Human PD-1 Research Articles

Augmented Antitumor Effect of Unripe Rubus coreanus Miquel Combined with Oxaliplatin in a Humanized PD-1/PD-L1 Knock-In Colorectal Cancer Mouse Model.

Immune checkpoint inhibitors (ICIs) have been shown to be extraordinarily effective in patients with colorectal cancer (CRC). However, the current ICIs still have adverse effects and limited efficacy of ICI monotherapy. We used a natural product to overcome the vulnerability of ICIs and tried a combination therapy with oxaliplatin to enhance the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) blockade anticancer effect. In the present study, we evaluated the T cell-mediated antitumor immunity with Unripe Rubus coreanus Miquel extract (RCE), which exerts anticancer properties via PD-1/PD-L1 blockade, combined with oxaliplatin in a co-culture cell model and allograft tumor humanized PD-1 mice. We found that RCE plus oxaliplatin apparently activates hPD-1 tumor-infiltrating CD8+ T cells, resulting in elevations of released interleukin-2 (IL-2) and granzyme B (GrB), and kills hPD-L1 MC38 CRC cells. RCE plus oxaliplatin considerably reduced tumor growth in humanized PD-1/PD-L1-expressing mouse MC38 CRC allograft. Moreover, RCE plus oxaliplatin remarkably increased the infiltration of CD8+ T cells in tumor tissues, as well as increasingly produced GrB of tumor-infiltrating CD8+ T cells in the tumor microenvironment. Our study delineated combination therapy with RCE as a PD-1/PD-L1 blockade and oxaliplatin to improve the response to immune checkpoint blockade therapy in conjunction with standard chemotherapy regimens in CRC.

33P Immune checkpoint inhibitor-induced cardiotoxicity is driven through inflammation, autophagy and stress

Immune checkpoint inhibitors (ICIs) are novel drugs with profound anti-cancer activity but also with adverse events. We investigated the cardiotoxic effects of ipilimumab (IPI, anti-CTLA-4), pembrolizumab (PEM, anti-PD-1) and avelumab (AVE, anti-PD-L1), in primary murine ventricular cardiomyocytes (mAVCs) and spleenocytes, which mainly consist of T- and B- lymphocytes. C57BL6/J mice were sacrificed and primary mAVCs and spleenocytes were isolated. Primary cells were incubated with IPI, PEM and AVE at a concentration range of 0-100 μg/ml for 24h and cell viability was assessed by MTT assay. Furthermore, spleenocytes were treated with IPI, PEM and AVE for 24h and their conditioned medium was added onto the mAVCs for 24h. For the confirmation of PEM binding on the murine PD-1, human and murine PD-1 extracellular domains (ED) were produced and PEM binding was confirmed by circular dichroism (CD). Human IgG4 was used as a negative control. Only IPI led to cytotoxicity in primary spleenocytes and was therefore excluded from the study, as ICIs should not induce cytotoxicity in immune cells (IC). Incubation of mAVCs with PEM and AVE conditioned media, revealed that only PEM could induce IC-dependent cytotoxicity at 50 and 100 μg/ml. The latter was accompanied by an increase in Tnfa, Inos, Rela, Ifng mRNA expression in primary spleenocytes after PEM treatment and Tnfa, Tgfb, Inos, Rela, Ifng as well as autophagy markers Lc3b, Lc3a, Becn2, Atg5 and Endoplasmic Reticulum (ER) stress markers Canx and Ddit3 increase in the primary mAVCs after treatment with PEM conditioned medium. The latter were confirmed with Western Blot analysis. We then investigated whether PEM binds on the murine PD-1 or whether the observed effects are due to an off-target effect. CD experiments showed that PEM binds on the murine PD-1 ED in a similar manner as with the human PD-1 ED used as the positive control, which supports the use of the murine model for the extraction of translational results. The binding of PEM on the murine PD-1 ED was also confirmed by in silico protein-protein docking experiments. PEM induces IC-dependent cytotoxicity on primary mAVCs via induction of inflammation, autophagy and ER-stress.

Human PD-1 agonist treatment alleviates neutrophilic asthma by reprogramming T cells

Neutrophilic asthma is associated with disease severity and corticosteroid insensitivity. Novel therapies are required to manage this life-threatening asthma phenotype. Programmed cell death protein-1 (PD-1) is a key homeostatic modulator of the immune response for T-cell effector functions. We sought to investigate the role of PD-1 in the regulation of acute neutrophilic inflammation in a murine model of airway hyperreactivity (AHR). House dust mite was used to induce and compare neutrophilic AHR in wild-type and PD-1 knockout mice. Then, the therapeutic potential of a human PD-1 agonist was tested in a humanized mouse model in which the PD-1 extracellular domain is entirely humanized. Single-cell RNA sequencing and flow cytometry were mainly used to investigate molecular and cellular mechanisms. PD-1 was highly induced on pulmonary T cells in our inflammatory model. PD-1 deficiency was associated with an increased neutrophilic AHR and high recruitment of inflammatory cells to the lungs. Consistently, PD-1 agonist treatment dampened AHR, decreased neutrophil recruitment, and modulated cytokine production in a humanized PD-1 mouse model. Mechanistically, we demonstrated at the transcriptional and protein levels that the inhibitory effect of PD-1 agonist is associated with the reprogramming of pulmonary effector T cells that showed decreased number and activation. PD-1 agonist treatment is efficient in dampening neutrophilic AHR and lung inflammation in a preclinical humanized mouse model.

Expression of Recombinant CTLA-4 and PD-L1 Proteins Fused with Thioredoxin, and Determination of Their Ligand-Binding Activities.

The use of chimeric proteins that selectively interact with various immune cell receptors to treat oncology patients has increased. One effective way to obtain recombinant proteins is to use the E. coli expression system. However, in eukaryotic protein production in E. coli, several difficulties arise that can be solved by fusing the target protein with thioredoxin. Thioredoxin can enhance solubility, but its large size can lead to an erroneous assessment of protein solubility, folding, and activity. The present study examined the ligand-binding activity of PD-L1, and CTLA-4 receptors fused with thioredoxin. The de novo synthesized genes of the extracellular domains of the PD-L1 and CTLA-4 were cloned into the pET28 and pET32 expression plasmids and used to transform E. coli BL21 cells. Purified recombinant proteins were characterized by western blotting, LC-MS/MS spectrometry, and ELISA. Amino acid sequence comparisons of the recombinant proteins obtained by LC-MS/MS with the SwissProt database resulted in the highest comparison scores from 4950 to 13396. The binding efficiencies of recombinant human B7-1 Fc to rCTLA-4 and rTrx-CTLA-4 proteins in ELISA did not differ significantly. Similar results were obtained with recombinant rhesus monkey PD-1 hFc against rPD-L1 and rTrx-PD-L1. Recombinant proteins specifically reacted with recombinant human B7-1 Fc and recombinant rhesus monkey PD-1 hFc. The fusion of thioredoxin with recombinant proteins through linkers slightly affected the activity of the extracellular domains of CTLA-4 and PD-L1.

Penpulimab, an Fc-Engineered IgG1 Anti-PD-1 Antibody, With Improved Efficacy and Low Incidence of Immune-Related Adverse Events.

BackgroundIgG4 anbibodies are deficient in stability and may contribute to tumor-associated escape from immune surveillance. We developed an IgG1 backbone anti-programmed cell death protein-1 (PD-1) antibody, penpulimab, which is designed to remove crystallizable fragment (Fc) gamma receptor (FcγR) binding that mediates antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and proinflammatory cytokine release.MethodsAggregation of different anti-PD-1 antibodies was tested by size exclusion chromatography, and melting temperature midpoint (Tm) and aggregation temperature onset (Tagg) were also determined. The affinity constants of penpulimab for PD-1 and human FcγRs were measured by surface plasmon resonance and biolayer interferometry. ADCC and ADCP were determined in cellular assays and antibody-dependent cytokine release (ADCR) from human macrophages was detected by ELISA. Binding kinetics of penpulimab to human PD-1 was determined by Biacore, and epitope/paratope mapping of PD-1/penpulimab was investigated using x-ray crystallography. Additionally, patients from six ongoing trials were included for analysis of immune-related adverse events (irAEs).ResultsPenpulimab demonstrated better stability and a lower level of host-cell protein residue compared with IgG4 backbone anti-PD-1 antibodies. As expected, penpulimab exhibited no apparent binding to FcγRIa, FcγRIIa_H131, FcγRIIIa_V158 and FcγRIIIa_F158, elicited no apparent ADCC and ADCP activities, and induced no remarkable IL-6 and IL-8 release by activated macrophages in vitro. Penpulimab was shown in the co-crystal study to bind to human PD-1 N-glycosylation site at N58 and had a slower off-rate from PD-1 versus nivolumab or pembrolizumab. Four hundred sixty-five patients were analyzed for irAEs. Fifteen (3.2%) patients had grade 3 or above irAEs. No death from irAEs was reported.ConclusionsIgG1 backbone anti-PD1 antibody penpulimab has a good stability and reduced host cell protein residue, as well as potent binding to the antigen. Fc engineering has eliminated Fc-mediated effector functions of penpulimab including ADCC, ADCP and reduced ADCR, which may contribute to its more favorable safety profile.Clinical Trial Registration www.ClinicalTrials.gov, identifier: AK105-101: NCT03352531, AK105-201: NCT03722147, AK105-301: NCT03866980, AK105-202:NCT03866967, AK105-203: NCT04172571, AK105-204: NCT04172506.

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 590: Humanized PD-1/LAG-3 mice for preclinical evaluation of combination therapy

Abstract Lymphocyte activation gene 3 (LAG-3) is a member of the immunoglobulin superfamily which shares homolog with CD4 and binds to MHC II with higher affinity. It is expressed on activated T cells, regulatory T cells (Tregs), natural killer cells and plasmacytoid dendritic cells, and inhibits their activation. Blocking LAG-3 activity is expected to provide therapeutic benefits in the treatment of cancer, especially when combined with PD-1 blockade. Due to the low homology between human and mouse LAG-3 and PD-1 and in order to enable increased translatability of preclinical in-vivo efficacy evaluation of immune checkpoint antibodies, we generated double humanized mice by replacing the extracellular domain of mouse LAG-3 and PD-1 with the corresponding human sequences. Homozygous double humanized mice express chimeric LAG-3 and PD-1 at comparable levels to mouse LAG-3 and PD-1 respectively in wildtype mice. When treated with human LAG-3 antibody (anti-LAG-3) or human PD-1 antibody (anti-PD-1), CT26 tumor growth was inhibited in a dose-dependent manner, proving that both humanized proteins are functional. However, not all tumor types respond to anti-LAG-3 or anti-PD-1 the same. To identify the suitable tumor models for preclinical efficacy evaluation of anti-LAG-3 and anti-PD-1 combo therapy, we engrafted several syngeneic tumor models onto our mice. The growth of subcutaneously engrafted MC38 colon tumor was mildly inhibited by anti-PD-1, but strongly inhibited in the presence of anti-LAG-3. Similarly, B16F10 melanoma tumor showed modest growth inhibition when treated with anti-PD-1 or anti-LAG-3 separately, while combination therapy significantly repressed tumor growth. However, neither monotherapies of anti-LAG-3 and anti-PD-1 nor combination treatment effectively inhibited tumorigenesis of LLC1 derived lung cancer, indicating that this tumor is likely insensitive to PD-1 and LAG-3 blockade. Collectively, these data indicate that double humanized PD-1 and LAG-3 mice are a powerful tool to evaluate the therapeutic efficacies of anti-hPD-1 and anti-hLAG-3 treatment in different types of tumor in vivo. Citation Format: Feifei Duan, Xiaoliu Yang, Cunxiang Ju, Jing Zhao, Xiang Gao, Santi Suryani Chen. Humanized PD-1/LAG-3 mice for preclinical evaluation of 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 590.

Abstract 1641: Evaluating in vivo efficacy of bi-functional fusion protein anti-PD-1-IL21 in humanized B-hIL21R mice and B-hPD-1/hIL21R mice

Abstract Interleukin 21 (IL21) binding to its receptor IL21R, results in downstream activation of multiple signaling molecules, including JAK1, JAK3, STAT1, and STAT3. Intriguingly, IL21 has been implicated in both positively and negatively regulating immune responses in a cell-type specific manner, suggesting drug candidates that target IL21/IL21R signaling has therapeutic potential. Specifically, recent reports suggest that antibodies targeting PD-1 fused to IL-21 has a synergistic anti-tumor effect. To generate a suitable animal model for in vivo assessment of a human anti-PD-1-IL21 fusion protein, Biocytogen developed humanized PD-1 and IL21R mice (B-hIL21R mice and B-hPD-1/hIL21R mice) by replacing the murine Il21r and PD-1 gene with the human IL21R and PD-1 counterparts via homologous recombination. Our data shows that human IL21R was detectable in sorted cells from homozygous B-hIL21R and B-hPD-1/hIL21R mice, while human PD-1 was detectable in B-hPD-1/hIL21R mice compared to wild type mice. Furthermore, STAT3 phosphorylation in splenocytes was successfully induced with mouse IL21 or human IL21 in homozygous B-hIL21R mice. In addition, percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD8+ T cells, CD4+ T cells and Tregs in homozygous B-hIL21R mice and B-hPD-1/hIL21R mice were similar to those in wild type mice, demonstrating that introduction of human IL21R and PD-1 genes in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. To evaluate in vivo efficacy of anti-mPD-1-hIL21 and anti-hPD-1-hIL21 fusion proteins, mouse colon adenocarcinoma MC38 cells were subcutaneously implanted into homozygous B-hIL21R mice (female, 6-7-week-old, n=6) or B-hPD-1/hIL21R mice (female, 6-7-week-old, n=6). Mice were grouped and treated when tumor volume reached approximately 100 mm3. Results showed that anti-PD-1-hIL21 fusion proteins were efficient at inhibiting tumor growth in B-hIL21R and B-hPD-1/hIL21R mice. Altogether, B-hIL21R and B-hPD-1/hIL21R mice are promising humanized mouse models for preclinical in vivo studies to assess novel drug candidates. Keywords: IL21/IL21R, humanized mice, fusion protein Citation Format: Ruili Lyu, Zhixia Li, Leila Kokabee, Jiawei Yao, Yanhui Nie, Chaoshe Guo. Evaluating in vivo efficacy of bi-functional fusion protein anti-PD-1-IL21 in humanized B-hIL21R mice and B-hPD-1/hIL21R mice [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 1641.

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 5615: YH008, a PD-1-CD40 bispecific antibody, inhibits tumor growth in vivo through PD-1-dependent activation of CD40 signaling

Abstract PD-1/PD-L1 inhibition immunotherapy is very popular in cancer treatment and has become one of the most pursued therapeutic strategies in the field. Combination treatment using anti-PD-1/PD-L1 agents with other immunomodulators brings additional benefits, such as preventing refractory effects towards PD-1/PD-L1 antibodies, and better inhibitory effects on tumor growth. Biocytogen developed YH008, a tetravalent anti PD-1/CD40 Fc-silenced IgG1 bispecific antibody (BsAb) that agonizes CD40 in a PD-1-dependent manner. Previously, YH008 demonstrated dose-dependent inhibitory efficacy against B16F10 engrafted tumors in PD-1/CD40 double humanized mice (established by Biocytogen: B-hPD1/CD40). Interestingly, in humanized CD40 mice that do not express human PD-1 expression, YH008 did not show tumor inhibition activity against the MC38 engrafted tumor. This observation indicates that YH008 activates the CD40 pathway in a PD-1 dependent manner. When compared with an anti-PD-L1/CD40 BsAb, YH008 showed superior tumor growth inhibition (TGI) against MC38 engrafted tumors in a triple humanized PD-1/PD-L1/CD40 mouse model (B-hPD-1/hPD-L1/hCD40). YH008 was observed to promote the proliferation and activation of dendritic cells, as well as increase CD8+/Treg ratio in B16F10 tumor-infiltrating lymphocytes (TILs). Furthermore, YH008 showed a superior liver toxicity profile compared to a selicrelumab analog. At a high dose of 100mg/kg, YH008 did not elevate liver enzymes in the blood of B-hPD1/hCD40 mice. Similarly, no significant adverse events were observed in a cynomolgus dose-escalation study. YH008 is currently in pre-clinical development with anticipated IND application in 2022. Citation Format: Baihong Liu, Hao Yang, Li Hui, Benny Yang. YH008, a PD-1-CD40 bispecific antibody, inhibits tumor growth in vivo through PD-1-dependent activation of CD40 signaling [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 5615.

Abstract 2804: Generation of RNA aptamers for measuring fractional occupancy of PD1 by PDL1

Abstract Background: The limitations of current biomarkers for predicting outcome to anti-PD1 therapy, such as measuring tumor PDL1, are well-known. Evaluation of the PD1-PDL1 interaction as a potential biomarker has not been explored because of the lack of a suitable assay. We previously reported on a novel technology called the LIRECAP (Ligand-Receptor Complex-binding Aptamer) assay that quantifies the fractional occupancy of a receptor by its ligand (PMID: 31383650). LIRECAP assay is based on two RNA aptamers with differential binding to the unoccupied receptor vs. the ligand-receptor complex, where the ratio of aptamer binding reflects fractional occupancy. Here, we report the generation of RNA aptamers for use in a LIRECAP assay to quantify PD1-PDL1 interactions in tumor. Methods: SELEX: RNA aptamers were enriched using a variation of SELEX that consisted of pre-clearing followed by a positive selection against human PD1, PDL or the PD1-PDL1 complex. Target-bound aptamers were extracted, amplified with SEL2 primers and transcribed. After eight rounds, enriched aptamers were sequenced and lead aptamers were identified. Assessing target binding: Dynabeads were coated with recombinant PD1 alone or PDL1 alone. Complex-coated beads were made by incubating PD1-bound beads with PDL1. Beads were formalin-fixed to mimic biospecimens and prevent PD1-PDL1 dissociation. Aptamers were added to beads, unbound aptamers washed off and bound aptamers extracted and quantified by SYBR green-based RT-qPCR using SEL2 primers. Binding of each aptamer was normalized to the binding of unselected aptamer library (Rd0). Results: (1) A progressive enrichment of unique sequences was seen in all the three SELEX (PD1, PDL1, Complex) with maximum enrichment reached between rounds 4 and 5. (2) Binding of aptamers: Most of the lead candidates showed predominant binding to the targets against which they were enriched. (a) Three out of four lead aptamers from the PD1 SELEX showed significantly higher binding to unoccupied PD1 than to PD1-PDL1 complex (p&amp;lt;0.05). (b) Two out of five lead aptamers from the PDL1 SELEX showed significantly higher binding to unoccupied PDL1 compared to the complex (p&amp;lt;0.05). (c) Four out of five lead aptamers from the Complex SELEX showed significantly higher binding to PD1-PDL1 complex than to unoccupied PD1 (p&amp;lt;0.05). None of these aptamers showed cross-reactivity to unoccupied PDL1. Conclusions and Significance: Novel RNA aptamers with differential specificity towards PD1, PDL1 or PD1-PDL1 complex have been successfully created for use in a LIRECAP assay that will be used to quantify the fractional occupancy of PD1 by PDL1 in tumor biospecimens. Studies assessing whether the resulting assay can predict response to anti-PD1 therapy are expected to begin shortly. Citation Format: Suresh Veeramani, Travis Fischer, William H. Thiel, George J. Weiner. Generation of RNA aptamers for measuring fractional occupancy of PD1 by PDL1 [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 2804.

Abstract 5049: Prediction of pharmacokinetics and receptor occupancy of a bispecific antibody against CD40 and PD-L1

Abstract Background: Currently bispecific monoclonal antibodies gain traction in pharmacology as a way to simultaneously target different surface molecules on immune and tumor cells and modulate their activity. While antibodies against a single target, such as CD40 on antigen-presenting cells (APCs) and PD1 on T cells, are widely used in clinic to treat various forms of cancer, bispecific antibodies are still in the early stages of clinical development. The aim of this study was to predict pharmacokinetics (PK), receptor occupancy (RO) and optimal dose for a bispecific antibody YH008 against CD40 and PD1 using the PBPK/RO model. Methods: The PBPK/RO model describes distribution of an antibody (mAb) between physiological compartments, transport across the endothelium, binding with CD40 on dendritic cells (DCs), B cells, and macrophages (Mph) and PD1 on CD4+ and CD8+ T cells in the immunological synapses (ISs). The model takes into account number of cells expressing target molecules and their ligands, expression levels, clearance of the antibody. Two scenarios of mAb binding with CD40 were considered: a case when the mAb competes with CD40L for the binding site and a case when the mAb binds to a different site. Binding affinities of YH008 for human CD40 and PD1 were taken from [1]. The model was validated using PK data on monospecific antibodies against CD40 and PD1. ROs of PD1 on CD4+ and CD8+ T cells and CD40 for Mph, DCs, B cells and percentages of the trimer complex (CD40-YH008-PD1) were calculated. Optimal dose of YH008 was selected on the basis of maximal exposure/area under curve (AUC) of the trimers in ISs between APCs and CD8+ T cells in the tumor. Results: Model simulations show good agreement with the experimental PK and RO data for monospecific mAbs against PD1 (Pembrolizumab) and CD40 (Selicrelumab, BI 655064). For the bispecific antibody, the simulations show that competition for the CD40 binding site between CD40L and the mAb does not significantly affect PK and RO in blood or tissue. The model predicts 99% RO on antigen-presenting cells (APCs) and 98% RO on T cells in blood at dose 80 mg. In the tumor, 95% RO on APCs and 85% RO on T cells, as well as 5-to-6-fold decrease in the number of PD1-PDL1 complexes in ISs, are achieved at dose 240 mg. The dose dependence for YH008 was calculated, and the optimal dose based on the numbers of the trimers and signaling complexes in the ISs was estimated. Conclusions: The PBPK/RO model for a bispecific mAb based on physiological levels of immune cells, target molecule expression, and binding parameters for YH008 antibody against CD40 and PD1 predicts PK and RO in peripheral blood and tumor. The model shows that competition for CD40 binding site between the mAb and CD40L does not significantly affect PK and RO. The optimal dose for maximal effect of YH008 was estimated. Citation Format: Alexandra Diakonova, Oleg Demin, Dmitry Shchelokov, Oleg Demin. Prediction of pharmacokinetics and receptor occupancy of a bispecific antibody against CD40 and PD-L1 [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 5049.

Abstract 5526: AK131, an anti-PD1/CD73 bispecific antibody for cancer immune therapy

Abstract Background: AK131 is a bispecific antibody derived from AK105 (the Akeso PD-1 antibody) and AK119 (the Akeso CD73 antibody). AK119 is a dual mechanism antibody (2021SITC poster #750) that blocks the generation of adenosine and stimulates antigen specific B cell activation. Adenosine is a key immune suppressive molecule thought to contribute to resistance to PD-1 antibody therapy. Both CD73 and PD-1 are targets with high expression in the tumor microenvironment. Moreover, a growing body of literature evidence supports an important role B cells play in tumor immunity. Thus we developed a bispecific PD-1/CD73 antibody with the capacity to relive immune checkpoint control of T cells, activate B cells, and eliminate immune suppression by adenosine, for immune therapy of cancer. Method: Binding assays of AK131 to antigens, and antigen expressing cells were performed by using ELISA, Fortebio, and FACS assays. In-vitro bioactivity assays of AK131 including the investigation of neutralizing activity to PD-1 by reporter gene assay, activation of T cells by detecting secretion of IFN-γ and IL-2 by ELISA, inhibition of CD73 enzymatic activity by cell-based enzyme assay, activation of B cells by measuring CD69, CD83, HLA-DR and IgM expression, as well as endocytosis of CD73 by cellular assay. In-vivo anti-tumor activity of AK131 was further confirmed by C57BL/6-hPD-1/hPD-L1/hCD73 transgenic mouse MC38-hPDL1-hCD73 tumor syngeneic model. Result: AK131 effectively bound to human PD-1 and CD73 with high affinity. The interaction between PD-1 and its ligand PD-L1 was blocked by AK131 in reporter gene assay. In cellular assays, AK131 effectively promoted the secretion of IFN-γ and IL-2 by T cells in co-culture of PBMCs and Raji-PDL1 cells. Moreover, AK131 inhibited CD73 enzymatic activity and induced endocytosis of CD73. AK131 enhanced the expression of CD69, CD83, HLA-DR and IgM which are the activation marker of B cell, in a adenosine-independent manner. In in-vivo assay, AK131 successfully inhibited tumor growth in C57BL/6-hPD-1/hPD-L1/hCD73 transgenic mouse MC38-hPDL1-hCD73 tumor syngeneic model. Conclusion: AK131 is a bi-specific antibody targeting both PD-1 and CD73. In pre-clinical studies, AK131 showed potent in-vitro and in-vivo activities. In summary, AK131 effectively blocked PD-1/PDL-1 interaction, promoted the activation of T cells and B cells, inhibited the CD73 enzymatic activity, and induced CD73 endocytosis. AK131 also demonstrated robust in-vivo anti-tumor activity in mouse model. These observations, addition to other preclinical study results, supports the development of AK131 as an anti-tumor agent 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. AK131, an anti-PD1/CD73 bispecific antibody for cancer immune 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 5526.

Abstract LB221: Development of PD-L1-targeted sialidase as a novel cancer immunotherapeutic approach

Abstract Sialoglycans have emerged as a critical glyco-immune checkpoint that impairs antitumor response by inhibiting innate and adaptive immunity. We have previously reported that Bi-Sialidase - an engineered human sialidase-Fc fusion - potentiates antitumor immune response by cleaving terminal sialic acids from sialoglycans (desialylating) on tumor cells and immune cells. Furthermore, we have shown that a tumor-targeted sialidase, a heterodimeric molecule consisting of one chain of sialidase-Fc and a second chain of a HER2-targeting antibody (trastuzumab), leads to more efficient desialylation of tumor cells than the non-targeted Bi-Sialidase and demonstrates antitumor activity in trastuzumab-resistant and low HER2-expressing tumor models. To evaluate the potential of targeted desialylation of both tumor cells and immune cells, we designed and characterized a PD-L1-targeted sialidase in preclinical models, since PD-L1 is expressed on both tumor cells and immune cells. Furthermore, the design of the PD-L1-targeted sialidase enables the combinatorial blockade of two orthogonal immune checkpoint pathways, inhibiting both the PD-1/PD-L1 axis and immunosuppressive sialoglycans. We generated a humanized anti-human PD-L1 antibody with comparable PD-1/PD-L1 blockade potency to the existing anti-PD-L1 drugs, atezolizumab and avelumab. Subsequently, we constructed a bifunctional heterodimeric molecule, consisting of one chain of sialidase-Fc of a third generation of engineered human sialidase (Neu 2) and a second chain of the in-house generated anti-PD-L1 antibody. The PD-L1-targeted sialidase maintained its potency for inhibiting the PD-1/PD-L1 axis as compared to its parental anti-PD-L1 antibody, and demonstrated improved desialylation of PD-L1-expressing tumor cells and immune cells in vitro. We tested the PD-L1-targeted sialidase in transgenic mouse tumor models that express human PD-1 and PD-L1 replacing their murine counterparts, since the parental PD-L1 antibody doesn’t cross-react with the mouse antigen. In the transgenic human PD-L1-expressing mouse colon carcinoma CT26 subcutaneous tumor model, the PD-L1-targeted sialidase exhibited enhanced efficacy compared to the Bi-Sialidase or the anti-PD-L1 antibody. Furthermore, the PD-L1-targeted sialidase demonstrated a dose-dependent tumor growth inhibition and modulation of immune cell infiltration. In conclusion, these results suggested that PD-L1-targeted sialidase offers a promising cancer immunotherapeutic approach, which simultaneously inhibits immunosuppressive sialoglycans and the PD-1/PD-L1 axis through targeted delivery of engineered human sialidase to PD-L1-expressing tumor cells and immune cells. Citation Format: Jenny Che, Lihui Xu, Wayne Gatlin, Robert LeBlanc, Lizhi Cao, James Broderick, Li Peng. Development of PD-L1-targeted sialidase as a novel cancer immunotherapeutic approach [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 LB221.

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 3510: SOT201 is a novel targeted IL-15Rbg agonist to alleviate PD-1-mediated immune cell suppression and potentiate anti-tumor efficacy

Abstract SOT201 is a novel immunocytokine consisting of a monoclonal humanized, Fc silenced antibody against PD-1 fused to a covalent RLI-15 complex of a human IL-15 mutein linked to the high-affinity binding site of the IL-15Rα, the sushi+ domain. SOT201 is developed for immunotherapeutic treatment of various types of cancers. The activity of SOT201 is based on spatiotemporal reinvigorating of anti-tumor immune responses by disrupting co-inhibitory T-cell signaling by blocking PD-1 and synergistically activating adaptive as well as innate immunity by IL-15-mediated signaling via the IL-2/IL-15βγ receptor on T cells, NK, NKT, and γδ T cells. SOT201 showed a superior potentiation of T cell stimulation over pembrolizumab in mixed lymphocyte reaction in vitro. Studies in cynomolgus monkeys showed that decreased affinity of IL-15 mutein in SOT201 for its IL-15Rβγ is well optimized to ensure favorable pharmacokinetic properties while inducing strong CD8+ T cell and NK cell activation and expansion. Synergistic action on CD8+ T cell activation of both anti-PD-1 and RLI-15 moieties was confirmed using mouse surrogate SOT201 molecules in vivo. Strong anti-tumor efficacy after SOT201 treatment was achieved in a human PD-1 transgenic mouse model implanted with the MC38-hPD-L1 cell line. These data represent a promising therapeutic candidate molecule leveraging the synergistic concerted action of anti-PD-1 blockage and simultaneous immune cell activation directed preferentially to the high PD-1+ T cell tumor environment. The therapeutic potential of SOT201 is currently being prepared for evaluation in a Phase I clinical study in metastatic advanced cancer patients as well as for PD-1 resistant/refractory patients as our clinical stage IL-2/IL-15Rβγ agonist SOT101 already showed a clinical benefit in these patients in its ongoing Phase I study. Citation Format: Irena Adkins, Zuzana Antosova, Klara Danova, Kamila Hladikova, Katerina Augustynkova, Katerina Sajnerova, Nada Podzimkova, Pavel Marasek, Guy de Martynoff, David Bechard, Ulrich Moebius, Radek Spisek. SOT201 is a novel targeted IL-15Rbg agonist to alleviate PD-1-mediated immune cell suppression and potentiate anti-tumor efficacy [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 3510.

Abstract 2870: Dose selection investigations and combination strategies of NM21-1480, a PD-L1/4-1BB/HSA trispecific MATCH3 therapeutic clinical candidate

Abstract Antagonistic molecules targeting the PD-1/PD-L1 axis have shown excellent activity in the clinic. However, the majority of patients do not respond to the therapy due to multifaceted reasons implicating a non-effective activation of the immune system in those patients. The co-stimulatory molecule 4-1BB has been shown to be a key signalling component of T cells and the combination of 4-1BB activation and PD-1/PD-L1 antagonism has been shown to be highly active in preclinical models. Systemic application of first-generation anti-4-1BB antibodies however have resulted in dose limiting hepatic toxicities. We have generated and are currently clinically investigating a novel 4-1BB/PD-L1/HSA trispecific MATCH3 immunomodulatory drug candidate (NM21-1480) that agonizes 4-1BB conditionally upon PD-L1 binding and concomitantly blocks the interaction between PD-1 and PD-L1. Here we investigate the impact of dose dependency in vitro and the translation of this to in vivo studies to aid the selection of optimal doses for our ongoing clinical study of NM21-1480 in cancer patients. In human PD-L1 tumor bearing mice, triple knock-in for human PD-1, human PD-L1 and human 4-1BB, we have investigated the impact of dose titrations of NM21-1480. We observe a dose dependent effect of NM21-1480 on tumor growth, systemic exposure, tumor exposure and pharmacodynamic biomarkers of immune cell activation. We also demonstrate the formation of a memory response in treated mice through rechallenge of the mice with tumor. We also investigate the effect of combination therapy of NM21-1480 with immune-oncology targeted therapeutics both in vitro and in vivo. In particular, we demonstrate that the combination of NM21-1480 with anti-CD3 T cell engagers is highly effective in T cell activation and tumor control. These data highlight the potential of NM21-1480 for the treatment of cancer patients and enable greater understanding of the relationship between dose, tumor exposure, immune activation and tumor growth inhibition. Citation Format: Daniel Snell, Tea Gunde, Stefan Warmuth, Peter Lichtlen, Linlin Liu, Shu-wen Teng, Lan Zhang, ChaoHsuan Pan, Peiqi Li, Julia Tietz, Matthias Brock, Alexandre Simonim, Christian Hess, Christopher Weinert, Maria Johansson, Bithi Chatterjeee, Nicole Bassler, Niels Kirk, Catia Mendes, Robin Heiz, Naomi Flueckiger, Dania Diem, Dana Mahler, Belinda Wickihalder, Simone Muntwiler, Sandro Wagen, Elmar vom Baur, Archie N. Tse, David Urech. Dose selection investigations and combination strategies of NM21-1480, a PD-L1/4-1BB/HSA trispecific MATCH3 therapeutic clinical candidate [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 2870.

GNUV201, a novel human and mouse cross-reactive PD-1 monoclonal antibody for cancer immunotherapy.

e14509 Background: Several PD-1 antibodies have been recently approved as anti-cancer therapies which work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. However, cross-reactive anti-PD-1 antibodies to both human and pre-clinically relevant animal models are still required to predict more precisely the efficacy and toxicity in humans, especially in various combination settings of anti-PD-1 with other treatments. Methods: Therefore, we have developed an antibody named GNUV201, a highly selective and interspecies cross-reactive humanized monoclonal antibody, with conventional hybridoma technology. Results: GNUV201 equally binds to mouse and human PD-1 (EC50= 32 pM and 28 pM, respectively), while marketed Keytruda and Opdivo bind only to human PD-1. According to the results of co-crystal structure and alanine-scanning mutagenesis to examine the interaction mode between GNUV201 and human PD-1, the epitope recognized by GNUV201 is on the “FG loop” region of human PD-1, which is well conserved in both human and mouse PD-1, supporting GNUV201’s inter-species cross-reactivity, and this position is distinct from those of Keytruda (“C’D loop”) and Opdivo (N-term). Notably, the structural feature in which the protruding epitope loop fits into GNUV201’s binding pocket supports its enhanced binding affinity due to slower dissociation (8.5 times slower than Keytruda in SPR). Furthermore, GNUV201 shows stronger binding affinity at pH 6.0 (KD = 0.9 nM at pH 6.0, 5.8 times smaller KD than at pH 7.4) which mimics more closely the hypoxic and acidic tumor micro-environments (TME), especially due to further improved Koff. This phenomenon is not observed with other anti-PD-1 competitors, implying that GNUV201 achieves better target occupancy in tumors compared to its marketed competitors. GNUV201 was also shown to successfully block the interaction of hPD-1 with hPD-L1 at similar single digit nM levels compared with Keytruda and Opdivo in vitro and strongly suppress tumor growth in syngeneic models. In particular, tumor growth regressed in B16F11 (mouse melanoma) and was inhibited in MC38 (mouse colon cancer) syngeneic models and MC38/hPD-1 knock-in mouse models during treatment, when compared with the original tumor size. Conclusions: In summary, we expect GNUV201 to be an excellent antibody candidate whose efficacy and toxicity in the human system can be predicted from preclinical study results due to its human-mouse cross-reactivity and whose efficacy can be further improved with better target occupancy due to its slower dissociation and preferential binding in low pH tumor microenvironments. We are currently engineering GNUV201 to further improve antigen binding kinetics, which could lead to enhanced efficacy and pharmacokinetics in humans, and to develop the best-in-class PD-1 antibody based on superior comparative data from on-going efficacy studies.

Initial efficacy and biomarker analysis of a phase 1b study targeting IL-1β and PD-1 with chemotherapy in metastatic pancreatic cancer (PanCAN-SR1).

e16287 Background: Pancreatic ductal adenocarcinoma (PDA) has been refractory to therapeutic targeting of the immune microenvironment. In preclinical work, IL-1β was upregulated in PDA tumors, and in mouse models, IL-1β expression led to activation of pancreatic stellate cells and immunosuppression. We hypothesize that blockade of IL-1β and PD-1 will result in significant alterations in immune and fibroblast subsets within the PDA microenvironment. Methods: We conducted an open-label multicenter Phase Ib study evaluating gemcitabine and nab-paclitaxel with canakinumab (ACZ885), a high-affinity human anti-interleukin-1β (IL-1β) mAb, and spartalizumab (PDR001), a mAb directed against human PD-1. 10 subjects with untreated metastatic PDA and RECIST measurable disease were enrolled. The primary objective was to identify a recommended phase II/III dose by evaluating the incidence of DLTs in the first 56 days. All subjects underwent baseline and on-study tissue and blood collection for extensive exploratory correlative studies. Secondary objectives including safety and tolerability and preliminary assessment of clinical activity. Results: 10 subjects were enrolled between November 2020 and March 2021. 6 out of 10 were evaluable for the dose confirmation. In the dose confirmation analysis there were no dose limiting toxicities (DLTs) and the recommended Phase II/III dose was established as; gemcitabine (1000 mg/m2 IV) and nab-paclitaxel (125 mg/m2 IV) on day 1,8,15; canakinumab (250 mg via subcutaneous injection) and spartalizumab (400 mg IV) on day 1; of each 28 day cycle. Adverse events were consistent with those seen with chemotherapy and were predominately hematologic. A preliminary efficacy analysis confirms 1 PR and 7 pts with SD. Two of the 7 pts have been treated for 11 and 12 cycles, respectively, without progression. Activation of CD8 T cells in peripheral blood and increased serum levels of IFN-induced chemokines CXCL9/10 were observed in patient samples. Using an in vitro suppression assay, we showed that baseline serum, from patients deriving clinical benefit, could induce myeloid derived suppressor cells, but that this effect was abrogated with treatment. Single cell transcriptional profiling also revealed treatment-dependent shifts in T cell activation state and myeloid cells in the tumors of patients experiencing clinical benefit. Conclusions: In this Phase Ib study, we established the Phase II/III dose of canakinumab and spartalizumab with chemotherapy in first line metastatic PDA. Translational studies suggest putative biomarkers that may help identify responding pts. This novel combination is being evaluated in a randomized Phase II/III study through the Precision Promise clinical trial network. Clinical trial information: NCT04581343.

Oncolytic virus expressing PD-1 inhibitors activates a collaborative intratumoral immune response to control tumor and synergizes with CTLA-4 or TIM-3 blockade

BackgroundOncolytic viruses (OVs) are capable to inflame the tumor microenvironment (TME) and elicit infiltrating tumor-specific T cell responses. However, OV treatment negatively alters the cancer-immune set point in tumors to...