Cynomolgus Research Articles

A Minimal PBPK/PD Model with Expansion-Enhanced Target-Mediated Drug Disposition to Support a First-in-Human Clinical Study Design for a FLT3L-Fc Molecule

FLT3L-Fc is a half-life extended, effectorless Fc-fusion of the native human FLT3-ligand. In cynomolgus monkeys, treatment with FLT3L-Fc leads to a complex pharmacokinetic/pharmacodynamic (PK/PD) relationship, with observed nonlinear PK and expansion of different immune cell types across different dose levels. A minimal physiologically based PK/PD model with expansion-enhanced target-mediated drug disposition (TMDD) was developed to integrate the molecule’s mechanism of action, as well as the complex preclinical and clinical PK/PD data, to support the preclinical-to-clinical translation of FLT3L-Fc. In addition to the preclinical PK data of FLT3L-Fc in cynomolgus monkeys, clinical PK and PD data from other FLT3-agonist molecules (GS-3583 and CDX-301) were used to inform the model and project the expansion profiles of conventional DC1s (cDC1s) and total DCs in peripheral blood. This work constitutes an essential part of our model-informed drug development (MIDD) strategy for clinical development of FLT3L-Fc by projecting PK/PD in healthy volunteers, determining the first-in-human (FIH) dose, and informing the efficacious dose in clinical settings. Model-generated results were incorporated in regulatory filings to support the rationale for the FIH dose selection.

Striking the Balance with a PD-L1×4-1BB Bispecific Antibody.

Antibody-based immune checkpoint blockade therapy has revolutionized the field of cancer immunotherapy, yet its efficacy remains limited in immunologically cold tumors. Combining checkpoint inhibitors with costimulatory agonists improves tumoricidal activity of T cells but also can lead to off-target hepatotoxicity. Although bispecific antibodies confer tumor selectivity to alleviate undesirable adverse effects, toxicity concerns persist with increased dosing. In this issue of Cancer Research, Yuwen and colleagues introduce ATG-101, a tetravalent PD-L1×4-1BB bispecific antibody with high programmed death ligand 1 (PD-L1) affinity and low 4-1BB affinity, aiming to mitigate hepatotoxicity. ATG-101 demonstrates PD-L1-dependent 4-1BB activation, leading to selective T-cell activation within the tumor microenvironment. ATG-101 exhibits potent antitumor activity, even in large, immunologically cold, and monotherapy-resistant tumor models. Single-cell RNA sequencing reveals significant shifts of immune cell populations in the tumor microenvironment from protumor to antitumor phenotypes following ATG-101 treatment. In cynomolgus monkeys, no serious cytokine storm and hepatotoxicity are observed after ATG-101 treatment, indicating a broad therapeutic window for ATG-101 in cancer treatment. This study highlights the potential of tetravalent bispecific antibodies in cancer immunotherapy, with implications for various antibody-based treatment modalities across different fields. See related article by Yuwen et al., p. 1680.

Molecular Detection and Characterization of Hemotropic Mycoplasma in Assamese Macaques (Macaca assamensis) of Northern Thailand

Hemotropic mycoplasmas, also known as hemoplasmas, are parasitic bacteria that infect red blood cells, potentially leading to varying degrees of anemia across numerous mammalian species, including nonhuman primates. The present study aims to investigate the prevalence of hemoplasma infection and identify the species involved among free-ranging Assamese macaques (Macaca assamensis) inhabiting northern Thailand. A total of 133 blood samples were collected from Assamese macaques in Chiang Rai province, Thailand, and subjected to screening for hemoplasma infection utilizing nested PCR amplification targeting the 16S rRNA gene. Positive samples were subsequently analyzed through nucleotide sequencing and phylogenetic analysis for putative species identification. Current study results revealed that 17.3% (23/133; 95% CI 11.29-24.81) of Assamese macaques tested positive for hemoplasma infection using the nested PCR assay. Partial 16S rRNA sequences derived from hemoplasma isolates in Assamese macaques exhibited 99% homology, forming a cluster within the same phylogenetic clade as “Candidatus Mycoplasma haematomacacae,” previously identified in long-tailed macaques, rhesus macaques, and Japanese macaques. These findings suggest the presence of “Ca. M. haematomacacae” not only in long-tailed macaques and rhesus macaques but also in Assamese macaques in Thailand. To our knowledge, this marks the first molecular detection of “Ca. M. haematomacacae” in Assamese macaques in Thailand. These results hold significance as they enhance our understanding of hemoplasma infection distribution among macaque populations in Thailand.

Preliminary investigation into long-term stress by isolated captivity-related changes of reproduction hormones in Cynomolgus monkey.

Stress profoundly affects physical and emotional well-being, extending its physiological influence to the female menstrual cycle, impeding the hypothalamus-pituitary-gonadal (HPG) axis, and affecting fertility by suppressing sex-stimulating hormones. In this study, we meticulously analyzed menstrual cycles and corresponding hormonal fluctuations in three female Cynomolgus monkeys. The preliminary findings indicated lower-than-normal levels of cortisol, follicle-stimulating hormone (FSH), and estradiol. Anovulatory bleeding occurred in one monkey, which could be linked to stress. In contrast to cortisol, alkaline phosphatase (ALP), which is correlated to cortisol levels, was consistently elevated in menstruating monkeys, suggesting its potential as a stress indicator. The non-menstruating group exhibited stress-related weight loss, emphasizing the observed ALP trends. Non-menstruating monkeys may experience more stress than menstruating monkeys. The implications of this study extend beyond the confines of primate studies and offer a valuable method for enhancing the welfare of female Cynomolgus monkeys.

Pilot PET study of vaginally administered bioadhesive nanoparticles in cynomolgus monkeys: Kinetics and safety evaluation

AbstractLong‐lasting vaginal dosage forms could improve the therapeutic efficacy of vaginal microbicides, but achieving long‐term delivery to the vaginal canal has been a significant challenge. To advance understanding of vaginal dosage retention and biodistribution, we describe a method of noninvasive imaging with 89Zr‐labeled bioadhesive nanoparticles (BNPs) in non‐human primates. We additionally examined the safety of repeated BNP application. BNPs administered vaginally to cynomolgus monkeys were still detected after 24 h (1.7% retention) and 120 h (0.1% retention). BNPs did not translocate to the uterus or into systemic circulation. Analysis of inflammatory biomarkers in the vaginal fluid and plasma suggest that BNPs are safe and biocompatible, even after multiple doses. BNPs are a promising delivery vehicle for vaginally administered therapeutics. Further studies using the non‐human primate imaging materials and methods developed here could help advance clinical translation of BNPs and other long‐lasting vaginal dosage forms.

Discovery of potent small-molecule inhibitors of lipoprotein(a) formation.

Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and apolipoprotein(a) (apo(a))1,2. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (KIV) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs. 3-7). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) KIV7-8. We identify compounds that bind to apo(a) KIV7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a).

Student’s knowledge on primates: Long-tailed macaque educonservation efforts through primatology course

Within 15 years, the population of Macaca fascicularis has declined rapidly. In early 2022, the IUCN Red List classified them as “Endangered”, though they were considered a species of “Least Concern” in 2008. Land alteration, illegal hunting, and human-macaque conflict often halt long-tailed macaque conservation efforts. Through the Primatology course, anthropology students are taught the importance of macaque conservation and its complications. This preliminary study aims to reveal anthropology students’ knowledge regarding the long-tailed macaque’s status quo and its conservation efforts, pre- and post- their enrolment in the course. Employing a descriptive qualitative approach, this study conducted in-depth interviews with 10 consenting informants—active undergraduate anthropology students. After the transcription process, the data was analyzed by dividing it into multiple subchapters and reviewed with existing literature. More than half of the informants’ knowledge about Macaca fascicularis and primates, in general, is quite satisfactory, though they are still unaware of their current conservation status. According to the informants, cultural and religious factors are closely related to long-tailed macaque conservation, shown in Balinese Hindu temples, Javanese wayang, and topeng monyet. The study concluded that primatology is important because it raises awareness about human’s closest relatives, their ecological significance, and their conservation measures. Adoption or adaptation of the ethnoprimatological framework in biology education as a means of sociocultural exploration and primate conservation is highly recommended. Further research on ethnoprimatology in Indonesia is much needed since Indonesia has many cultural values that might assure the people about contemporary conservation efforts

Abstract PO3-18-04: MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer

Abstract Objectives: ADCs have emerged as a major advance in contemporary medical oncology. The receptor tyrosine kinase (RTK) EphA5 possesses the classical receptor kinase topology with an extracellular-binding domain, a single-pass transmembrane domain, and a cytoplasmic kinase domain. We have previously shown that EphA5 is highly expressed in non-small cell lung cancer and contributes to DNA-damage repair and radiation therapy resistance. Others have reported EphA5 expression in gastric, ovarian, and pancreatic cancers. Here, we investigated the expression of EphA5 in breast cancer (BC) and assessed EphA5 as a potential cancer-specific target using a novel ADC designated MBRC-101. Methods: MBRC-101 is composed of a humanized anti-EphA5 IgG1 kappa monoclonal antibody conjugated to the small molecule microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease cleavable valine-citrulline (vc) ThioBridge linker (ThioBridge™-Glu-[Val-Cit-PAB-MMAE] PEG [24u]). MBRC-101 averages a drug-to-antibody ratio (DAR)=4. The specificity profile of the anti-EphA5 monoclonal antibody was characterized using a Membrane Proteome Array (MPA). Receptor-mediated antibody internalization was determined by real-time live-cell analysis; cell killing assays used both live-cell analysis of cell confluence and the CellTiter-Glo® luminescent cell viability assay. Expression of EphA5 in archival human breast tumor tissue sections was evaluated by immunohistochemistry (IHC) using a commercial polyclonal antibody against human EphA5. In vivo efficacy studies used patient-derived xenograft (PDX) models of BC. Results: IHC demonstrated robust and selective EphA5 expression in 87% (20 of 23) of triple negative breast cancer (TNBC) and 88% (23 of 26) of hormone receptor-positive (HR+) patient tumors tested. EphA5 expression was not detected in adjacent, non-malignant breast tissue. Antibody-specificity profile testing showed that the anti-EphA5 antibody bound exclusively to EphA5 and did not cross-react with other members of the Eph receptor/ephrin ligand family. Binding of the anti-EphA5 antibody to EphA5 on the cell surface triggered rapid internalization and processing of the EphA5/antibody complex through the endosomal-lysosomal system. Consistently, MBRC-101 was cytotoxic to EphA5-expressing cells and its activity was concentration-dependent and commensurate to the levels of EphA5 on the cell surface. In PDX murine models of TNBC, once weekly administrations of intravenous (IV) MBRC-101 showed dose-dependent, robust, and reproducible anti-tumor activity in vivo. Partial tumor responses were observed at doses of 2.5 mg/Kg IV and complete and nearly complete tumor responses starting at 5 mg/Kg IV. Doses up to 10 mg/Kg IV were well-tolerated in tumor-bearing mice with no observable weight loss. Pre-clinical safety confirmed MBRC-101 is also well tolerated in rats and in cynomolgus monkeys. Conclusions: These results support EphA5 as a new and promising membrane-associated molecular target for the design and development of ADC-based therapies against hormone-positive breast cancer and TNBC and support the conduct of a Phase 1/1b study of MBRC-101 in patients with breast cancer. Citation Format: Fernanda Staquicini, Fenny Tang, Vanessa de Oliveira, Daniela Staquicini, Yongjian Wu, Kirstin Barnhart, J Kellogg Parsons, Wadih Arap, Isan Chen, Renata Pasqualini. MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-04.

Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage

Clinical studies using suspensions or sheets of human pluripotent cell-derived retinal pigment epithelial cells (hiPSC-RPE) have been conducted globally for diseases such as age-related macular degeneration. Despite being minimally invasive, cell suspension transplantation faces challenges in targeted cell delivery and frequent cell leakage. Conversely, although the RPE sheet ensures targeted delivery with correct cell polarity, it requires invasive surgery, and graft preparation is time-consuming. We previously reported hiPSC-RPE strips as a form of quick cell aggregate that allows for reliable cell delivery to the target area with minimal invasiveness. In this study, we used a microsecond pulse laser to create a local RPE ablation model in cynomolgus monkey eyes. The hiPSC-RPE strips were transplanted into the RPE-ablated and intact sites. The hiPSC-RPE strip stably survived in all transplanted monkey eyes. The expansion area of the RPE from the engrafted strip was larger at the RPE injury site than at the intact site with no tumorigenic growth. Histological observation showed a monolayer expansion of the transplanted RPE cells with the expression of MERTK apically and collagen type 4 basally. The hiPSC-RPE strip is considered a beneficial transplantation option for RPE cell therapy.

Pharmacokinetics and safety of LEAD-452, an EGFR-specific 4-1BB-agonistic trimerbody in non-human primates

LEAD-452 is a humanized bispecific EGFR-targeted 4-1BB-agonistic trimerbody with a unique trimeric configuration compared to other 4-1BB-specific antibodies that are currently in development. Indeed, enhanced tumor-specific costimulation and very remarkable safety and efficacy profiles have been observed in mouse models. Here, we conducted for the first time a preclinical pharmacokinetic and toxicity study in non-human primates (NHP) (Macaca fascicularis). LEAD-452 exhibits comparable binding affinity for human and macaque targets, indicating its pharmacological significance for safety testing across species. The NHP were administered LEAD-452 in a series of ascending doses, ranging from 0.1 mg/kg to 10 mg/kg, and repeated doses up to 20 mg/kg. The administration of LEAD-452 was found to be clinically well tolerated, with no major related adverse effects observed. Furthermore, there have been no reported cases of liver toxicity, thrombocytopenia, and neutropenia, which are commonly associated with treatments using conventional anti-4-1BB IgG-based antibodies. In addition, neither IgM nor IgG-based anti-drug antibodies were detected in serum samples from NHP during the study, regardless of the dose of LEAD-452 administered. These results support the clinical development of LEAD-452 for the treatment of solid tumors.

Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques

HTLV-1 infection occurs by cell-to-cell transmission and can induce fatal adult T-cell leukemia (ATL). Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of ten vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the two vaccinated macaques without NAb induction and ten unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.

Biogeography of intestinal mucus-associated microbiome: Depletion of genus Pseudomonas is associated with depressive-like behaviors in female cynomolgus macaques

IntroductionDepression is a debilitating and poorly understood mental disorder. There is an urgency to explore new potential biological mechanisms of depression and the gut microbiota is a promising research area. ObjectivesOur study was aim to understand regional heterogeneity and potential molecular mechanisms underlying depression induced by dysbiosis of mucus-associated microbiota. MethodsHere, we only selected female macaques because they are more likely to form a natural social hierarchy in a harem-like environment. Because high-ranking macaques rarely displayed depressive-like behaviors, we selected seven monkeys from high-ranking individuals as control group (HC) and the same number of low-ranking ones as depressive-like group (DL), which displayed significant depressive-like behaviors. Then, we collected mucus from the duodenum, jejunum, ileum, cecum and colon of DL and HC monkeys for shotgun metagenomic sequencing, to profile the biogeography of mucus-associated microbiota along duodenum to colon. ResultsCompared with HC, DL macaques displayed noticeable depressive-like behaviors such as longer duration of huddle and sit alone behaviors (negative emotion behaviors), and fewer duration of locomotion, amicable and ingestion activities (positive emotion behaviors). Moreover, the alpha diversity index (Chao) could predict aforementioned depressive-like behaviors along duodenum to colon. Further, we identified that genus Pseudomonas was consistently decreased in DL group throughout the entire intestinal tract except for the jejunum. Specifically, there were 10, 18 and 28 decreased Pseudomonas species identified in ileum, cecum and colon, respectively. Moreover, a bacterial module mainly composed of Pseudomonas species was positively associated with three positive emotion behaviors. Functionally, Pseudomonas was mainly involved in microbiota derived lipid metabolisms such as PPAR signaling pathway, cholesterol metabolism, and fat digestion and absorption. ConclusionDifferent regions of intestinal mucus-associated microbiota revealed that depletion of genus Pseudomonas is associated with depressive-like behaviors in female macaques, which might induce depressive phenotypes through regulating lipid metabolism.

HPN328, a Trispecific T Cell-activating Protein Construct Targeting DLL3-Expressing Solid Tumors.

Delta-like ligand 3 (DLL3) is expressed in more than 70% of small cell lung cancers (SCLCs) and other neuroendocrine-derived tumor types. SCLC is highly aggressive and limited therapeutic options lead to poor prognosis for patients. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a CD3 binder for T cell engagement, an albumin binder for half-life extension, and a DLL3 binder for tumor cell engagement. In vitro assays, rodent models and non-human primates were used to assess the activity of HPN328. HPN328 induces potent dose-dependent killing of DLL3-expressing SCLC cell lines in vitro concomitant with T cell activation and cytokine release. In an NCI-H82 xenograft model with established tumors, HPN328 treatment led to T cell recruitment and anti-tumor activity. In an immunocompetent mouse model expressing a human CD3ε epitope, mice previously treated with HPN328 withstood tumor rechallenge, demonstrating long-term anti-tumor immunity. When repeat doses were administered to cynomolgus monkeys, HPN328 was well tolerated up to 10 mg/kg. Pharmacodynamic changes, such as transient cytokine elevation, were observed, consistent with the expected mechanism of action of T cell engagers. HPN328 exhibited linear pharmacokinetic in the given dose range with a serum half-life of 78 to 187 hours, supporting weekly or less frequent administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A phase 1/2 clinical trial is currently underway testing safety and efficacy in patients with DLL3 expressing malignancies.

Six months of voluntary alcohol consumption in male cynomolgus macaques reduces intracortical bone porosity without altering mineralization or mechanical properties

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = −0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Targeting CEACAM5-positive solid tumors using NILK-2401, a novel CEACAM5xCD47 κλ bispecific antibody.

Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.

Adult Human, but Not Rodent, Spermatogonial Stem Cells Retain States with a Foetal-like Signature.

Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling, it has been proposed that undifferentiated human spermatogonia can be subclassified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc) RNA-sequencing libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat, and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate 'state 0B' with a suppressive transcriptomic programme that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells-which are mitotically arrested-can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mice (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive lifespan while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes.

Complete Genomic Assembly of Mauritian Cynomolgus Macaque Killer Ig-like Receptor and Natural Killer Group 2 Haplotypes.

Mauritian-origin cynomolgus macaques (MCMs) serve as a powerful nonhuman primate model in biomedical research due to their unique genetic homogeneity, which simplifies experimental designs. Despite their extensive use, a comprehensive understanding of crucial immune-regulating gene families, particularly killer Ig-like receptors (KIR) and NK group 2 (NKG2), has been hindered by the lack of detailed genomic reference assemblies. In this study, we employ advanced long-read sequencing techniques to completely assemble eight KIR and seven NKG2 genomic haplotypes, providing an extensive insight into the structural and allelic diversity of these immunoregulatory gene clusters. Leveraging these genomic resources, we prototype a strategy for genotyping KIR and NKG2 using short-read, whole-exome capture data, illustrating the potential for cost-effective multilocus genotyping at colony scale. These results mark a significant enhancement for biomedical research in MCMs and underscore the feasibility of broad-scale genetic investigations.

Inhibition kinetics of acetylcholinesterase and butyrylcholinesterase from various species by 2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP)

The aerotoxic syndrome has been associated with exposure to tricresyl phosphate (TCP), which is used as additive in hydraulic fluids and engine lubricants. The toxic metabolite 2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP) is formed from the TCP isomer tri-ortho-cresyl phosphate (TOCP) in vivo and is known to react with the active site serine in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) resulting in the inhibition of the enzymes. Previous in vitro studies showed pronounced species differences in the inhibition kinetics of cholinesterases by organophosphorus compounds (OP), which must be considered in the development of relevant animal models for the investigation of OP poisoning and the aerotoxic syndrome. The present study was designed to investigate the inhibition kinetics of human, Cynomolgus monkey, pig, mini pig, guinea pig, mouse, and rat AChE as well as BChE by CBDP under standardized conditions. There were similar rate constants for the inhibition (ki) of human, Cynomolgus monkey and mouse AChE by CBDP. In contrast, the ki values obtained for guinea pig, mini pig, pig, and rat AChE were 2.8- to 5.9-fold lower than that of human AChE. The results of the present study confirmed CBDP as one of the most potent inhibitors of human BChE, indicating a ki value of 3.24 ± 0.33 ×108M−1min−1, which was about 1,140-fold higher than that of human AChE. Accordingly, a markedly more pronounced inhibition rate of BChE from the species guinea pig, mini pig, pig, rat, Cynomolgus monkey, and mouse by CBDP was found as compared to those of AChE from the respective sources, indicating 2.0- to 89.6-fold higher ki values.

Preclinical development of EXT608, an investigational parathyroid hormone derivative with extended half-life for the treatment of hypoparathyroidism.

Hypoparathyroidism, a deficiency of parathyroid hormone (PTH), results in hypocalcemia, hyperphosphatemia, and hypercalciuria. The disease is poorly controlled by calcium and vitamin D supplements or native PTH(1-84) replacement therapy. A version of PTH is being developed using D-VITylation technology, whereby vitamin D is conjugated to a therapeutic peptide, which confers a long plasma half-life by virtue of binding to the abundant vitamin D binding protein (DBP). D-VITylation of PTH caused no reduction in activity at the PTHR1 receptor, and resulted in a plasma elimination half-life of 7-15h in rats and 24-32h in cynomolgus monkeys. Analysis of steady-state pharmacokinetics as a function of dose showed flat profiles with smaller peak:trough ratios at low doses, indicative of slower subcutaneous absorption. In thyroparathyroidectomized (TPTx) rats, PTH(1-34)-vitamin D conjugates restored serum calcium and phosphate levels into the normal range over the 24h dosing period, and increased bone turnover markers and reduced bone mineral density. Urinary calcium was initially elevated, but normalized by the end of treatment on day 27. In healthy monkeys, a single dose of PTH(1-34)-vitamin D conjugates elevated serum calcium levels above the normal range for a period of 24-48h while simultaneously reducing urinary calcium. Therefore, the lead compound, EXT608, is a promising candidate as a therapeutic that can truly mimic the endogenous activity of PTH and warrants further study in patients with hypoparathyroidism.