Binding Of Programmed Cell Death-1 Research Articles

The multiple arrays of a PD1-derived peptide on chromatin specifically bind to PD-L1 and induce doxorubicin resistance in cancer cell lines

Programmed cell death-1 (PD-1) and its ligand 1 (PD-L1) have been extensively studied to block the activation of the PD-1 axis immune checkpoint pathway on T cells. However, recent evidence suggests that PD-1-independent PD-L1 pathways in cancer cells and macrophages are important in cellular proliferation and survival of those cell types but the underlying mechanism is little understood. To recapitulate the binding of multiple PD-1 to the high levels of PD-L1 expression on cancer cell membranes, recombinant histones carrying a PD-1 receptor-derived peptide (PDR) were prepared and used to assemble a PDR-displayed nucleosome array. PDR-displayed chromatin showed physiologically spaced nucleosomes, unaffected by N-terminal histone tails and biotinylated DNA modifications. PDR-displayed chromatin exhibited selective binding to the breast cancer cell line with high PD-L1 expression, MDA-MB-231, where saturated binding occurred within 3 h, comparable to well-known antigen-antibody reactions. Notably, PDR-displayed chromatin enhanced resistance to doxorubicin in PD-L1-overexpressed cancer cells, revealing a PD-L1 level-dependent effect on cell survival upon chemotherapy. Our study sheds light on the potential of PDR-displayed chromatin as a tool to induce chemoresistance in cancer cells without employing anti-PD-L1 antibodies or soluble PD-1 receptors. Further investigation into the underlying signaling pathways and therapeutic applications is warranted, positioning PDR-displayed chromatin as a valuable tool for understanding PD-L1-mediated intracellular signaling pathways in cancer cells with high PD-L1 expression.

Unveiling the Etiopathogenic Spectrum of Hypophysitis: A Narrative Review.

Hypophysitis, a rare inflammatory disorder of the pituitary gland, has seen an uptick in reported cases in recent years. Our objective is to summarize the most recent research on the etiopathogenesis, molecular mechanisms, and genetics of both primary and secondary hypophysitis. Primary autoimmune hypophysitis (PAH): During the acute phase of the disease, the pituitary gland in enlarged due to the infiltration of T and B lymphocytes. The chronic phase is characterized by progressive and irreversible pituitary atrophy. APA may play a role in the management, diagnosis, and prognosis of PAH. Specific autoantibodies such as anti-GH, anti-PIT-1, and anti-T-PIT have been found in patients with hypophysitis and hypopituitarism. A recent study suggested that a mechanism of escaping clonal deletion and mounting an immune response against self antigens can explain the unusual nature of the immune response observed in PAH patients. A cytokine array shows the presence of gamma-interferon and interleukin-17. Patients carrying mutations in the PIT1 or PROP1 genes may present PAH. Individuals carrying the HLA DQ8 haplotype are four times more likely to develop PAH. Immune checkpoint inhibitors induce hypophysitis (IIHs): IIHs is an increasingly frequent toxicity of in patients on treatment with inhibitors targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death-1 (PD-1). ICIs inhibit the CTLA-4 pathway, leading to overactivation of T lymphocytes. The binding of PD-1/PD-L1 suppresses the activity of T cells, promotes the conversion of T-helpers into T-regulatory cells, and activates pro-survival signaling pathways in cancer cells. Cytokines play a crucial role in IIHs. B-cell infiltration has been observed in IIHs, suggesting that antibody-mediated pituitary injury may contribute. Genetic polymorphisms of CTLA-4 and PD-1 genes can increase the risk of IIHs. HLA alleles may also be involved in the onset of IIHs; this HLA association presents a possible alternative mechanistic hypothesis. IIHs may also be linked to a paraneoplastic syndrome triggered by ectopic expression of pituitary specific antigens. SARS-CoV-2-related hypophysitis: Recently, the literature has reported occurrences of hypophysitis associated with the SARS-CoV-2 virus; long COVID-19 may also present as infundibulo-neuro-hypophysitis. The virus enters the central nervous system because of its distinct interaction with angiotensin-converting enzyme receptors via spike proteins binding the capillary endothelium, and it directly damages the pituitary cells. The effect of SARS-CoV-2 can occur indirectly through inflammation and the release of cytokines. The exact mechanism remains ambiguous. The available data on endocrine complications associated with the SARS-CoV-2 vaccine are scant. Nonetheless, isolated cases of hypophysitis have been documented. Treatment of hypophysitis: Glucocorticoids are the cornerstone in managing primary hypophysitis, given their targeted action on inflammation. A better understanding of the etiopathogenesis and molecular mechanism of hypophysitis can lead to more effective and personalized treatment strategies.

Regulation of PD-L1 Expression by Nuclear Receptors.

The suppression of excessive immune responses is necessary to prevent injury to the body, but it also allows cancer cells to escape immune responses and proliferate. Programmed cell death 1 (PD-1) is a co-inhibitory molecule that is present on T cells and is the receptor for programmed cell death ligand 1 (PD-L1). The binding of PD-1 to PD-L1 leads to the inhibition of the T cell receptor signaling cascade. PD-L1 has been found to be expressed in many types of cancers, such as lung, ovarian, and breast cancer, as well as glioblastoma. Furthermore, PD-L1 mRNA is widely expressed in normal peripheral tissues including the heart, skeletal muscle, placenta, lungs, thymus, spleen, kidney, and liver. The expression of PD-L1 is upregulated by proinflammatory cytokines and growth factors via a number of transcription factors. In addition, various nuclear receptors, such as androgen receptor, estrogen receptor, peroxisome-proliferator-activated receptor γ, and retinoic-acid-related orphan receptor γ, also regulate the expression of PD-L1. This review will focus on the current knowledge of the regulation of PD-L1 expression by nuclear receptors.

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

Novel phthalimides regulating PD-1/PD-L1 interaction as potential immunotherapy agents.

Programmed cell death 1(PD-1)/programmed cell death ligand 1(PD-L1) have emerged as one of the most promising immune checkpoint targets for cancer immunotherapy. Despite the inherent advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has fallen behind that of antibody drugs. Based on docking studies between small molecule inhibitor and PD-L1 protein, changing the chemical linker of inhibitor from a flexible chain to an aromatic ring may improve its binding capacity to PD-L1 protein, which was not reported before. A series of novel phthalimide derivatives from structure-based rational design was synthesized. P39 was identified as the best inhibitor with promising activity, which not only inhibited PD-1/PD-L1 interaction (IC50=8.9nmol/L), but also enhanced killing efficacy of immune cells on cancer cells. Co-crystal data demonstrated that P39 induced the dimerization of PD-L1 proteins, thereby blocking the binding of PD-1/PD-L1. Moreover, P39 exhibited a favorable safety profile with a LD50>5000mg/kg and showed significant invivo antitumor activity through promoting CD8+ T cell activation. All these data suggest that P39 acts as a promising small chemical inhibitor against the PD-1/PD-L1 axis and has the potential to improve the immunotherapy efficacy of T-cells.

DELIVERY OF ANTI-PD-1 GENE WITH RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV) AS PREVENTIVE AND CURATIVE THERAPY OF INFECTIOUS DISEASES IN CHILDHOOD : LITERATURE REVIEW

Infections in children are common and are the highest cause of hospitalization in children, especially in children aged 0-4 years. The binding of PD-1 (Programmed Cell Death-1) glycoprotein on its ligand in CD4+ and CD8+ T cells activates a pathway that results in T cell dysfunction. Inserting the anti-PD1 gene into rAAV opens opportunities for preventing and treating infections in children. This literature review aims to determine the potential of anti-PD1 gene rAAV as a new modality for preventing and treating pathogen infections in children. The procedure of searching for literature to answer questions was carried out through online searching of journals in the last ten years. The use of anti-PD-1 has shown to increase the immune response against certain viral, bacterial and parasitic infections. Using rAAV as an anti-PD-1 gene vector has great potential to be a preventive or curative therapy for various infections in children. Further research and development are needed to determine the viral model, dose, indications, and contraindications to the use of the rAAV-transmitted anti-PD-1 gene for the treatment of infectious diseases in children.

Finding and characterizing a catalytic antibody light chain, H34, capable of degrading the PD-1 molecule.

Programmed cell death 1 (PD-1) is an immune checkpoint molecule regulating T-cell function. Preventing PD-1 binding to its ligand PD-L1 has emerged as an important tool in immunotherapy. Here, we describe a unique human catalytic antibody light chain, H34, which mediates enzymatic degradation of human PD-1 peptides and recombinant human PD-1 protein and thus functions to prevent the binding of PD-1 with PD-L1. H34 degraded one half of the PD-1 molecules within about 6 h under the experimental conditions. Investigating the acquisition of the catalytic function by H34, which belongs to subgroup I and lacks a Pro95 residue in CDR-3, revealed the importance of this sequence, as a Pro95-reconstituted mutant (H34-Pro95(+)) exhibited very little catalytic activity to cleave PD-1. Interestingly, EDTA inhibited the catalytic activity of H34, which could work as a metallo-protease. Zn2+ or Co2+ ions may work as a cofactor. It is meaningfull that H34 was obtained from the human antibody gene taken from a healthy volunteer, suggesting that we potentially have such unique molecules in our body.

Tumor-derived exosomes in the PD-1/PD-L1 axis: Significant regulators as well as promising clinical targets.

Programmed cell death-1 (PD-1) is a negative coreceptor mainly expressed on the surface of activated T cells. The binding of PD-1 to its ligand PD-L1 significantly induces non-reactivity of T cells to maintain the balance of autoimmunity and immune tolerance. It is reported that tumor cells highly express PD-L1 to restrict cellular immune response, which is one of the most important mechanisms for tumor to mediate immune escape. Cancer immunotherapy targeting PD-1/PD-L1 has achieved remarkable success so far. Tumor-derived exosomes (TEXs) are lipid bilayer vesicles released by tumor cells in an endosome-dependent manner, mediating communication between tumor cells and adjacent cells in the tumor microenvironment. Through signals transmitted by TEXs, tumor can alter the biological characteristics of these cells to promote tumor growth and metastasis. Recent studies have demonstrated that TEXs not only carry tumor-derived PD-L1, but are also closely related to PD-1/PD-L1 expression on target cells. The primary focus of this review will be on how TEXs regulate the PD-1/PD-L1 axis to promote tumor progression, and the promising clinical applications targeting TEXs and exosomal PD-L1.

Dual-function chimeric antigen receptor T cells targeting c-Met and PD-1 exhibit potent anti-tumor efficacy in solid tumors.

Purpose Programmed cell death 1 (PD-1), which is upregulated under the continuous induction of the tumor microenvironment, causes chimeric antigen receptor (CAR)-T cell hypofunction via interaction with programmed death ligand 1 (PD-L1). This study aimed to construct CAR-T cells that are resistant to PD-1 inhibition to improve the effect of CAR-T cells in solid tumors. Methods We constructed a type of dual-function CAR-T cell that targets tumor-associated antigen c-Met and blocks the binding of PD-1 with PD-L1. The expression of c-Met, PD-L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR-T cells were determined using lactate dehydrogenase release assay, enzyme-linked immunosorbent assay, and flow cytometry, respectively. The levels of p-Akt, p-MAPK, caspase-3, and Bcl2 were detected by western blot. The in vivo anti-tumor effect was evaluated using tumor xenograft models. Results Dual-function CAR-T cells could mediate enhanced active signals upon encountering target antigens and had targeted cytotoxicity to target cells. However, the cytotoxicity of c-Met-CAR-PD-1+ T cells was impaired due to the interaction of PD-1 with PD-L1. By blocking the binding of PD-1 and PD-L1, the novel dual-function CAR-PD-1+ T cells could maintain cytotoxicity to PD-L1+ tumor cells. In tumor tissue, the dual-function CAR-T cells showed lower inhibitory receptor expression and lower differentiation characteristics, which resulted in potent anti-tumor effects and prolonged survival in PD-L1+ tumor xenograft models compared to single-target CAR-T cells. Conclusion These results confirm that the novel dual-function CAR-T cells exhibit stronger anti-tumor activity against solid tumors than traditional single-target CAR-T cells and present a new approach that enhance the activity of CAR-T cells in solid 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.

Construction and expression of chimeric antigen receptors targeting epidermal growth factor receptor (EGFR) and programmed cell death ligand-1(PD-L1)

Objective To construct an expression system of lentivirus vector encoding epidermal growth factor receptor-specific chimeric antigen receptor (EGFR-CAR) and programmed cell death ligand-1 (PD-L1) antibody. Methods Human PD-L1-Fc protein was used to immunize BALB/c mice. Cell-fusion and subcloning were performed to screen stable hybridoma strains with high secretion of PD-L1-specific antibodies, which were identified by both ELISA and Western blot. The activity of the antibodies in blocking the binding of programmed cell death-1 (PD-1) to PD-L1 was determined by fluorescence-activated cell sorting (FACS). Antibody affinity was analyzed by Fortebio Octet96. A single-chain variable fragment (scFv) was further constructed after antibody full-length sequencing and humanization using CDR grafting method. Meanwhile, the genes encoding the light and heavy chain variable regions (VL and VH) were cloned from a hybridoma secreting antibody against human EGFR by 5′ RACE technology to construct scFv gene. The expression of scFv was confirmed using pcDNA3.1 vector. EGFR-CAR containing CD137 intracellular function domain and PD-L1-scFv was ligated using 2A gene. The synthetic single molecule was cloned into pLVX-EF1a-IRES-ZsGreen1 lentivirus expression vector, and then transfected into 293T cells using Lenti-X Packaging Single Shots (VSV-G) to prepare infectious virus. Expression of CAR on cell surface and the soluble form of PD-L1-scFv in the supernatant of transfected 293V cells were detected by FACS and ELISA. Results A PD-L1 antibody named 11E3 with high ligand-receptor blocking performance was obtained. The humanized antibody showed a stable affinity (2.67×10-10 mol/L) after directly grafting the mouse CDRs (CDR1, CDR2 and CDR3) to human frameworks. EGFR-scFv was effectively expressed in a form of Fc-fusion. Secretory CAR (CTZ0431-1) and membrane CAR (CTZ0431-2) expression plasmids were constructed using lentivirus vector containing EGFR-CAR and PD-L1-scFv. The infection efficiency in 293V cells was around 10%. EGFR-scFv on the cell membranes and PD-L1-scfv in the culture supernatants were detected after 293V cells were infected with CTZ0431-1. EGFR-scFv and PD-L1-scfv were expressed on the cell membranes of 293V cells infected with CTZ0431-2. The expression rate of CAR in LV-CART46407-1-transfected activated T cells was 39.3%. Conclusions The lentivirus vectors co-expressing EGFR-CAR with moderate binding affinity and PD-L1-scFv with high binding affinity were successful constructed, which provided an essential tool for investing EGFR- and PD-L1 double targeted CAR-T cell therapy against solid tumor. Key words: EGFR-CAR; PD-L1-scFv; Membrane expression; Secretory expression; Synthetic biotechnology

Aberrant upregulation of PDK1 in ovarian cancer cells impairs CD8+ T cell function and survival through elevation of PD-L1

ABSTRACTBlockade of the programmed cell death 1(PD-1)/PD-1 ligand-1(PD-L1) pathway has been exploited therapeutically in many cancer types. Upregulation of PD-L1 in tumor cells contributes to malignancy through suppression of the T cell-mediated antitumor response. Pyruvate dehydrogenase kinase 1 (PDK1), a glycolytic gate-keeping enzyme, is also known to promote tumor development. Here, we have uncovered a mechanism of regulation of PD-L1 by PDK1 through activation of c-Jun-NH2-kinase (JNK)-c-Jun in ovarian cancer cells. Elevated PDK1 expression was correlated with that of PD-L1 in the TCGA ovarian cancer dataset and ovarian cancer tissue array. Overexpression of PDK1 in ovarian cancer cells impaired CD8+ T cell function by suppressing IFN-γ secretion through the PD-1/PD-L1 pathway. Conversely, knockdown of PDK1 in ovarian cancer cells relieved suppression of CD8+ T cell function. CD8+ T cell apoptosis induced by binding of PD-1 with PD-L1 was increased after co-culture with ovarian cancer cells overexpressing PDK1, while depletion of PDK1 exerted the opposite effect. In vivo experiments revealed synergistic improved overall survival and enhanced inhibition of tumor growth upon co-treatment with dichloroacetate (DCA), a PDK inhibitor, and PD-L1 antibody, accompanied by increased IFN-γ secretion by monocytes infiltrating tumor islets. Moreover, PDK1 expression and CD8+ T cell infiltration were inversely correlated in the ovarian cancer tissue array. Our collective findings provide a novel explanation of how PDK1 contributes to upregulation of PD-L1 in ovarian cancer and highlight its potential as a target therapeutic molecule that cooperates with the immune checkpoint blockade.

A new tool in the cancer immunotherapy toolbox?

Programmed cell death-1 (PD-1), a member of the CD28 superfamily, is primarily expressed on the surface of T cells. The normal function of PD-1 is to dampen the immune activity and promote self-tolerance of activated CD8 + cytotoxic T cells by suppressing T cell inflammatory reactions. Binding of PD-1 with its ligand PD-L1 (or B7-H1) or PD-L2 (or B7-DC) excessively leads to the exhaustion of CD8 + cytotoxic T cells (1). In most of patients with advanced cancers, CD8 + cytotoxic T cells are exhausted owing to the engagement of PD-1 by the aberrantly expressed PD-L1 on tumor cells and/or nurse cells in the tumor microenvironment.

An optimized protocol for the in vitro generation and functional analysis of human PD1/PD-L1 signal

Programmed cell death-1 (PD1) is an inhibitory receptor expressed on the activated T and B cells. Binding of PD1 to its ligands, PD-L1 and PD-L2 has led to deliver an inhibitory signal into the activated T cells. Recently, blocking PD1/PD-L1 pathway has emerged as a new treatment paradigm across a broad spectrum of malignancies. Remarkable clinical responses of monoclonal antibodies specific for PD-1 or its ligands in patients with many different types of cancer, attracted several pharmaceutical companies and researchers to investigate the agents that block PD1/PD-L1 signal. The safety and efficacy of the agents are needed to examine in the preclinical studies. In this study, we optimized a facile and cost-effective protocol for in vitro generation and functional analysis of human PD1/PD-L1 pathway. Activation of CD8 + CD279 + T cell was performed by anti-CD3 and D28 antibodies and the recombinant PD-L1 was used for inactivation of T cells through PD1/PD-L1 pathway. In this protocol, T-cell cytokine production (IL-2 and IFN-γ) and proliferation assay confirmed that a measurable PD1/PD-L1 signal was generated. We expected that in vitro PD1/PD-L1 signal that has been optimized in this study will serve as a valuable protocol for preclinical studies involving PD1/PD-L1 pathway.

Abstract 1484: Combined treatment with anti-LAG-3 and anti-PD-1 fully human monoclonal antibodies inhibits tumor growth in immunocompetent double-humanized LAG-3/PD-1 mice

Abstract Lymphocyte-activation gene 3 (LAG-3) receptor is expressed on activated CD4 and CD8 T cells, γδ T cells, Treg, NK, NKT, B and plasmacytoid dendritic cells. LAG-3 binds to major histocompatibility complex class II (MHC II) and delivers inhibitory signals that regulate T cell proliferation and cytokine production. LAG-3 blockade augments T cell proliferation and activation. Programmed cell death 1 (PD-1) receptor upon binding to its ligands PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273) also delivers an inhibitory checkpoint signal that is critical for the establishment and maintenance of peripheral T cell tolerance. PD-1 signaling plays a critical role in the tumor microenvironment by allowing tumors to escape immune surveillance. We tested in vivo activity of anti-mouse PD-1 and anti-mouse LAG-3 antibodies in several preclinical syngeneic tumor models and showed that combination treatment with both antibodies resulted in an additive anti-tumor effect compared to either single antibody treatment. TaqMan analysis in the MC38 tumor model demonstrated CD8+ T cell expansion in both the draining lymph nodes and spleens of mice in the combination treatment group. Anti-human LAG-3 antibody is a fully human monoclonal antibody developed for cancer immunotherapy. It binds with high affinity to human LAG-3 and blocks LAG-3/MHC II interaction. Fully human monoclonal antibody REGN2810 binds with high affinity to human PD-1 and blocks PD-1 interaction with PD-L1 and PD-L2. Double humanized LAG-3/PD-1 mice were engineered using VelociGene® technology to replace the extracellular domains of mouse Pdcd1 and Lag3 genes with the corresponding regions of human PD-1 and human LAG-3 genes. To validate humanized protein expression, we examined PD-1 and LAG-3 protein expression on T cells after anti-CD3/anti-CD28 antibody stimulation. We confirmed binding of human LAG-3 and PD-1 to the corresponding mouse ligands by cell adhesion assay for human LAG-3 and mouse MHC II interactions and by SPR-Biacore for human PD-1 and mouse PD-L1 interactions, respectively. Combination of REGN2810 and anti-hLAG-3 antibodies in MC38.ova tumor model in double humanized LAG-3/PD-1 mice, which allows testing of clinical antibodies that do not cross to mouse receptors, demonstrated improved efficacy, including reduced tumor growth and improved survival, compared to REGN2810 and anti-hLAG-3 monotherapies. Robust anti-tumor efficacy of REGN2810 and anti-hLAG-3 combination in preclinical setting supports their clinical development as a combination cancer immunotherapy. Citation Format: Elena Burova, Omaira Allbritton, Chandrika Taduriyasas, Venus Lai, William Poueymirou, Nicholas Papadopoulos, Douglas MacDonald, William Olson, Markus Mohrs, Ella Ioffe, Gavin Thurston. Combined treatment with anti-LAG-3 and anti-PD-1 fully human monoclonal antibodies inhibits tumor growth in immunocompetent double-humanized LAG-3/PD-1 mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1484.

77. Clinical Scale Zinc Finger Nuclease (ZFN)-Driven Gene-Editing of PD-1 in Tumor Infiltrating Lymphocytes (TIL) for the Potential Treatment of Metastatic Melanoma

Multiple inhibitory pathways are exploited by a number of cancers to block the body's immune response which may limit the effectiveness of adoptive cell transfer (ACT). Programmed cell death-1 (PD-1) is a member of the CD28 superfamily and is expressed on activated T cells. Importantly, its ligands, PDL-1 and PDL-2 are expressed on a variety of tumor cells, including melanoma. The binding of PD-1 to PDL-1 inhibits T cell effector function, and represents an important mechanism for PDL-1 expressing tumors to evade the host immune response to cancer. PD-1 thus represents an attractive target for gene-editing of tumor targeted T cells prior to ACT. Here we describe the elimination of PD-1 expression in tumor infiltrating lymphocytes (TIL) by genome-editing using zinc finger nucleases (ZFNs) directed against the PDCD1 gene at a scale sufficient for patient treatment. Using the MaxCyte GT Flow Transfection System to deliver mRNA encoding the PD-1 specific ZFNs, the clinical scale TIL production process yielded efficient modification of the PDCD1 gene locus (mean = 74.8% of alleles, n=3, range = 69.9 – 84.1%), which resulted in a 76% reduction in PD-1 surface-expression. Importantly, the PD-1 modified TIL products displayed an effector memory phenotype and expanded approximately 500 – 2000 fold during a rapid cell expansion. In addition, PD-1 modified and unmodified TILs showed equivalent levels of engraftment in a NSG-mouse model. PD-1 modified TILs demonstrated improved in vitro T cell effector functions relative to controls (statistically significant greater TNFα, GM-CSF and IFNγ cytokine secretion when co-cultured with MART-1+ tumor cells). Preliminary safety evaluations showed that PD-1 modified TILs maintained IL-2 dependent growth in vitro, and no tumors were observed in an NSG-mouse tumorigenicity study. These data support the further study of the efficacy and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma.

Clinical scale zinc finger nuclease (ZFN)-driven gene-editing of PD-1 in tumor infiltrating lymphocytes (TIL) for the potential treatment of metastatic melanoma

Multiple inhibitory pathways exist to block the immune response to cancer potentially limiting the effectiveness of adoptive cell transfer (ACT). Programmed cell death-1 (PD-1) is a member of the CD28 superfamily and is expressed on activated T cells. Its ligands, PDL-1 and PDL-2 are expressed on a variety of tumor cells, including melanoma. The binding of PD-1 to PDL-1 inhibits T cell effector function, and represents an important mechanism for PDL-1 expressing tumors to evade the host immune response to cancer. PD-1 thus represents an attractive target for gene-editing of tumor-targeted T cells prior to ACT. To this end, our aim was to eliminate PD-1 expression in tumor infiltrating lymphocytes (TIL) by genome-editing using zinc finger nucleases (ZFNs) directed against the PD-1 gene at a scale sufficient for patient treatment. Using the MaxCyte GT Flow Transfection System to deliver mRNA encoding the PD-1 ZFNs, we show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9 - 84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Importantly, the PD-1 gene-edited TIL product displayed an effector memory phenotype and expanded approximately 500 - 2000 fold during a rapid cell expansion in vitro while retaining T cell effector function. Thus further study to determine the safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.