-1 and PD-L1 antibodies have been approved for various cancers. Unlike antibodies, small molecule inhibitors may offer potential advantages in oral dosing, adjustable control of drug exposure, improved tumor penetration and many other aspects. Therefore, small molecule antagonist which can efficiently abolish interaction of PD-1 and PD-L1 may render a novel alternative treatment approach with potentially improved clinical benefits. ABSK043, an oral small molecule inhibitor of PD-L1 discovered by us, has entered into clinical Phase I in 2021. Preclinically, ABSK043 has shown significant tumor growth inhibition as a single agent in multiple models. To explore potential synergy of ABSK043 with other therapeutic agents, in vivo combination experiments were conducted. In this work, we present in vivo combination results for ABSK043 with several therapeutic agents such as a VEGFR inhibitor axitinib, or carboplatin, a key component of the widely used CRC standard-of care chemotherapy treatment regimen, as well as other immune-oncology agents. These combinations demonstrated strong in vivo synergy in tumor mouse syngeneic models. Taken together, these data for the first time demonstrated combination synergy of a small molecule PD-L1 inhibitor with other agents and pave the road for potential clinical evaluation of combination treatment of ABSK043 with these agents in patients. Citation Format: Haiyan Ying, Yongxian Zhang, Fei Yang, Wenqun Xin, Jiaqi Song, Zhui Chen. A potent and selective small molecule antagonist of PD-L1 ABSK043 demonstrates significant anti-tumor efficacy in combination with other agents in preclinical models [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 LB530.

Abstract 5520: AK129, an anti-PD1/LAG-3 bi-specific antibody for cancer therapy

Abstract Background: AK129 is a bi-specific antibody acts by co-blocking two immune checkpoint molecules co-expressed on T cells, PD-1 and LAG-3. Signaling through PD-1/PD-L1 exerts major effects on cytokines production by T cells, inhibiting production of IFN-γ and IL-2. LAG-3 is a cell surface molecule expressed on effector T cells and Tregs, which plays a vital role in regulating T cell function to promote tumoral immune escape. LAG-3 and PD-1 mediate different signaling pathways, but they may act synergistically to cause effector T cell inactivation and exhaustion. AK129 is being investigated as a cancer immunotherapeutic agent. Methods: The antigen binding activity of AK129 to PD-1 and LAG-3 were measured by ELISA and FACS. In reporter gene assay, the bioactivities of AK129 to block the interaction of PD-1 with PD-L1 and to neutralize LAG-3-mediated activity were determined. The bioactivity of AK129 to enhance IL-2 and IFN-γ production in PBMCs via blockade of PD-1 and PD-L1/2 interaction were determined by using cellular assays. In in-vivo pharmacology studies, anti-tumor activity of AK129 was investigated in BALB/c-hPD1/hLAG3 mice bearing CT26.WT tumor model. Results: AK129 showed good antigen binding to PD-1 and LAG-3. AK129 potently inhibited the binding of LAG-3 to its ligand MHC-II, and the binding of PD-1 to its ligand PD-L1, thus blocking downstream immune suppression. At the individual target level, AK129 demonstrated a similar PD-1 blocking activity compared to penpulimab, and similar LAG-3 blocking activity compared to relatlimab. Importantly, at the whole cell level, the enhancement effect of AK129 is stronger than the combination of penpulimab and relatlimab, as evidenced by significantly higher level of IL-2 and IFN-γ secretion from PBMCs. AK129 showed favorable anti-tumor activity in BALB/c-hPD1/hLAG3 mice bearing CT26.WT tumor model, with tumor growth inhibition values reaching 99.27% and 89.48% in high dose (20 mg/kg) and low dose (4 mg/kg) group of AK129, respectively. Conclusion: AK129 is a dual checkpoint blocking bispecific antibody with design features similar to the PD-1/CTLA-4 bispecific antibody AK104, which demonstrated efficacy similar to PD-1 and CTLA-4 antibody combo and much improved safety profile. AK129 has demonstrated robust anti-tumor activity in both in vivo and in vitro pre-clinical studies, and with enhanced T cell activity compared to PD-1 and LAG-3 antibody combo. The efficacy and safety of AK129 await testing in the clinic. Citation Format: Zhaoliang Huang, Xinghua Pang, Tingting Zhong, Chunshan Jin, Na Chen, Dennis Xia, Peng Zhang, Max Wang, Michelle Xia, Baiyong Li. AK129, an anti-PD1/LAG-3 bi-specific antibody for cancer therapy [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 5520.

Abstract 4245: QL301, a PD-L1 dependent 4-1BB agonist with enhanced preclinical anti-tumor efficacy and minimal liver toxicity

Abstract 4-1BB (CD137, TNFRSF9) is a potent co-stimulatory receptor found on T and NK cells. Activation of 4-1BB requires receptor clustering, which is naturally mediated by the endogenous trimeric 4-1BB ligand. Cross-linking via agnostic monoclonal antibodies can also activate 4-1BB but has generally resulted in unwanted side effects, mainly liver toxicity. To address the shortcomings of 4-1BB agonists, we have developed QL301, a PD-L1 x 4-1BB bispecific antibody that conditionally activates 4-1BB only when concurrently engaged to PD-L1, an immune checkpoint mediator elevated in the immuno-suppressive tumor microenvironment. The IgG1 backbone of QL301 was mutated to eliminate Fc gamma receptor-dependent functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), thereby preventing depletion of effector immune cells and non-specific activation of 4-1BB through Fc gamma receptor binding. In addition, QL301 blocks the suppressive interaction of PD-1 with PD-L1. Therefore, through a combination of 4-1BB agonism and PD-L1 blockade, QL301 provides both a co-stimulatory signal and the release of a suppressive signal to reinvigorate anti-tumor immune response. In vitro, QL301 induced robust cytokine release and expansion of CD8+ T cells conditional on the presence of tumor or antigen presenting cells expressing PD-L1. In xenograft and transgenic mice models, QL301 had potent anti-tumor effect that was superior to PD-L1 monoclonal antibodies. Tumor regression correlated with the expansion of CD8+ T cells. In addition, QL301 potentiated tumor cell killing mediated by an EGFR targeted CD3 bispecific antibody, providing a basis for combination therapy. In non-human primates, QL301 was well tolerated up to 30 mg/kg. There was no chronic elevation of serum AST and ALT levels, and histopathology analysis showed minimal inflammation of liver tissue. A phase 1 clinical trial to evaluate the safety, tolerability, and early efficacy of QL301 is currently ongoing. Citation Format: Shenda Gu, Irene Tang, Shirley Mihardja, Wei Wei Prior, Hieu V. Tran, Allan Chan, Anna McClain, Aaron Kurtzman, Shihao Chen, Youguang Luo, Xiaoyan Kang, Xiaoran Wu, Qingmei Zheng, Guodong Jia. QL301, a PD-L1 dependent 4-1BB agonist with enhanced preclinical anti-tumor efficacy and minimal liver toxicity [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 4245.