Comparative transcriptome analysis between rhesus macaques ( Macaca mulatta) and crab-eating macaques ( M. fascicularis).

Understanding gene expression variations between species is pivotal for deciphering the evolutionary diversity in phenotypes. Rhesus macaques ( Macaca mulatta, MMU) and crab-eating macaques ( M. fascicularis, MFA) serve as crucial nonhuman primate biomedical models with different phenotypes. To date, however, large-scale comparative transcriptome research between these two species has not yet been fully explored. Here, we conducted systematic comparisons utilizing newly sequenced RNA-seq data from 84 samples (41 MFA samples and 43 MMU samples) encompassing 14 common tissues. Our findings revealed a small fraction of genes (3.7%) with differential expression between the two species, as well as 36.5% of genes with tissue-specific expression in both macaques. Comparison of gene expression between macaques and humans indicated that 22.6% of orthologous genes displayed differential expression in at least two tissues. Moreover, 19.41% of genes that overlapped with macaque-specific structural variants showed differential expression between humans and macaques. Of these, the FAM220A gene exhibited elevated expression in humans compared to macaques due to lineage-specific duplication. In summary, this study presents a large-scale transcriptomic comparison between MMU and MFA and between macaques and humans. The discovery of gene expression variations not only enhances the biomedical utility of macaque models but also contributes to the wider field of primate genomics.

Full-length 16S rDNA sequencing based on Oxford Nanopore Technologies revealed the association between gut-pharyngeal microbiota and tuberculosis in cynomolgus macaques.

Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex (Mtbc), which develops from asymptomatic latent TB to active stages. The microbiome was purposed as a potential factor affecting TB pathogenesis, but the study was limited. The present study explored the association between gut-pharyngeal microbiome and TB stages in cynomolgus macaques using the full-length 16S rDNA amplicon sequencing based on Oxford Nanopore Technologies. The total of 71 macaques was divided into TB (-) control, TB (+) latent and TB (+) active groups. The differential abundance analysis showed that Haemophilus hemolyticus was decreased, while Prevotella species were increased in the pharyngeal microbiome of TB (+) macaques. In addition, Eubacterium coprostanoligenes in the gut was enriched in TB (+) macaques. Alteration of these bacteria might affect immune regulation and TB severity, but details of mechanisms should be further explored and validated. In summary, microbiota may be associated with host immune regulation and affect TB progression. The findings suggested the potential mechanisms of host-microbes interaction, which may improve the understanding of the role of microbiota and help develop therapeutics for TB in the future.

The HER2-directed antibody-drug conjugate DHES0815A in advanced and/or metastatic breast cancer: preclinical characterization and phase 1 trial results.

Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.

The multilevel extensive diversity across the cynomolgus macaque captured by ultra-deep adaptive immune receptor repertoire sequencing.

The extent to which AIRRs differ among and within individuals remains elusive. Via ultra-deep repertoire sequencing of 22 and 25 tissues in three cynomolgus macaques, respectively, we identified 84 and 114 novel IGHV and TRBV alleles, confirming 72 (85.71%) and 100 (87.72%) of them. The heterogeneous V gene usage patterns were influenced, in turn, by genetics, isotype (for BCRs only), tissue group, and tissue. A higher proportion of intragroup shared clones in the intestinal tissues than those in other tissues suggests a close intra-intestinal adaptive immunity network. Significantly higher mutation burdens in the public clones and the inter-tissue shared IgM and IgD clones indicate that they might target the shared antigens. This study reveals the extensive heterogeneity of the AIRRs at various levels and has broad fundamental and clinical implications. The data generated here will serve as an invaluable resource for future studies on adaptive immunity in health and diseases.

MHC-DRB alleles with amino acids Val11, Phe13, and the shared epitopes promote collagen-induced arthritis and a rapid IgG1 response in Filipino cynomolgus macaques.

Macaques are useful animal models for studying the pathogenesis of rheumatoid arthritis (RA) and the development of anti-rheumatic drugs. The purpose of this study was to identify the major histocompatibility complex (MHC) polymorphisms associated with the pathology of collagen-induced arthritis (CIA) and anti-collagen IgG induction in a cynomolgus macaque model, as MHC polymorphisms affect the onset of CIA in other animal models. Nine female Filipino cynomolgus macaques were immunized with bovine type II collagen (b-CII) to induce CIA, which was diagnosed clinically by scoring the symptoms of joint swelling over 9 weeks. MHC polymorphisms and anti-b-CII antibody titers were compared between symptomatic and asymptomatic macaques. Four of 9 (44%) macaques were defined as the CIA-affected group. Anti-b-CII IgG in the affected group increased in titer approximately 3 weeks earlier compared with the asymptomatic group. The mean plasma IgG1 titer in the CIA-affected group was significantly higher (p < 0.05) than that of the asymptomatic group. Furthermore, the cynomolgus macaque MHC (Mafa)-DRB1*10:05 or Mafa-DRB1*10:07 alleles, which contain the well-documented RA-susceptibility five amino acid sequence known as the shared epitope (SE) in positions 70 to 74, with valine at position 11 (Val11, V11) and phenylalanine at position 13 (Phe13, F13), were detected in the affected group. In contrast, no MHC polymorphisms specific to the asymptomatic group were identified. In conclusion, the presence of V11 and F13 along with SE in the MHC-DRB1 alleles seems essential for the production of IgG1 and the rapid induction of severe CIA in female Filipino cynomolgus macaques.

Adaptation of HLA testing to characterize the cynomolgus macaque MHC polymorphisms and alloantibody signatures.

Nonhuman primates are the closest animal models to humans with respect to genetics and physiology. Consequently, a critical component of immunogenetics research relies on drawing inferences from the cynomolgus macaque to inform human trials. Despite the conserved organization of the Major Histocompatibility Complex (MHC) between cynomolgus macaques and humans, MHC genotyping of cynomolgus macaques is challenging due to high rates of copy number variants, duplications, and rearrangements, particularly at the MHC class I loci. Furthermore, the limited availability of commercial reagents specific to cynomolgus macaques that can be used to characterize anti-MHC class I and class II antibody (Ab) specificities in cynomolgus macaques presents a major bottleneck in translational research. Here we successfully characterized cynomolgus macaque Mafa class I and class II serologic specificities in 86 animals originating from various geographical regions using the complement dependent cytotoxicity (CDC) assay with human HLA class I and class II monoclonal antibody (mAb) typing trays. Further, we successfully induced and characterized anti-Mafa class I and class II alloantibody specificity using HLA single antigen bead assays. We also subsequently tracked the alloAb burden in the animals during treatment with anti-B lymphocyte stimulator (BLyS) treatment. Altogether, these methods can be easily used in translational research to serotype MHC class I and class II specificity in macaques, characterize their alloAb specificity, and evaluate the efficacy of novel therapeutic modalities in depleting circulating alloAbs in these animals.

Characterizing bacterial and fungal communities along the longitudinal axis of the intestine in cynomolgus monkeys.

IMPORTANCE: Gut microbiota varies along the gastrointestinal (GI) tract and exerts profound influences on the host's physiology, immunity, and nutrition. Given that gut microbes interact with the host closely and the gastrointestinal function differed from the small to the large intestine, it is essential to characterize the gut biogeography of the microbial community. Here, we focused on intestinal bacteria and fungi in cynomolgus monkeys and determined their spatial distribution along the GI tract by performing 16S and 18S rRNA gene sequencing. The composition and function of bacterial and fungal communities differed significantly at different biogeographic sites of the intestine, and the site-specific correlations between intestinal bacteria and fungi were revealed. Thus, our studies characterized the gut biogeography of bacteria and fungi in NHPs and revealed their site-specific correlations along the GI tract.

Comparative analysis of gut microbiota between common (Macaca fascicularis fascicularis) and Burmese (M. f. aurea) long-tailed macaques in different habitats.

The environment has an important effect on the gut microbiota-an essential part of the host's health-and is strongly influenced by the dietary pattern of the host as these together shape the composition and functionality of the gut microbiota in humans and other animals. This study compared the gut microbiota of Macaca fascicularis fascicularis and M. f. aurea in mangrove and island populations using 16S rRNA gene sequencing on a nanopore platform to investigate the effect of the environment and/or diet. The results revealed that the M. f. fascicularis populations that received anthropogenic food exhibited a higher richness and evenness of gut microbiota than the M. f. aurea populations in different habitats. Firmicutes and Bacteroidetes were the two most abundant bacterial phyla in the gut microbiota of both these subspecies; however, the relative abundance of these phyla was significantly higher in M. f. aurea than in M. f. fascicularis. This variation in the gut microbiota between the two subspecies in different habitats mostly resulted from the differences in their diets. Moreover, the specific adaptation of M. f. aurea to different environments with a different food availability had a significant effect on their microbial composition.

Genotyping of Macaque Population on the Brain-Derived Neurotrophic Factor Gene Polymorphism by Mismatch Amplification Mutation Assay (MAMA)-PCR.

Human rs6265 (196G>A) polymorphism in the BDNF gene is associated with many clinically significant phenotypic manifestations. Rhesus monkey (Macaca mulatta) has a functionally significant rs309950446 ( 136G>A) polymorphism. To determine this polymorphism in macaques, we used mismatch amplification mutation assay (MAMA)-PCR method with non-complementary nucleotide to the template chain at the 3rd position from the 3'-end of the allele-specific primers (mismatch primers), which allowed the best discrimination of the alleles. Genotyping of male rhesus monkeys (n=178) and cynomolgus monkeys (Macaca fascicularis) (n=90) was carried out. The A/A, G/G, and G/A genotypes were found in 16, 34, and 50% rhesus macaques, respectively. In the cynomolgus macaques, the mutant polymorphic allele was not detected. The study results allow considering rhesus macaques as a potential biological model for assessment of the gen-environment interaction of the BDNF gene polymorphism.

Genome sequencing and application of Taiwanese macaque Macaca cyclopis.

Formosan macaque (Macaca cyclopis) is the only non-human primate in Taiwan Island. We performed de novo hybrid assembly for M. cyclopis using Illumina paired-end short reads, mate-pair reads and Nanopore long reads and obtained 5065 contigs with a N50 of 2.66 megabases. M. cyclopis contigs > = 10 kb were assigned to chromosomes using Indian rhesus macaque (Macaca mulatta mulatta) genome assembly Mmul_10 as reference, resulting in a draft of M. cyclopis genome of 2,846,042,475 bases, distributed in 21 chromosomes. The draft genome contains 23,462 transcriptional origins (genes), capable of expressing 716,231 exons in 59,484 transcripts. Genome-based phylogenetic study using the assembled M. cyclopis genome together with genomes of four other macaque species, human, orangutan and chimpanzee showed similar result as previously reported. However, the M. cyclopis species was found to diverge from Chinese M. mulatta lasiota about 1.8 million years ago. Fossil gene analysis detected the presence of gap and pol endogenous viral elements of simian retrovirus in all macaques tested, including M. fascicularis, M. m. mulatta and M. cyclopis. However, M. cyclopis showed ~ 2 times less in number and more uniform in chromosomal locations. The constrain in foreign genome disturbance, presumably due to geographical isolation, should be able to simplify genomics-related investigations, making M. cyclopis an ideal primate species for medical research.

Liver injury in cynomolgus monkeys following intravenous and intrathecal scAAV9 gene therapy delivery.

Hepatotoxicity associated with intravenous/intrathecal adeno-associated virus (AAV) gene therapy has been observed in preclinical species and patients. In nonhuman primates, hepatotoxicity following self-complementary AAV9 administration varies from asymptomatic transaminase elevation with minimal to mild microscopic changes to symptomatic elevations of liver function and thromboinflammatory markers with microscopic changes consistent with marked hepatocellular necrosis and deteriorating clinical condition. These transient acute liver injury marker elevations occur from 3-4 days post intravenous administration to ∼2 weeks post intrathecal administration. No transaminase elevation or microscopic changes were observed with intrathecal administration of empty capsids or a "promoterless genome" vector, suggesting that liver injury after cerebrospinal fluid dosing in nonhuman primates is driven by viral transduction and transgene expression. Co-administration of prednisolone after intravenous or intrathecal dosing did not prevent liver enzyme or microscopic changes despite a reduction of T lymphocyte infiltration in liver tissue. Similarly, co-administration of rituximab/everolimus with intrathecal dosing failed to block AAV-driven hepatotoxicity. Self-complementary AAV-induced acute liver injury appears to correlate with high hepatocellular vector load, macrophage activation, and type 1 interferon innate virus-sensing pathway responses. The current work characterizes key aspects pertaining to early AAV-driven hepatotoxicity in cynomolgus macaques, highlighting the usefulness of this nonclinical species in that context.

C9orf72 poly(PR) aggregation in nucleus induces ALS/FTD-related neurodegeneration in cynomolgus monkeys.

Poly(PR) is a dipeptide repeat protein comprising proline and arginine residues. It is one of the translational product of expanded G4C2 repeats in the C9orf72 gene, and its accumulation is contributing to the neuropathogenesis of C9orf72-associated amyotrophic lateral sclerosis and/or frontotemporal dementia (C9-ALS/FTD). In this study, we demonstrate that poly(PR) protein alone is sufficient to induce neurodegeneration related to ALS/FTD in cynomolgus monkeys. By delivering poly(PR) via AAV, we observed that the PR proteins were located within the nucleus of infected cells. The expression of (PR)50 protein, consisting of 50 PR repeats, led to increased loss of cortical neurons, cytoplasmic lipofuscin, and gliosis in the brain, as well as demyelination and loss of ChAT positive neurons in the spinal cord of monkeys. While, these pathologies were not observed in monkeys expressing (PR)5, a protein comprising only 5 PR repeats. Furthermore, the (PR)50-expressing monkeys exhibited progressive motor deficits, cognitive impairment, muscle atrophy, and abnormal electromyography (EMG) potentials, which closely resemble clinical symptoms seen in C9-ALS/FTD patients. By longitudinally tracking these monkeys, we found that changes in cystatin C and chitinase-1 (CHIT1) levels in the cerebrospinal fluid (CSF) corresponded to the phenotypic progression of (PR)50-induced disease. Proteomic analysis revealed that the major clusters of dysregulated proteins were nuclear-localized, and downregulation of the MECP2 protein was implicated in the toxic process of poly(PR). This research indicates that poly(PR) expression alone induces neurodegeneration and core phenotypes associated with C9-ALS/FTD in monkeys, which may provide insights into the mechanisms of disease pathogenesis.

Subtyping of Nonhuman Primate-Adapted Cryptosporidium hominis in Macaca Fascicularis and Macaca mulatta in Yunnan Province, Southwestern China.

Background: Cryptosporidium spp. are a type of protozoan parasite responsible for causing diarrheal illness worldwide. They infect a broad range of vertebrate hosts, including both non-human primates (NHPs) and humans. In fact, zoonotic transmission of cryptosporidiosis from NHPs to humans is frequently facilitated by direct contact between the two groups. However, there is a need to enhance the information available on the subtyping of Cryptosporidium spp. in NHPs in the Yunnan province of China. Materials and Methods: Thus, the study investigated the molecular prevalence and species of Cryptosporidium spp. from 392 stool samples of Macaca fascicularis (n = 335) and Macaca mulatta (n = 57) by using nested PCR targeting the large subunit of nuclear ribosomal RNA (LSU) gene. Of the 392 samples, 42 (10.71%) were tested Cryptosporidium-positive. Results: All the samples were identified as Cryptosporidium hominis. Further, the statistical analysis revealed that age is a risk factor for the infection of C. hominis. The probability of detecting C. hominis was found to be higher (odds ratio = 6.23, 95% confidence interval 1.73-22.38) in NHPs aged between 2 and 3 years, as compared with those younger than 2 years. Sequence analysis of the 60 kDa glycoprotein (gp60) identified six (IbA9 n = 4, IiA17 n = 5, InA23 n = 1, InA24 n = 2, InA25 n = 3, and InA26 n = 18) C. hominis subtypes with "TCA" repeats. Among these subtypes, it has been previously reported that the Ib family subtypes are also capable of infecting humans. Conclusion: The findings of this study highlight the genetic diversity of C. hominis infection among M. fascicularis and M. mulatta in Yunnan province. Further, the results confirm that both these NHPs are susceptible to C. hominis infection, posing a potential threat to humans.

Single-nucleus transcriptomics reveals a gatekeeper role for FOXP1 in primate cardiac aging.

Aging poses a major risk factor for cardiovascular diseases, the leading cause of death in the aged population. However, the cell type-specific changes underlying cardiac aging are far from being clear. Here, we performed single-nucleus RNA-sequencing analysis of left ventricles from young and aged cynomolgus monkeys to define cell composition changes and transcriptomic alterations across different cell types associated with age. We found that aged cardiomyocytes underwent a dramatic loss in cell numbers and profound fluctuations in transcriptional profiles. Via transcription regulatory network analysis, we identified FOXP1, a core transcription factor in organ development, as a key downregulated factor in aged cardiomyocytes, concomitant with the dysregulation of FOXP1 target genes associated with heart function and cardiac diseases. Consistently, the deficiency of FOXP1 led to hypertrophic and senescent phenotypes in human embryonic stem cell-derived cardiomyocytes. Altogether, our findings depict the cellular and molecular landscape of ventricular aging at the single-cell resolution, and identify drivers for primate cardiac aging and potential targets for intervention against cardiac aging and associated diseases.

Simulation and social network analysis provide insight into the acquisition of tool behaviour in hybrid macaques.

The pathways through which primates acquire skills are a central focus of cultural evolution studies. The roles of social and genetic inheritance processes in skill acquisition are often confounded by environmental factors. Hybrid macaques from Koram Island (Thailand) provide an opportunity to examine the roles of inheritance and social learning to skill acquisition within a single ecological setting. These hybrids are a cross between tool-using Burmese long-tailed (Macaca fascicularis aurea) and non-tool-using common long-tailed macaques (Macaca fascicularis fascicularis). This population provides an opportunity to explore the roles of social learning and inheritance processes while being able to exclude underlying ecological factors. Here, we investigate the roles of social learning and inheritance in tool use prevalence within this population using social network analysis and simulation. Agent-based modelling (ABM) is used to generate expectations for how social/asocial learning and inheritance structure the patterning in a social network. The results of the simulation show that various transmission mechanisms can be differentiated based on associations between individuals in a social network. The results provide an investigative framework for discussing tool use transmission pathways in the Koram social network. By combining ABM, network analysis, and behavioural data from the field we can investigate the roles social learning and inheritance play in tool acquisition of wild primates.

Brain-wide and cell-specific transcriptomic insights into MRI-derived cortical morphology in macaque monkeys.

Integrative analyses of transcriptomic and neuroimaging data have generated a wealth of information about biological pathways underlying regional variability in imaging-derived brain phenotypes in humans, but rarely in nonhuman primates due to the lack of a comprehensive anatomically-defined atlas of brain transcriptomics. Here we generate complementary bulk RNA-sequencing dataset of 819 samples from 110 brain regions and single-nucleus RNA-sequencing dataset, and neuroimaging data from 162 cynomolgus macaques, to examine the link between brain-wide gene expression and regional variation in morphometry. We not only observe global/regional expression profiles of macaque brain comparable to human but unravel a dorsolateral-ventromedial gradient of gene assemblies within the primate frontal lobe. Furthermore, we identify a set of 971 protein-coding and 34 non-coding genes consistently associated with cortical thickness, specially enriched for neurons and oligodendrocytes. These data provide a unique resource to investigate nonhuman primate models of human diseases and probe cross-species evolutionary mechanisms.

Mitonuclear Interactions and the Origin of Macaque Societies.

In most eukaryotes, aerobic respiration requires interactions between autosomally encoded genes (Ninteract genes) and mitochondrial DNA, RNA, and protein. In species where females are philopatric, contrasting distributions of genetic variation in mitochondrial and nuclear genomes create variation in mitonuclear interactions that may be subject to natural selection. To test this expectation, we turned to a group with extreme female philopatry: the macaque monkeys. We examined four genomic data sets from (1) wild caught and (2) captive populations of rhesus macaque, which is the most widely distributed nonhuman primate, and (3) the stump-tailed macaque and (4) a subspecies of longtail macaque, both of whose mitochondrial DNA is introgressed from a highly diverged ancestor. We identified atypically long runs of homozygosity, low polymorphism, high differentiation, and/or rapid protein evolution associated with Ninteract genes compared with non-Ninteract genes. These metrics suggest a subset of Ninteract genes were independently subject to atypically pervasive natural selection in multiple species. These findings suggest that natural selection on mitonuclear interactions could have influenced several aspects of macaque societies including species diversity, ecological breadth, female-biased adult sex ratio and demography, sexual dimorphism, and mitonuclear phylogenomics.

Complete sequencing of a cynomolgus macaque major histocompatibility complex haplotype.

Macaques provide the most widely used nonhuman primate models for studying the immunology and pathogenesis of human diseases. Although the macaque major histocompatibility complex (MHC) region shares most features with the human leukocyte antigen (HLA) region, macaques have an expanded repertoire of MHC class I genes. Although a chimera of two rhesus macaque MHC haplotypes was first published in 2004, the structural diversity of MHC genomic organization in macaques remains poorly understood owing to a lack of adequate genomic reference sequences. We used ultralong Oxford Nanopore and high-accuracy Pacific Biosciences (PacBio) HiFi sequences to fully assemble the ∼5.2-Mb M3 haplotype of an MHC-homozygous, Mauritian-origin cynomolgus macaque (Macaca fascicularis). The MHC homozygosity allowed us to assemble a single MHC haplotype unambiguously and avoid chimeric assemblies that hampered previous efforts to characterize this exceptionally complex genomic region in macaques. The high quality of this new assembly is exemplified by the identification of an extended cluster of six Mafa-AG genes that contains a recent duplication with a highly similar ∼48.5-kb block of sequence. The MHC class II region of this M3 haplotype is similar to the previously sequenced rhesus macaque haplotype and HLA class II haplotypes. The MHC class I region, in contrast, contains 13 MHC-B genes, four MHC-A genes, and three MHC-E genes (vs. 19 MHC-B, two MHC-A, and one MHC-E in the previously sequenced haplotype). These results provide an unambiguously assembled single contiguous cynomolgus macaque MHC haplotype with fully curated gene annotations that will inform infectious disease and transplantation research.

Primate gastrulation and early organogenesis at single-cell resolution.

Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, nonhuman primates are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we collected six Carnegie stage 8-11 cynomolgus monkey (Macaca fascicularis) embryos and performed in-depth transcriptomic analyses of 56,636 single cells. Our analyses show transcriptomic features of major perigastrulation cell types, which help shed light on morphogenetic events including primitive streak development, somitogenesis, gut tube formation, neural tube patterning and neural crest differentiation in primates. In addition, comparative analyses with mouse embryos and human embryoids uncovered conserved and divergent features of perigastrulation development across species-for example, species-specific dependency on Hippo signalling during presomitic mesoderm differentiation-and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the nonhuman primate research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.

Selection of a PD-1 blocking antibody from a novel fully human phage display library.

Programmed cell death protein 1 (PD-1) is an immunoregulatory target which is recognized by different monoclonal antibodies, approved for the therapy of multiple types of cancer. Different anti-PD-1 antibodies display different therapeutic properties and there is a pharmaceutical interest to generate and characterize novel anti-PD-1 antibodies. We screened multiple human antibody phage display libraries to target novel epitopes on the PD-1 surface and we discovered a unique and previously undescribed binding specificity (termed D12) from a new antibody library (termed AMG). The library featured antibody fragments in single-chain fragment variable (scFv) format, based on the IGHV3-23*03 (VH ) and IGKV1-39*01 (Vκ) genes. The D12 antibody was characterized by surface plasmon resonance (SPR), cross-reacted with the Cynomolgus monkey antigen and bound to primary human T cells, as shown by flow cytometry. The antibody blocked the PD-1/PD-L1 interaction in vitro with an EC50 value which was comparable to the one of nivolumab, a clinically approved antibody. The fine details of the interaction between D12 and PD-1 were elucidated by x-ray crystallography of the complex at a 3.5 Å resolution, revealing an unprecedented conformational change at the N-terminus of PD-1 following D12 binding, as well as partial overlap with the binding site for the cognate PD-L1 and PD-L2 ligands which prevents their binding. The results of the study suggest that the expansion of antibody library repertoires may facilitate the discovery of novel binding specificities with unique properties that hold promises for the modulation of PD-1 activity in vitro and in vivo.