Human Programmed Cell Death-1 Research Articles

Mechanism of PD-1/PD-L1 in Regulating cTfr/cTfh Balance in Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is frequent, an imbalance between helper cells (Th) and regulatory T cells (Treg) is the fundamental immunological cause of RA. This study investigates how recombinant human programmed cell death 1 (PD-L1) protein affects circulating T follicular helper (cTfh), circulating T follicular regulatory (cTfr), and their equilibrium. Magnetic bead sorting was used to select CD4+CXCR5+T cells from RA patients' and healthy individuals' peripheral blood mononuclear cells for in vitro growth. Recombinant human PD-L1 protein stimulated CD4+CXCR5+T cells. Cell counting kit 8 (CCK-8), flow cytometry surface labeling, ELISA, and RT-PCR were used to measure CD4+CXCR5+T cell proliferation inhibition, cTfh and cTfr frequencies, IL-21 expression, and PI3K, AKT, Bcl-6, and Blimp-1 mRNA levels. The recombinant human PD-L1 protein dose-dependently inhibited the proliferation of CD4+CXCR5+T cells in active RA peripheral blood. However, it has a weaker inhibitory effect on healthy peripheral blood CD4+CXCR5+T cells. PD-L1 protein decreased cTfh in active RA peripheral blood CD4+CXCR5+T overall cultured cells but did not affect cTfr; The cTfr/cTfh ratio increased but did not affect the frequency of cTfh and cTfr in healthy persons' cultured CD4+CXCR5+T cells. PD-L1 protein reduced IL-21 in CD4+CXCR5+T cell culture supernatant from active RA peripheral blood. Recombinant human PD-L1 protein lowered PI3K, AKT, and Bcl-6 mRNA in active RA peripheral blood CD4+CXCR5+T cell culture, including significant differences. But Blinmp-1 mRNA variations were neither substantial nor statistically different. PD-1/PD-L1 limits cTfh proliferation, differentiation, and activation via the PI3K/AKT signaling pathway regulates its immunological balance with cTfr, and corrects the cTfr/cTfh imbalance by controlling their interaction.

A Novel Monoclonal Antibody against PD-1 for the Treatment of Viral Oncogene-Induced Tumors or Other Cancer.

Programmed cell death 1 (PD-1) and programmed death-ligand 1 (PD-L1) interact to form an immune checkpoint fostering viral infection and viral oncogene-induced tumorigenesis. We generated a novel anti-human PD-1, humanized monoclonal antibody P1801 and investigated its pharmacologic, pharmacokinetic (PK), and pharmacodynamic properties. In vitro binding assays revealed that P1801 uniquely binds to human PD-1 and inhibits its interaction with PD-L1/2. It showed a minor effect on the induction of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). P1801 significantly induced the release of IL-2 from activated T-cells but not from nonactivated T-cells. A dose-dependent linear PK profile was observed for the cynomolgus monkeys treated with repeated doses of P1801 at 5 mg/kg to 200 mg/kg once weekly. A four-week repeat-dose toxicity study revealed that P1801 given weekly was safe and well tolerated at doses ranging from 5 to 200 mg/kg/dose. No pathological abnormalities were noted. In humanized PD-1 mice harboring human PD-L1-expressing colon tumor cells, P1801 administered intraperitoneally twice per week at 12 mg/kg significantly inhibited tumor growth and prolonged mouse survival. P1801 displayed unique binding properties different from pembrolizumab and nivolumab. Therefore, it showed distinctive immunological reactions and significant antitumor activities. We are initiating a Phase 1 clinical study to test its combination use with ropeginterferon alfa-2b, which also has antiviral and antitumor activities, for the treatment of cancer.

Real-world effectiveness and safety of recombinant human endostatin plus PD-1 inhibitors and chemotherapy as first-line treatment for EGFR/ALK-negative, advanced or metastatic non-small cell lung cancer

BackgroundThis study aimed to evaluate the effectiveness and safety of recombinant human endostatin (Rh-endostatin) plus programmed cell death 1 (PD-1) inhibitors and chemotherapy as first-line treatment for advanced or metastatic non-small cell lung cancer (NSCLC) in a real-world setting.MethodsThis was a retrospective study on patients with EGFR/ALK-negative, advanced or metastatic NSCLC. Patients received Rh-endostatin plus PD-1 inhibitors and chemotherapy every three weeks for 4 to 6 cycles. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were objective response rate (ORR), disease control rate (DCR), overall survival (OS), and safety.ResultsA total of 68 patients were included in this retrospective analysis. As of data cutoff (December 13, 2022), the median follow-up of 21.4 months (interquartile range [IQR], 8.3-44.4 months). The median PFS and OS was 22.0 (95% confidence interval [CI]: 16.6-27.4) and 31.0 months (95% CI: 23.4-not evaluable [NE]), respectively. The ORR was 72.06% (95% CI: 59.85-82.27%), and DCR was 95.59% (95% CI: 87.64-99.08%). Patients with stage IIIB/IIIC NSCLC had significantly longer median PFS (23.4 vs. 13.2 months), longer median OS (not reached vs. 18.0 months), and higher ORR (89.2% vs. 51.6%) than those with stage IV NSCLC (all p ≤ 0.001). The ORR was higher in patients with high PD-L1 expression (tumor proportion score [TPS] ≥ 50%) than in those with low PD-L1 expression or positive PD-L1 expression (75% vs. 50%, p = 0.025). All patients experienced treatment-related adverse events (TRAEs), and ≥ grade 3 TRAEs occurred in 16 (23.53%) patients.ConclusionsRh-endostatin combined with PD-1 inhibitors plus chemotherapy as first-line treatment yielded favorable effectiveness with a manageable profile in patients with advanced or metastatic NSCLC, representing a promising treatment modality.

The Relationship Between PD-1(rs2227981) and PD-L1(rs2890658) Polymorphisms and Urothelial Cell Carcinoma.

Background Urothelial cell carcinoma, which is believed to develop from the urothelium (transitional epithelium), is the most common aggressive tumor and accounts for the ten most prevalent cancers in the world. The risk factors for urothelial cell carcinoma are aging, smoking, gender, and genetic alternations. Programmed cell death1 (PD-1) has been widely described as a negative regulator of T-cells by sending inhibitory signals to the T-cell. Through PD-1 binding with PD-L1 (ligand for PD-1), an inhibitory signal is propagated to the T cell. The polymorphisms of PD-1 and PD-L1 lead to an efficient T-cell response and affect an anti-tumor reaction. The polymorphisms of PD-1 and PD-L1 could also affect the carcinogenesis of human cancer, including urothelial cell carcinoma. Therefore, in this study, we evaluated the relation between PD-1(rs2227981) and PD-L1(rs2890658) polymorphisms and the carcinogenesis of urothelial cell carcinoma. Materials and methods This study was conducted using 211 healthy controls and 256 cases of urothelial cell carcinoma among the Japanese population. The DNA samples were extracted from the peripheral white blood cells of each subject. The genotype was detected by using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Results C/T (Adjusted OR 1.55, 95% CI:1.02-2.35) and C/T+T/T (OR 1.46, 95% CI:1.01-2.12) genotypes of PD-1 rs2227981 were significant and risk factors for urothelial cancer.Male with A/A genotype in PD-L1 and CT genotype in PD-1 has a significant higher risk factor compared with other genotypes (Adjusted OR 1.83, 95% CI:1.05-3.21). Conclusions and discussion We found that C/T(PD-1) and "A/A (PD-L1) and C/T(PD-1)" were predominant in urothelial cell carcinoma cases. This indicates that C/T(PD-1) and "A/A (PD-L1) and C/T(PD-1)" genotypes could increase susceptibility to urothelial cell carcinoma. However, since our findings indicated that the effects of PD-1 and PD-L1 polymorphisms included discrepancies, additional research will be needed to evaluate the relationship between human cancer and PD-1 and PD-L1 polymorphisms. This is the first study that seeks to find the relation between PD-1(rs2227981) and PD-L1(rs2890658) polymorphisms concerning urothelial cell carcinoma among the Japanese population.

Develop a PD-1-blockade peptide to reinvigorate T-cell activity and inhibit tumor progress

Immune checkpoint inhibitors, particularly monoclonal antibodies blocking the programmed cell death 1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway, have been successfully utilized in the clinic. However, certain drawbacks associated with antibodies, such as high immunogenicity and poor tissue penetration, need to be addressed for their broader clinical application. Peptides, as low molecular weight alternatives, have garnered increasing interest in this field. In this study, we employed bacterial surface display technology to identify a PD-1-binding peptide, PBP. The PBP peptide exhibited moderate affinity for human PD-1 (hPD-1) and displayed cross-reactivity with mouse PD-1 (mPD-1). Molecular docking analysis revealed that the interaction residues of the PBP peptide with PD-1 played crucial roles in the formation of the PD-1/PD-L1 complex. A competing binding assay demonstrated that the peptide could interfere the interaction of PD-1 and PD-L1. Moreover, in vitro experiments showed that the PBP peptide could reinvigorate T cells inhibited by PD-L1. In an in vivo mouse model of CT26, the PBP peptide effectively suppressed tumor growth by enhancing T cell function. In conclusion, our results suggest that the PBP peptide exerts an anti-tumor effect by impeding the interplay between PD-1 and PD-L1, highlighting its potential as an alternative for tumor immunotherapy.

Structure-based small inhibitors search combined with molecular dynamics driven energies for human programmed cell death-1 (PD-1) protein

Human immune system is specialized in distinguishing normal cells from foreign particles mainly through proteins expressed on immune cells called ‘checkpoints’. Immune checkpoints work as a switch to activate and deactivate immune responses. T cells express one of the immune checkpoint, human programmed cell death-1 (PD-1), which normally operates as an off-switch function to protect the normal cell from T-cell attack. Binding of PD-1 to its ligand, the programmed cell death ligand (PD-L1/2) expressed on myeloid/cancer cells, induce downstream inhibitory signals, leading to tumor immune evasion. Targeting PD-1 or PD-L1 can boost the immune response against cancer cells. To design novel small molecule inhibitors for the PD-1, in silico structure-based screening on pharmacophoric points and molecular docking were performed. Based on the docking score and significant binding interaction with the crucial residues of PD-1 (Thr59, Glu61, Ser62, Glu84, Arg86 and Ala132), compounds were selected from the ZINC20 database, and their dynamic behavior and conformational stability were examined through molecular dynamic simulations. Besides, the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method was used to calculate the binding strength of each selected inhibitor complexed with PD-1. The binding energy calculations revealed that these selected inhibitors show a considerable affinity for PD-1. The selected novel inhibitors exhibit excellent drug-like and pharmacokinetic properties (absorption, distribution, metabolism, excretion and toxicity). In conclusion, the identified novel compounds (ZINC1443480030, ZINC1002854123, ZINC988238128, ZINC1481242350, ZINC1001739421, ZINC1220816434 and ZINC1167786692) from the current study can be validated in-vitro as potential PD-1 inhibitors and for discovery of novel drugs against PD-1 in the future. Communicated by Ramaswamy H. Sarma

The risks of hematological toxicities of nivolumab in cancer patients: A PRISMA-compliant meta-analysis.

Nivolumab is the human programmed cell death-1 (PD-1)-blocking antibody showing significant effect in many refractory cancers. However, little is known about its risks of hematological toxicities, rare but clinically serious and potentially life-threatening adverse events. We want to explore whether nivolumab can increase the risks of hematological toxicities compared with other immunotherapy or chemotherapy drugs. The databases of PubMed, Embase, Web of science, and CNKI were searched. We used the medical subject heading terms "Nivolumab" plus keyword "Nivolumab" to search studies published from August 1990 to October 2021. For the included articles, we calculated the relative risks and the corresponding 95% confidence intervals (CIs) for the risks of anemia, neutropenia, and leukopenia in patients treated with nivolumab versus control drugs. Five original articles on the nivolumab trials were identified with 2399 patients enrolled in this meta-analysis. The relative risks of anemia, neutropenia, and leukopenia were 0.343 (95% CI: 0.177-0.663; P = .001), 0.020 (95% CI: 0.008-0.053; P = .000), and 0.054 (95% CI: 0.015-0.191; P = .000), respectively. The PD-1 inhibitor-nivolumab did not increase the risk of anemia, neutropenia and leukopenia. It may enhance awareness about lower risks of hematological toxicities when choosing nivolumab as PD-1 inhibitor among clinicians.

The association between PD-1 gene polymorphisms and susceptibility to multiple sclerosis

Programmed cell death 1 (PD-1) is an immune checkpoint and has been reported to be associated with several autoimmune diseases. We aimed to investigate the association between human PD-1 gene (PDCD1) polymorphisms and multiple sclerosis (MS). This case-control study was conducted on 229 MS patients and 246 healthy controls. Genotyping of rs36084323 (PD-1.1 G/A), rs11568821 (PD-1.3 G/A) and rs2227981 (PD-1.5 C/T) polymorphisms was performed by PCR-RFLP technique. The frequency difference of PD-1.1 genotypes and alleles (−536 G/A) between patients and healthy controls was not significant. Regarding PD-1.3, the AA + AG genotype was found to be relatively higher in the control group. Concerning PD-1.5 (+7785 C/T), the frequency of T allele carriers (TT + CT) was relatively higher in MS patients, which was marginally insignificant (p = .07). PD-1 gene polymorphisms may be associated with MS; however, accurate conclusions require further studies with a larger number of samples.

Preclinical Characterization of GLS-010 (Zimberelimab), a Novel Fully Human Anti-PD-1 Therapeutic Monoclonal Antibody for Cancer.

BackgroundZimberelimab (GLS-010) is a novel fully human monoclonal immunoglobulin G4 (IgG4) against the programmed cell death-1 (PD-1) receptor.AimTo evaluate the affinity, competitive blocking capability, T cell activation effect, cytotoxic effector functions by Fc, preliminary anti-tumor activity, and pharmacokinetics of GLS-010.MethodsThe affinity of GLS-010 to PD-1 and the ability of GLS-010 to block the PD-L1/2 to PD-1 interaction on the cell surface were measured. An allogeneic mixed lymphocyte reaction was conducted to evaluate the inhibitory effect of GLS-010 on Tregs and stimulatory effect on T cell proliferation and activation. Pharmacodynamics and pharmacokinetics were evaluated in tumor-bearing mice and cynomolgus monkeys, respectively.ResultsThe equilibrium dissociation constant (KD) for the association between GLS-010 and PD-1 was 1.75×10-10 M. GLS-010 could effectively block the binding of PD-L1/2 to PD-1. GLS-010 showed statistically significant anti-tumor effects in the MC38 model in human PD-1 knock-in mice. The RO rate on in the low-, moderate-, and high-dose groups were 64.50%-48.53% in CD3+T, 58.87%-40.12% in CD8+T, and 66.26%-49.07% in CD4+T, respectively. With the increasing dose from 2 mg/kg to 18 mg/kg, the systemic exposure level of GLS-010 (AUC0-last) and C0 increased proportionally, while the proportion of AUC0-last was higher than the proportion of the increase in the dose.ConclusionsAs a fully human anti-PD-1 monoclonal antibody, GLS-010 has a high affinity to PD-1 and shows potent anti-tumor effects in vivo and in vitro. The results support that GLS-010 could be investigated in clinical trials in tumor patients.

1169TiP Penpulimab-based combination neoadjuvant/adjuvant therapy for patients with resectable locally advanced non-small cell lung cancer: A phase II clinical study (ALTER-L043)

Immunotherapy alone or combined with chemotherapy as neoadjuvant therapy has shown encouraging results. Penpulimab, a human IgG1 monoclonal antibody directed against human programmed cell death-1 (PD-1), was engineered to eliminate Fc-mediated effector function that compromises anti-tumor immune cell function, and to optimize receptor occupancy by improving duration of drug binding. As a promising multi-target tyrosine kinase inhibitor, Anlotinib significantly improved overall survival in advanced NSCLC patients in the phase III trial ALTER0303.

Penpulimab in combination with anlotinib as first-line treatment in advanced nonsquamous non-small-cell lung cancer.

e21072 Background: Penpulimab is a human IgG1 monoclonal antibody (mAb) directed against human programmed cell death-1 (PD-1). Penpulimab, with its unique binding epitope, was engineered to eliminate Fc-mediated effector function that compromises anti-tumor immune cell function, and to optimize receptor occupancy by improving duration of drug binding. As a promising multi-target tyrosine kinase inhibitor (TKI), anlotinib significantly improved overall survival in advanced NSCLC patients (pts) in the phase 3 trial ALTER0303. Antiangiogenesis therapy combined with PD-1/PD-L1 inhibitors has shown excellent efficacy in advanced NSCLC pts. This is the trial evaluating chemo-free combination of penpulimab plus anlotinib in treatment-naive advanced NSCLC pts regardless of PD-L1 expression (NCT03866980). Methods: Pts with previously untreated, stage IIIB/IIIC/IV non-squamous NSCLC without sensitizing mutation of the epidermal growth factor receptor (EGFR) gene or translocation of the anaplastic lymphoma kinase (ALK) gene were enrolled. Eligible pts received penpulimab 200mg Q3W in combination with anlotinib 12mg QD (2 weeks on 1 week off) until loss of clinical benefit or unacceptable toxicity. The primary endpoint was progression-free survival (PFS). Secondary endpoints included ORR, disease control rate (DCR), duration of response (DoR) and overall survival. Results: As of January 13, 2021, 26 pts had received the combination therapy of penpulimab plus anlotinib (median age 59.5yrs [range 30-71], 76.9% male, 76.9% ECOG PS 1), with a median treatment duration of 3.5 months. Of 21 pts who have had at least one tumor assessment, the ORR was 57.1% (12 PRs) and DCR was 90.5% (12 PRs, 7 SDs). Median PFS has not been reached, and eleven responders remain in response. Treatment-related adverse events (TRAEs) occurred in 53.8% of pts (≥G3 TRAEs occurred in 15.4% [4/26]). Treatment-related SAEs occurred in 15.4% [4/26], and 7.7% of pts [2/26] had drug interruption or discontinuation due to TRAEs. Most common TRAEs (≥15%) were ALT increased, AST increased, hyperthyroidism and hypertension (15.4% each). Conclusions: The combination of penpulimab plus anlotinib as first-line treatment for locally advanced/metastatic NSCLC showed the promising efficacy with a manageable safety profile, thereby suggesting that this combination therapy may be a viable chemo-free treatment strategy for locally advanced/metastatic NSCLC pts. Clinical trial information: NCT03866980.

P80.01 A Multicenter, Randomized, Phase 3 Trial of Penpulimab in Combination With Anlotinib or Chemotherapy as First-Line Treatment in Advanced NSCLC

Penpulimab, a human immunoglobulin G1 (IgG1) monoclonal antibody (mAb) directed against human programmed cell death-1 (PD-1), was engineered to optimize receptor occupancy by improving duration of drug binding, and to eliminate Fc-mediated effector function that compromises anti-tumor immune cell function. Anlotinib, an antiangiogenic multi-target tyrosine kinase inhibitor. Chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). A combination of the PD-1 checkpoint inhibitor and the antiangiogenic drug may improve clinical response in NSCLC by blocking the accumulation of immunosuppressive cells and promoting influx of effector T cells into tumors.

Biophysical and Immunological Characterization and In Vivo Pharmacokinetics and Toxicology in Nonhuman Primates of the Anti-PD-1 Antibody Pembrolizumab.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.

A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors.

Therapeutic monoclonal antibodies (mAbs), targeting tumor antigens, or immune checkpoints, have demonstrated a remarkable anti-tumor effect against various malignancies. However, high costs for mono- or combination therapies, associated with adverse effects or possible development of resistance in some patients, warrant further development and modification to gain more flexibility for this immunotherapy approach. An attractive alternative to passive immunization with therapeutic antibodies might be active immunization with mimotopes (B-cell peptides) representing the mAbs' binding epitopes, to activate the patient's own anti-tumor immune response following immunization. Here, we identified and examined the feasibility of inducing anti-tumor effects in vivo following active immunization with a mimotope of the immune checkpoint programmed cell death 1 (PD1), alone or in combination with a Her-2/neu B-cell peptide vaccine. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotopes, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effects were evaluated in a syngeneic tumor mouse model stably expressing human Her-2/neu. The identified PD1-derived mimotopes were shown to significantly block the mAbs' capacity in inhibiting the respective PD1/PD-L1 interactions. A significant reduction in tumor growth in vivo was observed following active immunization with the mPD1-derived mimotope, associated with a significant reduction in proliferation and increased apoptotic rates in the tumors. Particularly, combined vaccination with the mPD1-derived mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the vaccine's anti-tumor effect. Our results suggest active immunization with mimotopes of immune checkpoint inhibitors either as monotherapy or as combination therapy with tumor-specific vaccines, as a new strategy for cancer treatment.

Enhanced Therapeutic Efficacy of a Novel Oncolytic Herpes Simplex Virus Type 2 Encoding an Antibody Against Programmed Cell Death 1

The efficacy of immune checkpoint blockade therapy against immunologically “cold” tumors can be enhanced by applying the checkpoint inhibitors in combination with oncolytic viruses. Alternatively, the oncolytic virus construct has been modified to express factors that boost oncolytic virus function. We engineered a novel oncolytic herpes simplex virus 2 (HSV2) encoding an anti-human programmed cell death 1 (PD-1) monoclonal antibody (oHSV2-aPD1). This virus resulted in the detectable expression of a functional monoclonal antibody against human PD-1 by infecting eukaryotic cells. Therapeutic efficacy of oHSV2-aPD1 proved superior to unmodified oncolytic HSV2 treatment or PD-1 blockade alone and as effective as their combination in the poorly immunogenic melanoma models. Additionally, local oHSV2-aPD1 treatment induced a durable antitumor response and activated many immune effector cells and molecules both in the tumor microenvironment and in the systemic immune system. This provides support for combinatorial strategies involving local administration of an oncolytic HSV2 expressing a PD-1 inhibitor.

A small-sized protein binder specific for human PD-1 effectively suppresses the tumour growth in tumour mouse model

Immune checkpoint inhibitors have drawn a consider attention as an effective cancer immunotherapy, and several monoclonal antibodies targeting the immune checkpoint receptors, such as human programmed cell death-1 (hPD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), are clinically used for treatment of various cancers. Here we present the development of a small-sized protein binder which specifically binds to hPD‐1. The protein binder, which is composed of leucine-rich repeat (LRR) modules, was selected against hPD-1 through phage display, and its binding affinity was maturated up to 17 nM by modular evolution approach. The protein binder was shown to be highly specific for hPD-1, effectively inhibiting the interaction between hPD-1 and its ligand, hPD-L1. The protein binder restored T-cell function in vitro, and exhibited a strong anti-tumour activity in tumour mouse model, indicating that it acts as an effective checkpoint blockade. Based on the results, the developed protein binder specific for hPD-1 is likely to find a potential use in cancer immunotherapy.

Histologic patterns of liver injury induced by anti-PD-1 therapy.

BackgroundNivolumab and pembrolizumab—two monoclonal antibodies that block human programmed cell death-1 (PD-1)—have been successfully used to treat patients with multiple advanced malignancies. The histologic patterns of hepatic toxicity induced by anti-PD-1 treatment have not been well studied and the aim of this study was to explore them.MethodsEight patients with advanced malignancies who were treated with either nivolumab or pembrolizumab were identified from five institutions. These patients had no history of underlying liver disease and a viral hepatitis panel was negative in all patients.ResultsSeven of eight patients exhibited mild to moderate gastrointestinal symptoms such as abdominal pain, fatigue, nausea, vomiting, and jaundice after anti-PD-1 treatment. Significant elevations in liver-chemistry tests were detected in all patients. Six cases (6/8) demonstrated an acute lobular hepatitis pattern of histologic injury. The remaining two cases showed different histologic patterns of injury: steatohepatitis with mild cholestasis (1/8) and pure acute cholestatic injury (1/8). No case showed typical features of autoimmune hepatitis. The liver function recovered in all eight cases after cessation of anti-PD-1 agents and with immunosuppressive therapy.ConclusionsOur study suggests that screening patients for abnormal liver-function tests prior to anti-PD-1 therapy as well as periodic monitoring of liver-function tests are necessary to prevent severe liver injury. Rather than causing classical autoimmune hepatitis, PD-1 inhibitors appear to produce an immune-mediated nonspecific acute hepatitis. Drug cessation, without steroid therapy, may therefore be sufficient in some patients.

Computational Redesign of PD-1 Interface for PD-L1 Ligand Selectivity

Chronic or persistent stimulation of the programmed cell death-1 (PD-1) pathway prevents Tcells from mounting anti-tumor and anti-viral immune responses. Blockade of this inhibitory checkpoint pathway has shown therapeutic importance by rescuing Tcells from their exhausted state. Cognate ligands of the PD-1 receptor include the tissue-specific PD-L1 and PD-L2 proteins. Engineering a human PD-1 interface specific for PD-L1 or PD-L2 can provide a specific reagent and therapeutic advantage for tissue-specific disruption of the PD-1 pathway. We utilized ProtLID, a computational framework, which constitutes a residue-based pharmacophore approach, to custom-design a human PD-1 interface specific to human PD-L1 without any significant affinity to PD-L2. In subsequent cell assay experiments, half of all single-point mutant designs proved to introduce a statistically significant selectivity, with nine of these maintaining a close to wild-type affinity to PD-L1. This proof-of-concept study suggests a general approach to re-engineer protein interfaces for specificity.

Abstract 3822: Characterization of the first chicken-derived anti-PD-1 clinical stage antibody with a unique epitope and promising anticancer activity

Abstract Inhibition of immunologic checkpoints like Programmed Cell Death 1 (PD-1) has shown clinical efficacy in a broad range of cancers by improving or restoring T-cell activity. Anti-PD-1 antibodies show great promise in treating cancer malignances when administered alone or in combination with other immune activating approaches. However, high protein sequence identity between human and mammalian species used for antibody generation often disfavor generation of antibodies against functionally conserved epitopes, or prevents isolating antibodies cross reacting with ortholog species used for evaluating potential toxicity. Chickens are phylogenetically distant from mammals and are better at generating antibodies against epitopes that are conserved in mammals. Because chickens generate antibodies from a very restricted set of V-gene germline genes that are diversified by “gene conversion”, we envisioned that high throughput humanization of antibody frameworks was achievable by “mass CDR grafting” after recovering antibodies by immunization and B-cell cloning. Wild type chickens were immunized with PD-1 antigen, and a repertoire of 120 antibodies was generated with Symplex™ technology, by combining single B-cell FACS sorting and high throughput RT-PCR cloning of cognate VH and VL chains. The isolated PD-1 repertoire was cloned with an inert Fc backbone and humanized by a combination of in silico CDR grafting and gene synthesis. Humanized antibodies were expressed and screened for retained binding affinity and functionality in T-cell based assays. We successfully generated a humanized PD-1 antibody repertoire and found that most antibodies retained affinity and functionality similar to that of parental chicken antibodies. Furthermore, the antibody repertoire displayed broad binding epitope coverage on PD-1, often with strong pM affinity, and showed biophysical properties acceptable for drug development. Our lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding and downstream PD-1 signaling, resulting in elevated T-cell cytokine production in vitro. Moreover, Sym021 bound human PD-1 with much stronger affinity of 30 pM compared to clinical PD-1 mAbs nivolumab and pembrolizumab, while also cross reacting to cynomolgous and mouse PD-1. This enabled direct testing of Sym021 in syngenic mouse in vivo models and evaluation of preclinical toxicology in cynomolgus monkeys. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to clinical antibodies pembrolizumab and nivolumab. These results supported entry of PD-1 targeting Sym021 into clinical trials. Citation Format: Torben Gjetting, Monika Gad, Camilla Fröhlich, Maria C. Melander, Gunther Galler, Johan Lantto, Thomas Bouquin, Ivan D. Horak, Michael Kragh, Mikkel W. Pedersen, Klaus Koefoed. Characterization of the first chicken-derived anti-PD-1 clinical stage antibody with a unique epitope and promising anticancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3822.

Abstract 4558: In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091

Abstract The programmed cell death-1 (PD-1) receptor provides inhibitory checkpoint signals to activated T cells upon binding to its ligands, PD-L1 and PD-L2, which are expressed on antigen-presenting cells and cancer cells leading to suppression of T-cell effector function and tumor immune evasion. Blockade of the PD-1 axis using either anti-PD-1 or anti-PD-L1 approved monoclonal antibodies (mAbs) results in improved T-cell effector function and anti-tumor immune responses. Durable tumor responses occur in 15-30% of cancer patients. BI 754091, a humanized IgG4 mAb with high affinity against hPD-1 blocks the interaction between PD-1 and PD-L1 or PD-L2. BI 754091 was characterized in a panel of binding, blocking and functional cell-based assays. In addition, efficacy and safety was assessed in mice and in cynomolgus monkeys, respectively. The ability of BI 754091 to stimulate cytokine production in exhausted human T cells in vitro was tested in an autologous assay system with antigen-specific memory CD4+ T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of BI 754091 or isotype control. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on their surface. Furthermore, PD-L1 and PD-L2 were expressed on the dendritic cells. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure for T-cell activation. BI 754091 showed a potent dose dependent T-cell activation. The average fold increase of IFNγ was 7.9 as compared to isotype control, with an average EC50 of 0.9 nM. The in vivo activity of BI 754091 was determined in MC-38 tumor-bearing mice, using a mouse strain where parts of the extracellular domain of murine PD-1 was replaced by the corresponding human PD-1 domain (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice). A dose of 10 mg/kg BI 754091, given either as single treatment or in a twice weekly schedule, induced significant tumor growth inhibition (median TGI of 83% and 90%, respectively) and complete responses (CRs) in some tumors (3 CRs out of 10 and 2 CRs out of 10, respectively). BI 754091 binds to PD-1 from cynomolgus monkeys with comparable affinities as to human PD-1, thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754091 were well tolerated without adverse immune-related effects. BI 754091 is currently undergoing clinical investigations (NCT02952248). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Iñigo Tirapu, Sven Mostböck, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jürgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4558.