Primate Studies Research Articles

Echocardiographic characterization of age- and sex-associated differences in cardiac function and morphometry in nonhuman primates.

Aging per se is a major risk factor for cardiovascular diseases and is associated with progressive changes in cardiac structure and function. Rodent models are commonly used to study cardiac aging, but do not closely mirror differences as they occur in humans. Therefore, we performed a 2D echocardiographic study in non-human primates (NHP) to establish age- and sex-associated differences in cardiac function and morphometry in this animal model. M mode and 2D echocardiography and Doppler analyses were performed cross-sectionally in 38 healthy rhesus monkeys (20 females and 18 males), both young (age 7-12years; n = 20) and old (age 19-30years; n = 18). The diameters of the cardiac chambers did not differ significantly by age group, but males had larger left ventricular diameters (2.43 vs 2.06cm in diastole and 1.91 vs 1.49cm in systole, p = 0.0004 and p = 0.0001, respectively) and left atrial diameter (1.981 vs 1.732cm; p = 0.0101). Left ventricular mass/body surface area did not vary significantly with age and sex. Ejection fraction did not differ by age and females presented a higher ejection fraction than males (54.0 vs 50.8%, p = 0.0237). Diastolic function, defined by early to late mitral peak flow velocity ratio (E/A), was significantly lower in old rhesus monkeys (2.31 vs 1.43, p = 0.0020) and was lower in females compared to males (1.595 vs 2.230, p = 0.0406). Right ventricular function, evaluated by measuring the Tricuspid Annular Plane Systolic Excursion, did not differ by age or sex, and Right Ventricular Free Wall Longitudinal Strain, did not differ with age but was lower in males than in females (-22.21 vs -17.95%, p = 0.0059). This is the first echocardiographic study to evaluate age- and sex-associated changes of cardiac morphometry and function in young and old NHP. The findings of this work will provide a reference to examine the effect of age and sex on cardiac diseases in NHP.

Chst9 marks a spatially and transcriptionally unique population of Oprm1-expressing neurons in the nucleus accumbens

Opioids produce addictive, analgesic, and euphoric effects via actions at mu opioid receptors (µORs). The µOR is encoded by the Oprm1 gene and is expressed in multiple brain regions that regulate reward and motivation, such as the nucleus accumbens (NAc). Oprm1 expression in NAc medium spiny neurons (MSNs) mediates opioid place preference, seeking, and consumption. However, recent single nucleus RNA sequencing (snRNA-seq) studies have revealed that multiple subpopulations of NAc neurons express Oprm1 mRNA, making it unclear which populations mediate diverse behaviors resulting from µOR activation. Using published snRNA-seq datasets from the rat NAc, we identified a novel population of MSNs that express the highest levels of Oprm1 of any NAc cell type. Here, we show that this population is selectively marked by expression of Chst9, a gene encoding a carbohydrate sulfotransferase. Notably, Chst9+ neurons exhibited more abundant expression of Oprm1 as compared to other cell types, and formed discrete cellular clusters along the medial and ventral borders of the NAc shell subregion. Moreover, CHST9 mRNA was also found to mark specific MSN populations in published human and primate snRNA-seq studies, indicating that this unique population may be conserved across species. Together, these results identify a spatially and transcriptionally distinct NAc neuron population characterized by the expression of Chst9. The abundant expression of Oprm1 in this population and the conservation of these cells across species suggests that they may play a key functional role in opioid response and identify this subpopulation as a target for further investigation.

A pig kidney supporting human physiology

Because of the global shortage of donor kidneys, xenotransplantation emerges as a potential solution for individuals with kidney failure who face challenges in securing a suitable donor kidney. A study featured in this month's issue of Kidney International assesses the kidney physiology of a porcine kidney transplanted into a brain-dead human with kidney failure, demonstrating life-sustaining physiological function for 7 days. Together with preclinical nonhuman primate studies, decedent models provide complementary data for development of clinical kidney xenotransplantation.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

Visual perception of highly memorable images is mediated by a distributed network of ventral visual regions that enable a late memorability response.

Behavioral and neuroscience studies in humans and primates have shown that memorability is an intrinsic property of an image that predicts its strength of encoding into and retrieval from memory. While previous work has independently probed when or where this memorability effect may occur in the human brain, a description of its spatiotemporal dynamics is missing. Here, we used representational similarity analysis (RSA) to combine functional magnetic resonance imaging (fMRI) with source-estimated magnetoencephalography (MEG) to simultaneously measure when and where the human cortex is sensitive to differences in image memorability. Results reveal that visual perception of High Memorable images, compared to Low Memorable images, recruits a set of regions of interest (ROIs) distributed throughout the ventral visual cortex: a late memorability response (from around 300 ms) in early visual cortex (EVC), inferior temporal cortex, lateral occipital cortex, fusiform gyrus, and banks of the superior temporal sulcus. Image memorability magnitude results are represented after high-level feature processing in visual regions and reflected in classical memory regions in the medial temporal lobe (MTL). Our results present, to our knowledge, the first unified spatiotemporal account of visual memorability effect across the human cortex, further supporting the levels-of-processing theory of perception and memory.

Differential induction of donor-reactive Foxp3+ regulatory T cell via blockade of CD154 vs CD40

Recently published studies in both murine models and a meta-analysis of non-human primate renal transplant studies showed that anti-CD154 reagents conferred a significant survival advantage over CD40 blockers in both animal models and across multiple organs. Here we sought to compare the induction of donor-reactive forkhead box P3+-induced regulatory T cells (Foxp3+ iTreg) in mice treated with anti-CD154 versus anti-CD40 monoclonal antibodies (mAbs). Results indicated that while treatment with anti-CD154 mAb resulted in a significant increase in the frequency of donor-reactive CD4+ Foxp3+ iTreg following transplantation, treatment with anti-CD40 or Cd40 deficiency failed to recapitulate this result. Because we recently identified CD11b as an alternate receptor for CD154 during alloimmunity, we interrogated the role of CD154:CD11b interactions in the generation of Foxp3+ iTreg and found that blockade of CD11b in Cd40−/− recipients resulted in increased donor-reactive Foxp3+ iTreg as compared with CD40 deficiency alone. Mechanistically, CD154:CD11b inhibition decreased interleukin (IL)-1β from CD11b+ and CD11c+ dendritic cells, and blockade of IL-1β synergized with CD40 deficiency to promote Foxp3+ iTreg induction and prolong allograft survival. Taken together, these data provide a mechanistic basis for the observed inferiority of anti-CD40 blockers as compared with anti-CD154 mAb and illuminate an IL-1β-dependent mechanism by which CD154:CD11b interactions prevent the generation of donor-reactive Foxp3+ iTreg during transplantation.

Sitagliptin elevates plasma and CSF incretin levels following oral administration to nonhuman primates: relevance for neurodegenerative disorders

The endogenous incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) possess neurotrophic, neuroprotective, and anti-neuroinflammatory actions. The dipeptidyl peptidase 4 (DPP-4) inhibitor sitagliptin reduces degradation of endogenous GLP-1 and GIP, and, thereby, extends the circulation of these protective peptides. The current nonhuman primate (NHP) study evaluates whether human translational sitagliptin doses can elevate systemic and central nervous system (CNS) levels of GLP-1/GIP in naive, non-lesioned NHPs, in line with our prior rodent studies that demonstrated sitagliptin efficacy in preclinical models of Parkinson’s disease (PD). PD is an age-associated neurodegenerative disorder whose current treatment is inadequate. Repositioning of the well-tolerated and efficacious diabetes drug sitagliptin provides a rapid approach to add to the therapeutic armamentarium for PD. The pharmacokinetics and pharmacodynamics of 3 oral sitagliptin doses (5, 20, and 100 mg/kg), equivalent to the routine clinical dose, a tolerated higher clinical dose and a maximal dose in monkey, were evaluated. Peak plasma sitagliptin levels were aligned both with prior reports in humans administered equivalent doses and with those in rodents demonstrating reduction of PD associated neurodegeneration. Although CNS uptake of sitagliptin was low (cerebrospinal fluid (CSF)/plasma ratio 0.01), both plasma and CSF concentrations of GLP-1/GIP were elevated in line with efficacy in prior rodent PD studies. Additional cellular studies evaluating human SH-SY5Y and primary rat ventral mesencephalic cultures challenged with 6-hydroxydopamine, established cellular models of PD, demonstrated that joint treatment with GLP-1 + GIP mitigated cell death, particularly when combined with DPP-4 inhibition to maintain incretin levels. In conclusion, this study provides a supportive translational step towards the clinical evaluation of sitagliptin in PD and other neurodegenerative disorders for which aging, similarly, is the greatest risk factor.

Disruption of awake sharp-wave ripples does not affect memorization of locations in repeated-acquisition spatial memory tasks.

Storing and accessing memories is required to successfully perform day-to-day tasks, for example for engaging in a meaningful conversation. Previous studies in both rodents and primates have correlated hippocampal cellular activity with behavioral expression of memory. A key role has been attributed to awake hippocampal replay - a sequential reactivation of neurons representing a trajectory through space. However, it is unclear if awake replay impacts immediate future behavior, gradually creates and stabilizes long-term memories over a long period of time (hours and longer), or enables the temporary memorization of relevant events at an intermediate time scale (seconds to minutes). In this study, we aimed to address the uncertainty around the timeframe of impact of awake replay by collecting causal evidence from behaving rats. We detected and disrupted sharp wave ripples (SWRs) - signatures of putative replay events - using electrical stimulation of the ventral hippocampal commissure in rats that were trained on three different spatial memory tasks. In each task, rats were required to memorize a new set of locations in each trial or each daily session. Interestingly, the rats performed equally well with or without SWR disruptions. These data suggest that awake SWRs - and potentially replay - does not affect the immediate behavior nor the temporary memorization of relevant events at a short timescale that are required to successfully perform the spatial tasks. Based on these results, we hypothesize that the impact of awake replay on memory and behavior is long-term and cumulative over time.

Abstract 1897: Preclinical development of MGC028, an ADAM9-targeted, glycan-linked, exatecan-based antibody-drug conjugate for the treatment of solid cancers

Abstract Introduction: Antibody-drug conjugates (ADCs) seek to increase the therapeutic window of potent cytotoxic agents by linking them with monoclonal antibodies (mAbs) to selectively deliver cytotoxic payloads to tumor cells. The effectiveness and safety of ADCs rely on both the mAb specificity, and the linker-payload employed. Several approved ADCs using microtubule inhibitor payloads are impacted by ocular adverse events that have been observed both preclinically and in patients. A class of linker-payloads incorporating topoisomerase 1 inhibitors (TOP1i) has emerged recently as an effective alternative to tubulin inhibitor-based ADCs. To date, TOP1i ADCs have not been associated with dose-limiting ocular toxicity seen with microtubule inhibitor payloads. Here we report the preclinical development of an ADAM9 (a disintegrin and metalloprotease domain 9)-targeted ADC that incorporates a novel glycan-linked TOP1i. ADAM9, a member of the ADAM family of multifunctional type 1 transmembrane proteins, plays a role in tumorigenesis and cancer progression and is overexpressed in multiple cancers, making it an attractive target for cancer treatment. Methods: MGC028 incorporates the cleavable linker-payload, bicyclononyne carbamoyl sulfamide Val-Ala-PABC exatecan (SYNtecan E™), site-specifically conjugated at asparagine 297 of the heavy chain through enzymatic glycan remodeling and metal-free click chemistry using Synaffix’s GlycoConnect™ technology. In vivo efficacy studies were performed in immunodeficient mice with ADAM9-expressing human tumor cell-line (CDX) or patient-derived (PDX) xenografts. A non-human primate toxicology study was conducted in which MGC028 was administered by 15-minute IV infusion every two weeks at dose levels of 22.5 and 55 mg/kg for a total of two doses. Results: MGC028 exhibited specific, dose-dependent in vivo antitumor activity toward ADAM9-positive CDX models representing gastric, lung, pancreatic and colorectal cancer, and head and neck squamous cell carcinoma. Furthermore, MGC028 demonstrated antitumor activity toward ADAM9-positive PDX models of lung and pancreatic cancer, and cholangiocarcinoma. MGC028 was well tolerated in a repeat-dose non-human primate toxicology study, up to 55 mg/kg, the highest dose level tested. Observations were limited to mild, reversible increases in liver enzymes, without microscopic correlates, and decreased lymphoid cellularity in the thymus. In particular, ocular toxicities were not observed. Conclusions: MGC028 exhibited potent antitumor activity in in vivo models representing various solid cancer indications and was well tolerated in non-human primates at exposure levels exceeding those required for antitumor activity. Our findings support continued investigation of MGC028 as an ADC therapeutic for the treatment of ADAM9-expressing solid cancers. Citation Format: Juniper A. Scribner, Jennifer G. Brown, Thomas Son, Linda Jin, Carroll McKenzie, Viktoriya Nam, Curtis Bush, Dienis Quinonez, Delta Ford, Verlene Gonzalez, James Tamura, Sergey Gorlatov, Hua Li, Shelley Butler, Ezio Bonvini, Deryk Loo. Preclinical development of MGC028, an ADAM9-targeted, glycan-linked, exatecan-based antibody-drug conjugate for the treatment of solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1897.

Abstract 6703: Innovative mRNA strategies to encode CD19/CD3 T cell engagers in the treatment of B cell hematological malignancies

Abstract The advent of T Cell Engager (TCE) immunotherapies marks a transformative shift in cancer treatment paradigms, introducing a novel action mechanism. Blinatumomab, a bispecific CD19/CD3 TCE, has shown remarkable efficacy in treating refractory or relapsed B-cell Acute Lymphoblastic Leukaemia (B-ALL), including cases resistant to conventional chemotherapy, leading to its global approval for B-ALL management. Despite its pronounced clinical success, Blinatumomab faces challenges with patient adherence, primarily due to the protein’s short half-life. This necessitates continuous administration requested 24 hours injection throughout the treatment course. Additionally, the drug’s association with the induction of cytokine release syndrome (CRS), presumably linked to the hyperactivation of the cytokine network, presents a significant concern. To address these limitations, we leverage mRNA modality to develop innovative therapeutic mRNAs encoding CD19/CD3 TCE. These mRNA drugs hold the potential to improve protein exposure in vivo with a steady production of encoded protein in-situ. On the other hand, with the gradual expression of protein, mRNA shifted the transient, “pulse”-like pharmacokinetic (PK) feature of the protein drugs to a more “plateau”PK curve, which allows a natural priming of T cells to reduce the risk of CRS induction. In this work, we developed a series of mRNAs encoding CD19/CD3 TCE through our mRNA technology platform and LNP delivery platform. Comparative analyses demonstrated that the TCEs produced in vivo by these mRNAs match the structure and function of recombinantly expressed proteins. PK studies in both mice and non-human primates confirmed a favorable PK profile with over 10-fold increase in TCE exposure compared to their protein counterparts. This increased exposure correlated with superior antitumor efficacy in both ALL and lymphoma models, evidenced by the potent effects of single mRNA doses compared to multiple doses of protein-based treatments. Meanwhile IL6 cytokines induced in mRNA treated mice were significantly alleviated than those in protein treated animals, indicating a reduced risk of induction of CRS. One candidate mRNA demonstrated an optimal "plateau" pharmacokinetic profile, achieving extensive target cell eradication over 20 days with a single low dose of 0.001 mpk in non-human primates. An investigator-initiated clinical study with mRNA encoding CD19/CD3 TCE is underway in relapsed and/or refractory acute lymphoblastic leukemia patients. The preliminary data from the on-going study showed ABO2015 with an acceptable safety profile, and activation of peripheral T cells was observed in one patient with low dose. These findings underscore the significant clinical potential of mRNA-encoded CD19/CD3 TCE in treating B-cell malignancies, heralding a paradigm shift in the clinical application of protein-based T cell engagers. Citation Format: Xia Zhong, Liang Du, Zhenxing Yang, Xiaoli Xu, Jijun Yuan, Bo Ying. Innovative mRNA strategies to encode CD19/CD3 T cell engagers in the treatment of B cell hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6703.

Abstract 3138: Discovery of AT65474, a highly selective anti-CLDN6 ADC with a proprietary payload

Abstract CLDN6 is a membrane-bound oncofetal protein and a promising target for cancers such as ovarian and testicular cancer. CLDN6 shares high sequence homology with other CLDN family members which are expressed in normal tissues, making a high degree of selectivity required for CLDN6-targeting therapies. In particular, CLDN6 is closest to CLDN9 in sequence with a difference of only three residues in the extracellular loops, making it challenging to generate a highly selective antibody. We report the development of a humanized anti-CLDN6 antibody with high affinity and high selectivity for targeted cells which displays > 800-fold higher binding affinity for CLDN6 over CLDN9. The antibody was refined for use as an ADC and incorporates AxcynCYSTM technology for site-specific conjugation which can reproducibly achieve high DAR4 ratios of greater than 97% by HIC analysis. AT65474 is an ADC candidate that incorporates a proprietary payload derived from an FDA approved drug that was optimized in-house for increased potency and improved PK properties to limit systemic exposure. In vitro, the payload displays broad sub-nanomolar activity across a wide range of cancer cell lines including those resistant to paclitaxel, DM1, MMAE and DXd. The payload is conjugated via an enzyme-cleavable linker optimized for solubility, stability, and high drug to antibody ratios. We provide evidence characterizing AT65474 as a highly potent ADC with high DAR4 homogeneity and strong in vivo activity at low doses. Compared with a benchmark ADC in clinical development, AT65474 shows stronger binding, higher internalization, superior in vitro cytotoxicity, and stronger bystander killing effect. When evaluated in PA-1 and OV90 xenograft models of ovarian cancer, AT65474 displays significantly higher efficacy over the benchmark ADC. Furthermore, we show that AT65474 displays an ADCC effect that contributes to tumor suppression in the OV90 model. AT65474 was shown to be well tolerated in all xenograft mouse models and a pre-tox study in non-human primates. The combined data support further development of AT65474.​ Citation Format: Chen Zhong, You Xue Wu, Xi Yun Zhang, Yin Wen Cheng, Qian Wang, Ping Du, Shao Jun Liu, Bin Zou. Discovery of AT65474, a highly selective anti-CLDN6 ADC with a proprietary payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3138.

Abstract 3137: Development of AT2604, a highly efficacious ADC targeting alkaline phosphatases ALPP and ALPPL2​

Abstract Placental alkaline phosphatases, ALPP and ALPPL2, are highly homologous, membrane-bound proteins involved in fetal development. Despite having limited expression in normal tissues, they are highly differentiated in tumor cells making them ideal targets for antibody drug conjugate (ADC) development. AT2604 is an ADC that consists of a humanized IgG1 anti-ALPP/ALPPL2 antibody that displays specificity over other human alkaline phosphatases and is cross reactive to NHP ALPPL2 but not to murine ALPPL2. The anti-ALPP/ALPPL2 antibody shows high target binding affinity and internalization into ALPP and ALPPL2 positive cells.​ AT2604 utilizes AxcynCYSTM technology for site-specific conjugation to achieve a highly homogeneous (>97%) DAR4 ADC product. The payload is the clinically validated microtubule disrupting agent monomethyl auristatin E (MMAE) conjugated to the antibody via a protease-cleavable peptide linker approved for ADC use. When evaluated in mouse xenograft models of gastric (NCI-N87) and pancreatic (HPAC) cancer, AT2604 displayed strong tumor growth inhibition (>90%) at 1 mg/kg (QW3 × 2 dosing) in both models. Furthermore, a pre-tox study in non-human primates concluded AT2604 has a HNSTD of 10 mg/kg (QW3 × 3 dosing) and did not exhibit any non-reversible adverse effects; implying that AT2604 possesses an improved safety profile over other vedotin-based ADCs. Taken together, the data supports the continued development of AT2604 towards evaluation in human trials.​ Citation Format: Bryan K. Yeung, Xiao Hui Koh, Wei Li Ling, Weiqi Tan, You Xue Wu, Xi Yun Zhang, Hong Yee Tan, Shao Jun Liu, Chen Zhong, Bin Zou. Development of AT2604, a highly efficacious ADC targeting alkaline phosphatases ALPP and ALPPL2​ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3137.

Abstract 743: Using a novel NextGen linker system to generate a conditionally active biologic (CAB) anti-nectin4-ADC demonstrates improved efficacy in pancreatic PDX cancer models and improved tolerability and toxicity profile in non-human primates

Abstract Antibody-drug conjugates (ADCs) are a promising treatment for various forms of cancer. The treatment efficacy is often limited by on-target/off-tumor toxicity caused by the antigen expression in healthy tissue and by unwanted payload release outside the tumor caused by cleavage of the linker via circulating proteases. Our conditionally active biologics (CAB) technology addresses the on-target/off-tumor toxicity and assists in reducing the off-target/off-tumor toxicity by reducing binding to the target under normal physiological conditions (1). To eliminate the remaining off-target/off-tumor toxicity, we developed a novel linker with superior serum stability, solubility, and tumor-specific payload release. Conjugation can be accomplished using regular IgG backbones without the need for sequence modification. Here we report the in vitro and in vivo characterization of a NextGen linker system to generate a CAB anti-Nectin4-ADC that combines the advantages of CAB antibody pH selectivity with the new linker technology. In vivo efficacy data demonstrated complete tumor regression in several cell line derived xenograft models as well as superior efficacy to an enfortumab vedotin analogue in a patient derived pancreatic cancer model. PK and tox data from a toxicology study in non-human primates will be presented. In addition, data demonstrating the influence of linker technology on specific cancer models will also be presented. In conclusion, the NextGen Nectin4 CAB ADC represents a potentially more effective treatment with increased safety in the clinic. (1) Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ,and Short JM Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Proc Natl Acad Sci U S A 2021;118 Citation Format: Jing Wang, Jian Chen, Gerhard Frey, Haizhen Liu, Charles Xing, Kathryn Woodard, Hwai Chang, William J. Boyle, Jay M. Short. Using a novel NextGen linker system to generate a conditionally active biologic (CAB) anti-nectin4-ADC demonstrates improved efficacy in pancreatic PDX cancer models and improved tolerability and toxicity profile in non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 743.

Abstract 4702: MediLink’s TMALIN ADC linker technology: Tumor microenvironment specific extracellular and intracellular double cleavage mechanism for better efficacy and expanded target space

Abstract Antibody-drug conjugates (ADC) are a type of the fastest growing anticancer drug modalities. However, the traditional ADC technologies constrain on therapeutic development, requiring overexpressed targets, high internalizing antigens and efficient intracellular processing. To this end, MediLink’s TMALIN platform was developed, with an irreversible pyrimidine coupling anchor and a valyl dipropyl-lysyl glycinamide-methylene (-VK*G-NHCH2-O-) cleavable sequence. The TMALIN platform is featured as a high hydrophilicity linker-payload, remarkable stability in circulation, coupled with an efficient payload release mechanism which is achieved through the cleavage both extracellularly in the tumor microenvironment and intracellularly within lysosomes. A site-specific and homogeneous conjugation using TMALIN platform provides highly stable ADC in circulation. It is evidenced by less than 2% payload drop-off after a 21-day incubation in plasma under physiological conditions in vitro. This feature is observed in over 7 ADCs developed using the TMALIN platform. Additionally, the high stability was also illustrated by the pharmacokinetic profiles in NHP studies and clinical trials, with the overlapped curves of ADC and TAb in all TMALIN platform-based ADCs. Apart from the stability, a battery of in vitro and in vivo studies showed the linker-payload efficiently releases the payload in both tumor cells and the tumor microenvironment. Due to the high stability in circulation and efficient cleavage within tumors, the TMALIN platform ADCs have shown promising efficacy and good safety windows in preclinical studies. In xenograft mouse models, including those with low antigen expression, these ADCs have demonstrated tumor regressions and good tolerability. This highlights the potential of the TMALIN platform in improving the efficacy of ADCs and expanding their therapeutic applications. Furthermore, favorable safety profiles have been observed in clinical trials and/or pivotal non-human primate (NHP) studies for ADCs developed using the TMALIN platform. No drug-related adverse effects in organs were identified including the lungs, liver, or kidneys for those ADCs. In summary, the TMALIN platform addresses the unmet need of ADCs by releasing the payload extracellularly in tumors and tumor microenvironments on top of the known intracellular lysosomal cleavage mechanisms. This enables the advancement of ADC development, and has the potential to expand the field of ADC therapy and improve patient outcomes. Citation Format: Tongtong Xue, Liang Xiao, Qigang Liu, Shuai Song, Ailin Shan, Xiaozhou Zhang, Sasha Stann, Jiaqiang Cai. MediLink’s TMALIN ADC linker technology: Tumor microenvironment specific extracellular and intracellular double cleavage mechanism for better efficacy and expanded target space [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4702.

Abstract 5302: CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors

Abstract INTRODUCTION: CD137 is a TNF receptor superfamily (TNFRSF9) costimulatory receptor expressed by T cells and NK cells. Clustering-mediated CD137 signaling enhances immune cell survival, proliferation, cytokine production and memory formation. CD40 (previously TNFRSF5) is a related superfamily member expressed primarily on B cells and antigen-presenting cells (APCs) which upon clustering is also a key modulator of T cell responses. Mesothelin (MSLN) is highly upregulated on tumor cells in ovarian and pancreatic cancer and select other solid tumour histologies. To avoid systemic toxicity, CB699 is designed to be immunologically active in the presence of any two of its three binding targets, thus enhancing both immune-immune and tumour-immune crosstalk. We present here the preclinical pharmacology of CB699. EXPERIMENTAL PROCEDURES: Binding of CB699 to CD40, CD137 or MSLN was measured by surface plasmon resonance and by flow cytometry. CD137 and/or CD40 agonism, immune cell activation and cytokine secretion were evaluated in vitro using reporter gene assays and primary cell coculture assays in the presence/absence of MSLN-expressing tumor cells. The ability of CB699 to enhance tumor cell killing was characterized in 3D spheroid cocultures. CB699 toxicology and toxicokinetics was assessed in a dose range-finding study in cynomolgus macaques. CB699 was also assessed in vivo with a human CD34+ cell engrafted mouse model. RESULTS: CB699 binds to primary and immortalized cells that express CD40, CD137 or MSLN, with low nanomolar affinity. CB699 also binds to cells from primary disaggregated human solid tumors. CB699 induces CD137 reporter cell activity in cocultures containing cells expressing either CD40 or MSLN. Additionally, CB699 induces CD40 reporter cell activity in cocultures containing cells expressing either CD137 or MSLN. CB699 induces IL-2 secretion and upregulation of CD86 on B cells in primary cocultures containing healthy human PBMCs and MSLN expressing cancer cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB699 and was coupled with immune cell expansion. CB699 showed significant pharmacology in vivo with immune cell engrafted mice. Finally, CB699 was well tolerated in a dose range finding non-human primate toxicology study. CONCLUSIONS: CB699 is a novel MSLN-binding CD137 and CD40 dual agonist that selectively enhances immune cell activity by enhancing both T cell and APC signaling axes. CB699 is positioned to enter clinical development. Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Holly Spencer, Minjung Song, Timothy Wong, Phillip D. Bartlett, Kieran Williams, Lukas Jasaitis, Philip Bland-Ward. CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5302.

Abstract 7481: The allosteric STING agonist CRD3874 is systemically tolerated demonstrates systemic tolerability

Abstract Background: The activation of STING by CDN bind site targeting ligands leads to the generation of Type I interferons as well as pyroptosis and autophagy of the cell expressing STING due to STING’s ability to transport protons across the endoplasmic reticulum. CDN site targeting STING agonists tested in the clinic thus far have shown limited systemic tolerability as well as a tendency to kill the very immune cells that they are designed to activate. CRD3874 is an allosteric small-molecule human STING agonist with a unique binding mode that generates robust Type I interferon responses while simultaneously blocking STINGs proton transport activity that is associated with pyroptosis and autophagy. Methods: Allosteric binding was established using radioligand binding assays. Anti-cancer activity of CRD3874-SI by the IV route was evaluated in human STING KI C57/BL6 mice (genOway, France). Inflammasome and autophagy markers were studies in human and mouse cells. Results: In contrast with competitive STING agonists, CRD3874 potentiates the binding of radiolabeled cGAMP to STING. Treatment of immune cells with CRD3874 or other STING agonists, led to dose dependent increases in Type1 interferons and other pro-inflammatory cytokines. However, unlike other STING agonists, activation of STING by CRD3874 did not lead to induction of autophagy or Inflammasome markers in cells or mice. Intravenous administration of CRD3874-SI in human STING knock-in mice caused tumor regression and survival benefit in multiple murine tumor models. The compound was well tolerated at high doses in the primate GLP study when administered intravenously and caused dose and exposure dependent increases in cytokines. This profile of retaining the high efficacy of a STING agonist while demonstrating systemic safety is unique to CRD3874-SI. Conclusions: CRD3874 is a systemically administered STING agonist with promising single agent activity and an excellent IV safety profile in NHP. An investigator sponsored FIH Phase 1 trial with CRD3874-SI has been initiated at Memorial Sloan Kettering, NY, in sarcoma, MCC patients under the supervision of Dr. Ciara Kelly (NCT06021626). Citation Format: Monali Banerjee, Sandip Middya, Ritesh Shrivastava, Anindita Middya, Nagaswamy Mane, Nidhi Rawat, Thanilsana Soram, Debjani Chakraborty, Rajib Ghosh, Rubeena R. Mansuri, Dharmendra Yadav, Anuj Gautam, Anuj Singh, Kavita Puniya, David Pryde, Sourav Basu, Fabiane Sonego, Gaelle H. Martin, Kader Thiam, Arjun Surya. The allosteric STING agonist CRD3874 is systemically tolerated demonstrates systemic tolerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7481.

Abstract 2734: RC148, a novel bispecific antibody targeting PD-1 and VEGF for cancer immunotherapy

Abstract Vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic factors in modulating tumor microenvironment (TME), and the concentration of VEGF in TME is significantly higher than that of peripheral. Inhibiting VEGF can improve immunotherapy efficacy by modulating the TME, preventing angiogenesis and regulating immune cell populations. Combined anti-VEGF and anti-Programmed cell death protein 1 (PD-1)/Programmed cell death ligand (PD-L1) therapies have exerted potent antitumor effect in preclinic and clinic studies. Therefore, we developed a novel humanized IgG1 silenced bispecific antibody RC148 to target VEGF and PD-1. RC148 allows dimeric VEGF crosslinking and gradually enhances PD-1 binding activity, potentially reduces systemic toxicity of anti-VEGF therapy and enhances antitumor potency via VEGF trigged enrichment in tumor areas. In preclinical studies, RC148 blocks the binding of VEGF to VEGFR and PD-1 to PD-L1, prevents the growth and migration of human umbilical vein endothelial cells, enables efficient T cell activation, exhibited synergistic antitumor effects in rodent models and showed safety profiles in non-human primate studies. Citation Format: Jianmin Fang, Dong Li, Kaiyue Wu, Sisi Wang, Du Li, Guorui Zhao, Shenjun Li, Jing Jiang. RC148, a novel bispecific antibody targeting PD-1 and VEGF for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2734.

Abstract 6719: TriTCE Co-Stim: A next generation trispecific T cell engager platform with integrated CD28 costimulation, engineered to improve responses in the treatment of solid tumors

Abstract Bispecific T cell engagers (TCEs) have exhibited clinical successes in the treatment of hematological cancers, while treatment of solid tumors remains a challenge. Treatment of solid tumors with conventional CD3-engaging TCEs can result in limited T cell proliferation and recruitment to the tumor site, and treatment related T cell anergy, thus restricting the ability of bispecific TCEs to inhibit growth of these poorly infiltrated tumors and rapidly growing tumors. Next generation tumor-targeting, trispecific T cell engagers with integrated costimulation (TriTCE Co-Stim) have the potential to provide more durable responses and re-invigorate T cell responses by mediating integrated signaling through CD3 (signal 1) and co-stimulation through CD28 (signal 2). Superagonist anti-CD28 antibodies activate T cells but resulted in clinical toxicities with severe cytokine release syndrome (CRS). Therefore, achieving a balance between signal 1 and 2 is critical for optimal T cell activation and proliferation. Using our AzymetricTM and EFECTTM platforms, we generated heterodimeric TriTCE Co-Stim antibody formats with various geometries and affinities to optimize T cell response and widen the therapeutic window for the treatment of solid tumors. We have previously described screening and interrogation of different TriTCE Co-stim formats and paratope affinities, and the selection of a lead format with optimized CD3 and CD28 T cell activation. Here, we have further expanded our validation of the TriTCE Co-stim platform with our lead CLDN18.2 TriTCE co-stim format, to include a tolerability assessment in nonhuman primates.Our lead TriTCE Co-Stim format exhibited a favorable safety profile in vitro and mediated target-dependent induction of T cell activity, with enhanced proliferation, survival, and T cell-mediated cytotoxic potency compared to bispecific TCEs. In a predictive in vivo model of CRS, our lead TriTCE Co-Stim did not exhibit systemic toxicity or peripheral cytokine release. TriTCE Co-Stim mediated enhanced tumor growth regression in vivo with an increase of T cells within the tumor and no increase of T cells within the periphery. Repeat doses of the lead TriTCE Co-Stim molecule at 3 mg/kg were well-tolerated in nonhuman primates with no abnormal clinical signs. Minor decreases in body weight and food consumption were observed. Clinical pathology changes were consistent with a mild systemic inflammatory response. In summary, our lead TriTCE co-stim molecule has enhanced target-dependent antitumor activity vs. bispecific benchmarks and is tolerated a murine CRS model and repeat dose non-human primate study. These data suggest TriTCE Co-stim may provide tolerable and more durable antitumor responses and contribute to improved clinical outcomes. Citation Format: Lisa Newhook, Purva Bhojane, Kurt Stahl, Nichole E. Escalante, Peter Repenning, Diego Perez Escanda, Polly Shao, Maya C. Poffenberger, Alec Robinson, Kesha Patel, Alexandra Livernois, Chayne L. Piscitelli, Nicole Afacan, Paul A. Moore, Nina E. Weisser, Thomas Spreter von Kreudenstein. TriTCE Co-Stim: A next generation trispecific T cell engager platform with integrated CD28 costimulation, engineered to improve responses in the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6719.

Pump and sway: Wild primates use compliant supports as a tool to augment leaping in the canopy.

Despite qualitative observations of wild primates pumping branches before leaping across gaps in the canopy, most studies have suggested that support compliance increases the energetic cost of arboreal leaping, thus limiting leaping performance. In this study, we quantified branch pumping behavior and tree swaying in wild primates to test the hypothesis that these behaviors improve leaping performance. We recorded wild colobine monkeys crossing gaps in the canopy and quantitatively tracked the kinematics of both the monkey and the compliant support during behavioral sequences. We also empirically measured the compliance of a sample of locomotor supports in the monkeys' natural habitat, allowing us to quantify the resonant properties of substrates used during leaping. Analyses of three recordings show that adult red colobus monkeys (Piliocolobus tephrosceles) use branch compliance to their advantage by actively pumping branches before leaping, augmenting their vertical velocity at take-off. Quantitative modeling of branch resonance periods, based on empirical measurements of support compliance, suggests that monkeys specifically employed branch pumping on relatively thin branches with protracted periods of oscillation. Finally, an additional four recordings show that both red colobus and black and white colobus monkeys (Colobus guereza) utilize tree swaying to cross large gaps, augmenting horizontal velocity at take-off. This deliberate branch manipulation to produce a mechanical effect for stronger propulsion is consistent with the framework of instrumental problem-solving. To our knowledge, this is the first study of wild primates which quantitatively shows how compliant branches can be used advantageously to augment locomotor performance.

Bearded capuchin monkeys as a model for Alzheimer's disease.

The absence of a natural animal model is one of the main challenges in Alzheimer's disease research. Despite the challenges of using nonhuman primates in studies, these animals can bridge mouse models and humans, as nonhuman primates are phylogenetically closer to humans and can spontaneously develop AD-type pathology. The capuchin monkey, a New World primate, has recently attracted attention due to its skill in creating and using instruments. We analyzed one capuchin brain using structural 7T MRI and performed a neuropathological evaluation of three animals. Alzheimer-type pathology was found in the two of the capuchins. Widespread β-amyloid pathology was observed, mainly in focal deposits with variable morphology and a high density of mature plaques. Notably, plaque-associated dystrophic neurites associated with disruption of axonal transport and early cytoskeletal alteration were frequently found. Unlike in other species of New World monkeys, cerebral arterial angiopathy was not the predominant form of β-amyloid pathology. Additionally, abnormal aggregates of hyperphosphorylated tau, resembling neurofibrillary pathology, were observed in the temporal and frontal cortex. Astrocyte hypertrophy surrounding plaques was found, suggesting a neuroinflammatory response. These findings indicate that aged capuchin monkeys can spontaneously develop Alzheimer-type pathology, indicating that they may be an advantageous animal model for research in Alzheimer's disease.