Murine Sequences Research Articles

Structure-Based Engineering of Monoclonal Antibodies for Improved Binding to Counteract the Effects of Fentanyl and Carfentanil.

The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues (F/FAs) in both street mixtures and counterfeit pills. To expand current treatment options, drug-targeting monoclonal antibodies (mAbs) offer a viable therapeutic for both pre- and postexposure clinical scenarios. This study reports the isolation, in vitro characterization, and in vivo efficacy of two murine mAb families targeting fentanyl, carfentanil, or both. Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil, crystal structures of mAbs in complex with fentanyl or carfentanil were analyzed to identify key residues involved in ligand binding in murine versus humanized structures, and site-directed mutagenesis was used to verify their functional importance. The structural analysis identified a framework residue, Tyr36, present in the murine germline sequence of two mAbs, which was critical for binding to fentanyl and carfentanil. These studies emphasize the importance of structural considerations in mAb engineering to optimize mAbs targeting small molecules including opioids and other drugs of public health interest.

Abstract 113: Generation and validation of a novel humanized IL-31/IL-31RA/OSM/OSMR mouse model

Abstract Oncostatin M (OSM) and interleukin-31 (IL-31) are two pleiotropic cytokines that share a common signaling receptor subunit, the OSM receptor beta (OSMR). Both cytokines are released by monocytes, macrophages, dendritic cells and T lymphocytes in inflammatory situations, and upon binding to their respective receptors, induce the production of inflammatory cytokines (such as IL-6), chemokines, and other cellular growth factors during tissue repair, mainly via the JAK/STAT signal pathway. As OSM and IL-31 are reported to be involved in a variety of physiological and pathological processes, targeting IL-31 or OSM signaling is of therapeutic interest. To provide a novel preclinical platform for testing novel modulators of these signaling axes, a humanized IL-31/IL-31RA/OSM/OSMR mouse model was generated. First, single gene knock-in mice were constructed by gene editing technology; multigenic knock-in mice were then obtained by breeding, yielding a quadruple humanized model in which the murine cytokine sequences were replaced by full-length human cytokine sequences in situ, while the murine receptor sequences were replaced by chimeric sequences comprised of the human extracellular region and murine intracellular regions in situ. mRNA expression confirmed expression of IL-31, IL-31RA, and OSMR in this model, while ELISA confirmed expression of human OSM protein in the model without evidence of murine OSM expression. Next, the signaling capacity of the humanized OSMR receptor was confirmed: mouse embryonic fibroblasts (MEFs) isolated from humanized B-hOSM/hOSMR mice were capable of responding to human OSM to release IL-6, while wild-type MEFs could only respond to murine OSM. Subsequently, the ability of IL-31RA antibodies to alleviate scratching was tested in a chemically induced model of atopic dermatitis. Compared to mice receiving control antibodies, scratching frequency in humanized B-hIL31/hIL31RA/hOSMR mice treated with nemolizumab was attenuated (analog antibody was generated in-house). Finally, blood counts and flow cytometric profiling indicates that the humanized mice have a normal frequency of leukocytes and lymphocytes. Taken together, this quadruple humanized IL-31/IL-31RA/OSM/OSMR mouse model is a promising preclinical tool that can be used to evaluate novel modulators of this signaling pathway prior to clinical development. Citation Format: Linlin Wang, Lei Zhao, Meiqi Zhang, Zhi Zhang, Zhen Chen, Mari Kuraguchi, Jingjing Lian, Jiansu Zhang. Generation and validation of a novel humanized IL-31/IL-31RA/OSM/OSMR mouse model [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 113.

Abstract 5339: Establishment of a humanized IL5/IL5RA mouse model to evaluate modulators of eosinophilic inflammation in vivo

Abstract IL-5/IL-5R signaling plays a key role in the maturation, activation, survival, and recruitment of eosinophils to sites of inflammation. Targeting this pathway is an attractive therapeutic strategy for certain inflammatory diseases, such as asthma. Here, we generated a novel humanized IL5/IL5RA model (B-hIL5/hIL5RA) to examine the utility of human IL-5/IL5R-targeting therapeutic candidates. Specifically, the entire murine Il5 coding region was replaced by the corresponding human IL5 sequence, and the human IL5RA sequence coding the extracellular domain was inserted to replace the corresponding murine sequence. Successful generation of the model was confirmed by expression of human IL-5 by ELISA and IL5RA by flow cytometry. Furthermore, the proportion of eosinophils in the blood of B-hIL5/hIL5RA mice increased after stimulation with human IL5 recombinant protein, indicating that the humanized IL5RA protein is functional. Next, the ability of the humanized mice to establish asthma-like disease was tested using ovalbumin: following a 14 day sensitization period, mice were challenged with nebulized OVA daily from day 21 until the endpoint; treatment with mepolizumab or benralizumab analog was performed. Flow cytometric analysis of the bronchioalveolar fluid indicates that treatment with mepolizumab or benralizumab analog significantly lowered the number of eosinophils when compared to the isotype control in homozygous B-hIL5/hIL5RA mice. Blood counts and quantification of lung H&E sections also confirm that the treatments lowered the number of eosinophils in circulation and lung tissue, respectively. Taken together, these data indicate that B-hIL5/hIL5RA mice are a suitable preclinical model to test novel modulators of human IL-5/IL-5RA signaling. Citation Format: Suman Zhao, Shujin Zhang, Zhen Chen, Liya Yang, Mari Kuraguchi, Jiangfeng Yuan, Heiling Shen. Establishment of a humanized IL5/IL5RA mouse model to evaluate modulators of eosinophilic inflammation in vivo [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 5339.

Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone

Single-cell RNA sequencing has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-CAR, osteo-CAR, pre-osteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by Postn expression. Osteo-X, osteo-CAR, and pre-osteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in pre-osteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single cell RNA sequencing datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.

Oleanolic acid inhibits the tumor progression by regulating Lactobacillus through the cytokine-cytokine receptor interaction pathway in 4T1-induced mice breast cancer model

The therapeutic mechanism of oleanolic acid (OA) in breast cancer has been widely reported, but little has been known about the combined effects of transcriptome and gut microbiome. In this study, the phenotypic effect of oleanolic acid on mice was tested at the end of the administration cycle, and RNA sequencing on murine tumor tissue and 16S-rRNA sequencing on intestinal contents were conducted to analyze gene expression profiles and microbial diversity between the control group and OA treated group using 4T1-induced mice breast cancer model. As a result, it has been confirmed that oleanolic acid would play a significant inhibitory effect on the development of breast tumors in mice. Based on the integrative analysis of the transcriptomic and metagenomic data, it was found that the abundance of Lactobacillus in the intestinal flora of mice significantly increased in the OA group. Moreover, the up-regulation of Il10 had a significant effect on inhibiting the tumor progression, which played a role through cytokine-cytokine receptor interaction pathway.

Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme's self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

Heterobivalent Peptide Agonists Comprising Covalently Linked Protease Activated Receptor 1 and Protease Activated Receptor 3 Peptides Mimic the Cytoprotective Cell Signaling Activities of Activated Protein C

Introduction: Activated protein C (APC) exhibits both anticoagulant and cytoprotective cell signaling actions, including anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. Extensive in vitro and in vivo data for APC's cell signaling benefits identified requirements for two G-protein coupled receptors (GPCR), protease-activated receptor (PAR) PAR1 and PAR3 which are cleaved by APC at the noncanonical sites (R46 and R41, respectively). Agonism of PARs following proteolysis of the N-terminal tail is driven by the newly revealed N-terminus and related synthetic peptides, e.g., for APC, PAR1 peptides (P1) beginning with N47 or PAR3 peptides (P3) beginning with G42. We found that the non-canonical P1-47-66 and P3-42-65 peptides mimic APC in that each is anti-inflammatory by reducing NLRP3-driven caspase-1 activity in human THP-1 cells (Healy LD et al, J Thromb Haemost 2021;19:269-280). Remarkably, these two peptides act synergistically to attenuate activity of caspase-1 at low concentrations where either peptide alone has little activity. Thus, we undertook studies of P1 peptides that were covalently linked to P3 peptides for ability to mimic APC's anti-inflammatory and endothelial barrier stabilization actions. Methods: Monocytes (THP-1 cells) were differentiated into macrophages using PMA, then pretreated with APC or peptides prior to stimulation with lipopolysaccharide and adenosine triphosphate to induce caspase-1 whose activity was measured using a luminescent caspase-1 substrate. Endothelial barrier stability was monitored for transendothelial electrical resistance (TEER) using an electric cell-substrate sensing system (iCelligence apparatus) when cultured human EAhy926 or murine aortic endothelial cells (EC) were exposed to thrombin treatment (0.25 nM) plus or minus treatment with P1 and/or P3 peptides or G10-related peptides. Synthetic P1 and P3 sequence-derived peptides of various compositions were made, and bivalent P1:P3 peptides were made using oligo-Gly-peptide linkers containing from 4 to 14 Gly residues. Results: Using THP-1 cell anti-inflammatory assays for various peptides, studies showed that P1-47-55 was as potent as P1-47-66 and that the N-terminal N47 was essential. In anti-inflammatory activity assays, some P3 peptides shorter than P3-42-65 (e.g., P3-44-54, P3-51-65, and P3-51-59) were as active as P3-42-65. Remarkably, this is the first example for any PAR-agonist peptide that does not require a native-like sequence with an intact free alpha-amino N-terminus to agonize the PAR response. For bivalent P1:P3 studies, we covalently linked P3-47-55 to P3-51-65 using oligo-Gly linkers with 4-14 Gly residues. In anti-inflammatory activity assays, the maximal activity was seen for a 10-Gly linker. The bivalent peptide comprising P1-47-55-[Gly(10 residues)]-P3-51-65, designated as the G10 peptide, was much more potent than any P1 peptide or P3 peptide in anti-inflammatory THP-1 cell assays in dose-response studies. In TEER studies of thrombin-challenged EAhy926 EC where APC markedly attenuates thrombin-induced endothelial barrier dysfunction, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was much more potent than the P1-47-55 peptide or any combination of P1-47-55 with a P3 peptide. The P3 peptides alone were very weak. In TEER studies of thrombin-challenged murine aortic EC where murine APC markedly attenuates thrombin-induced endothelial barrier dysfunction, the G10 peptide comprising murine PAR sequences mimicked murine APC's protective actions. Conclusions: Covalent linkage of a PAR1-derived peptide agonist to a PAR3-derived peptide agonist using a 10-Gly-residue linker provided the first heterobivalent PAR peptide GPCR agonist. This heterobivalent G10 peptide potently mimicked APC's cytoprotective activities of anti-inflammation and endothelial barrier stabilization. Molecular mechanisms for G10 likely involve both orthosteric and allosteric effects for PAR1 and PAR3 which can form heterodimers. Given APC's beneficial cell signaling effects in many preclinical injury models and given the increasing usage of peptide-based drugs, studies of the G10 peptide for beneficial APC-mimicking effects may help to identify important directions for future translational research.

Haematopoietic loss of KDM6A impairs cardiac recovery post myocardial infarction via pro-inflammatory myeloid cells

Abstract Background Ischaemic heart failure (IHF) remains a leading cause of death worldwide. Clonal haematopoiesis has emerged as a major risk factor for IHF and patients with CH have worse outcomes after acute myocardial infarction (AMI). CH is characterized by clonal expansion of a haematopoietic stem cell that carries a leukaemogenic mutation. Recently, acquired loss-of-function mutations in the histone demethylase KDM6A were identified as CH-drivers in IHF patients. KDM6A is frequently mutated in human cancer and acts as a tumour suppressor in acute myeloid leukaemia. However, the role of KDM6A in the regulation of cardiac damage after MI is not understood. Purpose Investigate the effect of leukocyte specific loss of KDM6A in the development of IHF after AMI in mice and humans. Methods/Results A mouse model of haematopoietic specific loss of Kdm6a (VaviCre-Kdm6aflox) combined with a model of myocardial injury was employed to investigate the role of Kdm6a in leukocytes in post-MI cardiac recovery. Firstly, following 28 days (D) post-MI, mice lacking Kdm6a in haematopoietic cells (KDM6A KO) showed significantly worse cardiac ejection fraction compared to controls (controls 43.98 ± 2.55 % vs KDM6A KO 31.86 ± 3.23 % recovery from baseline, P=0.009). This was accompanied by an increase in cardiac scar size at D28 post-MI (controls 22.46±5.25% vs KDM6A KO 56.83±11.1 % area, P<0.0001). Moreover, KDM6A KO mice showed peripheral blood monocytosis (controls 33793±5644 vs KDM6A KO 76227±14035 monocytes/ml blood, P =0.008) at D3 post-MI, which was accompanied by an increased influx of CD11b+ myeloid cells in the injured heart (7804±1023 vs 11444±1436 CD11b+ cells/mg tissue, P=0.05), as well as by an increased expression of pro-inflammatory marker TNF-α (controls 1.01±0.14 vs KDM6A KO 2.16±0.45 fold change, P=0.04) in the ischaemic myocardium. These data were corroborated by single-cell RNA sequencing (scRNA-seq) profiling of CD45+ cardiac leukocytes 3D post-MI. Here, transcriptomically distinct subsets of macrophages/monocytes showed upregulation of pro-inflammatory markers such as Il1-β, Nlrp3, Cxcl2, and Nfkb1 (P < 0.05, Bonferroni correction). Finally, scRNA-seq of circulating blood cells from IHF patients carrying KDM6A CH-driver mutations showed elevated levels of pro-inflammatory genes such as IL32, IL7R and CCL5 in CD14+ classical monocytes (P < 0.05, Bonferroni correction) compared to non-carriers. Conclusions In summary, this study shows that loss of KDM6A in myeloid cells amplifies acute cardiac inflammation and impairs recovery of left ventricular function after AMI accompanied by increased pro-inflammatory myeloid cells in the heart. Further interrogation of genes identified in the aforementioned murine and human sequencing data and subsequent in vitro mechanistic studies will be essential to identify the mechanism by which loss of KDM6A results in altered leukocyte function and worse HF prognosis post-MI.

Phase 2 Study of CD19 CAR T Cells with a Fully Human Binder for Large B-Cell Lymphoma in Relapse or Progression after a Murine Binder-Bearing CD19 CAR T-Cell Therapy

BACKGROUND T cells engineered with a CD19-targeted chimeric antigen receptor (CD19 CAR T cells) have revolutionized the treatment of high-risk, relapsed, or refractory large B-cell lymphoma (LBCL), but durable responses are only achieved in 30-40% of patients (pts). Outcomes are dismal in case of disease recurrence after CD19 CAR T-therapy; in this situation we have previously reported low efficacy and limited duration of response after a second infusion of the same CD19 CAR T-cell product (Gauthier, Blood, 2022). We observed that repeat CD19 CAR T-cell infusions were impaired by CD8+ T-cell responses against murine amino acid sequences located in the single chain variable fragment (scFv) of the CAR, which led to rapid immune rejection of CAR T cells. We hypothesized that CAR T cells engineered with a fully human scFv could circumvent anti-murine scFv immune responses in pts previously exposed to murine scFv-bearing CAR T cells. We report our phase 2 clinical trial results investigating treatment with fully human scFv-bearing CD19 CAR T cells (JCAR021) in R/R LBCL pts with relapsed or progressive disease after murine scFv-bearing CD19 CAR T-cell therapy. METHODS Pts with R/R LBCL were enrolled at the Fred Hutchinson Cancer Center on a phase II study (NCT03103971). Key eligibility criteria included: i) relapsed disease after complete response (CR) to prior CD19-targeted non-JCAR021 CAR T-cell therapy or persistent disease after partial response to prior CD19-targeted non-JCAR021 CAR T-cell therapy; ii) CD19-positive disease since completing the prior CD19-targeted CAR T-cell therapy. Pts received lymphodepletion (LD) with cyclophosphamide 300 mg/m2/d and fludarabine 30 mg/m2/d for 3 days. Cytokine release syndrome (CRS) and neurotoxicity (NT) were graded according to the Lee 2014 criteria and CTCAE v4.03 criteria, respectively. Disease response was evaluated at day +28 after JCAR021 infusion per PET-CT Lugano criteria. The planned sample size and CAR T-cell dose were n=27 and 7x10 6 CAR T cells/kg, respectively. RESULTS Pt characteristics, and outcomes are shown in the Table. All eight screened pts were enrolled, received LD, and JCAR021 infusion. Median age at enrollment was 63 (range: 41-77). Prior to lymphodepletion, 4 pts (50%) had bulky disease (largest lesion ≥5cm). All pts had extranodal disease. We observed elevated LDH prior to lymphodepletion in 6 pts (75%). Median prior lines of therapy was 5 (range: 3-7). Prior CD19 CAR T-cell product type was lisocabtagene maraleucel, n=4 (50%); axicabtagene ciloleucel, n= 3 (37%); or tisagenlecleucel, n=1. Response to the prior CD19 CAR T-cell therapy was CR in 5 pts (62%) and PR in 3 pts (38%). Median time from the first CAR T-cell infusion to relapse or progression was 165 days (range 49-434). Median time from the first CAR T-cell infusion to JCAR021 infusion was 304 days (range: 202-603). JCAR021 was administered at the dose of 7x10 6 (n=6, 75%) or 2x10 6 CAR T-cells/kg (n=2, 25%; dose reduction due to grade 5 NT in subject #6). We observed all-grade CRS in 4 pts (50%; grade 1, n=2; grade 2, n=2 not requiring vasopressors) and NT in 1 patient (dose limiting toxicity, grade 5 NT from intracerebral hemorrhage with concurrent acute kidney injury and toxic encephalopathy). At day+28 responses were seen in 3 of 8 pts (37%; CR, n=2; PR, n=1), all of whom received 7x10 6 CAR T-cells/kg. Two pts in CR after JCAR021 had bulky disease and elevated LDH prior to LD. In both pts, the prior CD19 CAR T-cell therapy had induced CR lasting ≥ 6 months. In the two evaluable responders (death in CR, n=1), we observed disease relapse or progression within 3 months. Median overall survival was 105 days (95%CI, 92-NA). We measured expansion of JCAR021 CAR T-cells in all pts. Exploratory analyses showed lower in vitro CAR T-cell expansion during manufacturing (fold expansion of CD8+ CAR T-cells, p<0.001) and lower in vivo CAR T-cell expansion (peak CD8+ CAR-T, p=0.002; AUC day0-28, p=0.01; JCAR021 dose, 7x10 6/kg) compared to pts receiving JCAR021 as their first CD19 CAR-T product (CAR-naïve cohort). CONCLUSION In LBCL pts with disease relapse or progression after a first CD19 CAR T-cell therapy, we observed low response rates and lack of durable responses after treatment with the fully human scFv-bearing product JCAR021, prompting early study termination. Lower CAR T-cell expansion during manufacturing in CAR-exposed pts suggest pre-existing T-cell dysfunction as a potential mechanism of failure.

Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene

At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the Staphylococcus aureus nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 105 TU/mL (±3.34 × 104 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions.

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies.

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells' (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo-derived senescence signature (SenSig) using a foreign body response-driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and "cartilage-like" fibroblasts as senescent and defined cell type-specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34-CSF1R-TGFβR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.

Abstract 2735: Development of a novel, fully human anti-CD19 chimeric antigen receptor for in vivo delivery via CD8-targeted fusosome

Abstract Background: We have developed a novel gene therapy platform based on fusogen biology that allows targeted delivery of CAR transgenes to “resting” T cells through systemic administration of a CD8-targeted paramyxovirus-based viral vector (VV). Therapies utilizing autologous CAR T cells have demonstrated success using the murine CD19-specific scFv, FMC63. These murine sequences have the potential to incite T cell and antibody responses against the CAR, which may play a role in CAR T cell elimination. This scenario increases in likelihood in the context of in vivo CAR delivery, where immunosuppression via lymphodepletion is not required. To reduce the potential for CAR-specific immunogenicity, we developed a novel, fully human CD19-specific CAR for use with our in vivo delivery platform. Methods: For efficacy-based screening of human CD19 binders, healthy donor T cells were transduced with VSV-g pseudotyped lentivirus containing second-generation fully human CD19 CARs. FMC63 CARs were produced in a similar fashion and used as benchmark controls for initial characterization studies. CD8-targeted fusogen was generated through targeted mutation to ablate binding to its native receptor. This “blinded” fusogen was subsequently engineered to display a novel scFv that is specific for human CD8α. VV encoding either FMC63 or human CD19 CARs were pseudotyped with CD8-specific fusogen to produce CD8-targeted fusosomes. CD19 CAR efficacy was analyzed in vitro via luciferase assays against NALM-6 or CD19KO NALM-6 tumor lines. Systemic NALM-6 in vivo tumor models (IV injection) were performed in NSG mice. Results: Here we show, our fully human CD19 CAR is comparable to FMC63 CAR in vitro using both short-term luciferase cytotoxicity and long-term Incucyte tumor killing assays. Furthermore, human CD19 CAR T cells demonstrate similar levels of tumor control NALM-6 tumor bearing mice treated with ex vivo generated CD19 CAR+ cells (Mean AUC [n = 3 donors]: FMC63 = 1.26e4, huCD19 = 4.86e4). Our lead human CD19 CAR, like FMC63, lacked killing and cytokine production when cultured with CD19KO NALM-6 tumor cells, demonstrating CD19 antigen specificity. Furthermore, CD8-targeted in vivo delivery of our fully human CD19 CAR showed reduction in NALM-6 tumor load similar to FMC63 CAR (Mean AUC [n = 2 donors]: FMC63 = 1.59e5, huCD19 = 5.06e4). Summary: In vivo delivery of our fully human CD19 CAR with CD8-targeted fusosomes affords tumor clearance comparable to FMC63-CAR in mice bearing NALM-6 tumors. We believe this fully human CD19 CAR has the potential to reduce the risk of immunogenicity compared to a CD19 CAR with the murine scFv FMC63 potentially allowing increased expansion and persistence of CD19 CAR T cells following in vivo delivery in patients. Citation Format: Jeremy M. Kinder, Christie Ciarlo, Chanel Athena Estrada, Neal van Hoeven, Vandana Chaturvedi, Garrett Zipp, Aaron Lampano, Adam J. Johnson, Kutlu Elpek, Terry J. Fry, Aaron E. Foster. Development of a novel, fully human anti-CD19 chimeric antigen receptor for in vivo delivery via CD8-targeted fusosome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2735.

Abstract 4592: In vivo efficacy evaluation of anti-human VEGFR2 antibodies in humanized B-hVEGFR2 mice

Abstract Angiogenesis, vascular development, vascular permeability, and embryonic hematopoiesis is partly regulated by VEGF binding to VEGFR2, which forms receptor dimers and activates downstream signaling pathways. In addition to developmental cues, VEGFR2 is also associated with disease progression for psoriasis, rheumatoid arthritis, diabetic retinopathy, and more significantly, tumor growth and drug resistance. Given this, inhibition of VEGFR2 signaling is actively being studied, and multiple VEGFR2 inhibitors have entered various phases of clinical studies and have shown promising results. To evaluate in vivo efficacy of anti-human VEGFR2 antibodies, we generated a human VEGFR2 knock-in (B-hVEGFR2) mouse model by replacing the murine Vegfr2 extracellular domain sequence with the corresponding human sequence. We validated human VEGFR2 gene and protein expression in B-hVEGFR2 mice by RT-PCR and flow cytometry, respectively. Furthermore, we observed an anti-tumor effect with anti-human VEGFR2 antibodies in homozygous B-hVEGFR2 mice subcutaneously implanted with MC38 colon cancer cells. Taken together, we established a novel preclinical B-hVEGFR2 mouse model for evaluation of VEGFR2-targeted immunotherapy. Citation Format: Lei Zhao, Chengzhang Shang, Rebecca Soto, Chonghui Liu, Zhaoxue Yu. In vivo efficacy evaluation of anti-human VEGFR2 antibodies in humanized B-hVEGFR2 mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4592.

Presence of type IIB procollagen in mouse articular cartilage and growth plate is revealed by immuno-histochemical analysis with a novel specific antibody

Type II collagen is the major fibrillar collagen in cartilage. It is synthesized in the form of precursors (procollagens) containing N- and C-terminal propeptides. The two main isoforms of type II procollagen protein are type IIA and type IIB procollagens, generated in a developmentally regulated manner by differential splicing of the primary gene transcript. Isoform IIA contains exon 2 and is produced mainly by chondroprogenitor cells while isoform IIB lacks exon 2 and is produced by differentiated chondrocytes. Thus, expression of IIA and IIB isoforms are reliable markers for identifying the differentiation status of chondrocytes but their biological function in the context of skeletal development is still not yet fully understood. Specific antibodies against IIA and IIB procollagen isoforms are already available. In this study, a synthetic peptide spanning the junction between exon 1 and exon 3 of the murine sequence was used as an immunogen to generate a novel rabbit polyclonal antibody directed against procollagen IIB. Characterization of this antibody by Western-blotting analysis of murine cartilage extracts and ELISA tests demonstrated its specificity to the type IIB isoform. Furthermore, by immunohistochemical studies, this antibody allowed the detection of procollagen IIB in embryonic cartilage as well as in articular cartilage and growth plate of young adult mice. Interestingly, this is the first antibody that has allowed the detection of procollagen IIB at both the intra- and extracellular level. This antibody therefore represents an interesting new tool for monitoring the spatial and temporal distribution of IIB isoforms in skeletal tissues of mouse models and for tracking the trafficking and processing of type IIB procollagen.

CD84: A Novel Target for CAR T-Cell Therapy for Acute Myeloid Leukemia

Relapsed or refractory acute myeloid leukemia (AML) patients have scarce treatment options despite the recent emergence of novel targeted therapies. Chimeric antigen receptor (CAR) T-cell therapies have been approved for the treatment of B-cell malignancies and multiple myeloma, but it remains very challenging to treat myeloid malignancies with this therapeutic modality, in part due to the absence of specific tumor antigens in these indications. Antigens expressed on AML cells are usually shared with healthy hematopoietic stem/progenitor cells (HSPC) and targeting them can lead to life-threatening on-target/off-tumor toxicity. CD84 antigen is an immunoreceptor member of the SLAM family (SLAMF5), whose expression has been reported in certain immune cells and in B-cell malignancies. In this study, we show for the first time that CD84 is overexpressed in AML and present the first results of CD84-targeting CAR T-cells for the treatment of this disease. We analyzed AML primary samples and found that CD84 is overexpressed on the surface of AML cells, both de novo and at relapse, while lower expression was detected in CD34+ HSPC. Furthermore, immunohistochemistry analysis of healthy human tissues showed that CD84 is not expressed in the parenchyma of liver, kidney, lung, myocardium, skin, or brain (cerebral cortex and cerebellum), although expression was detected in tissue monocytes/macrophages. This expression pattern is similar to that of CD123, a clinical stage and safe CAR T target in AML. Anti-CD84 CAR T cells were engineered using both fully human and murine CD84-binding sequences. The sequences of the variable regions of the light (VL) and the heavy (VH) chain were used to design different single-chain variable fragments (scFv), changing the order (VLVH or vice versa) and the linker length. In total, we designed 21 lentiviral second-generation CARs with an anti-CD84 scFv, a CD8α hinge and transmembrane domain, a 4-1BB costimulatory and a CD3ζ signaling domain. Some constructs were discarded due to failed lentiviral transduction or target recognition; the following CAR T-cells were characterized in depth: one with a fully human scFv, CD84.01 (VHVL), and 5 with different murine scFv, CD84.02 (VLVH), CD84.03 (VHVL), CD84.04 (VHVL), CD84.05 (VHVL), and CD84.06 (VHVL). All these CAR T-cells displayed cytotoxic activity towards, and were able to proliferate and secrete cytokines in vitro when exposed to the CD84high Kasumi-1 and the CD84low U937 AML cell lines (Figure 1A). We then analyzed their cytotoxicity against monocytes, B cells and T cells purified from the same donor as the CD84 CAR T cells were generated; all CART cells exerted some cytotoxic activity against their own T cells, which was much lower than that against Kasumi-1 cells. To assess the myeloablative potential of CD84 CAR T cells, we carried out cytotoxic assays against CD34+ HSPCs purified from 1) donor cord blood; 2) directly from donor bone marrow samples, or 3) an apheresis product of a donor for an allogeneic hematopoietic stem cell transplant. We found that the cytotoxic effect against CD34+ HSPC was much lower than that against leukemic cells. Based on these in vitro results, the CARTs CD84.02 and CD84.03 were tested for their efficacy in a human NSG xenograft mouse model of AML (Kasumi-1). Although CD84.02 appeared to be more effective than CD84.03, both were able to control the disease for at least 43 days (Figure 1B), at which point no leukemic cells were detected in the bone marrow and spleen of CAR T-treated animals. In contrast, CD84 CAR T cells were found in these organs at the end of the experiment. In summary, here we report that CART cells directed against CD84 can eliminate AML cells both in vitro and in vivo. Our data support the therapeutic use of CD84 CAR T cells for relapsed/refractory (R/R) AML. However, due to its potential myelotoxicity, we encourage its first use in a clinical trial as a bridge therapy to allogeneic stem cell transplant for patients with R/R AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discovery of Schistosoma mekongi circulating proteins and antigens in infected mouse sera.

Schistosomiasis is a neglected tropical disease caused by an infection of the parasitic flatworms schistosomes. Schistosoma mekongi is a restricted Schistosoma species found near the Mekong River, mainly in southern Laos and northern Cambodia. Because there is no vaccine or effective early diagnosis available for S. mekongi, additional biomarkers are required. In this study, serum biomarkers associated with S. mekongi-infected mice were identified at 14-, 28-, 42-, and 56-days post-infection. Circulating proteins and antigens of S. mekongi in mouse sera were analyzed using mass spectrometry-based proteomics. Serine protease inhibitors and macrophage erythroblast attacher were down-regulated in mouse sera at all infection timepoints. In addition, 54 circulating proteins and 55 antigens of S. mekongi were identified. Notable circulating proteins included kyphoscoliosis peptidase and putative tuberin, and antigens were detected at all four infection timepoints, particularly in the early stages (12 days). The putative tuberin sequence of S. mekongi was highly similar to homologs found in other members of the genus Schistosoma and less similar to human and murine sequences. Our study provided the identity of promising diagnostic biomarkers that could be applicable in early schistosomiasis diagnosis and vaccine development.

Abstract 1652: A humanized mouse model of the promising immune checkpoint molecule B7-H4

Abstract B7-H4, a member of the B7-family of immune-regulatory ligands, has emerged in recent years as a promising target for immune checkpoint therapy development. B7-family ligands bind via extracellular tandem immunoglobulin-like domains to CD28-family receptors on lymphocytes to communicate co-stimulatory or co-inhibitory signals. B7-H4 remains an orphan ligand, although evidence from knockout mice suggests a co-inhibitory role and its expression on cancer cells and tumor-associated macrophages is associated with inhibition of proliferation, cell cycle progression, and cytokine secretion in T cells. B7-H4 is highly expressed in a variety of tumors (e.g., renal, gastric, breast, and ovarian carcinomas) and plays less well-appreciated roles in autoimmune diseases, viral infections, and maternal-fetal immunity. Negative regulation of T cell responses by B7-H4 has also been shown to induce tolerance of islet allografts in mouse models of Type I diabetes. A range of B7-H4-targeted immunotherapy agents have been successfully developed, including monoclonal antibodies, antibody-drug conjugates, B7-H4/CD3-targeting bispecific antibodies, and B7-H4-specific CAR-T cells. To evaluate the efficacy of a B7-H4 antibody, Biocytogen generated humanized B-hB7-H4 mice. Our targeting strategy was to replace the murine B7-H4 extracellular domain sequences with their corresponding human sequences. We verified mRNA and protein expression of human B7-H4 in the humanized B-hB7-H4 mice by RT-PCR and Western Blot, respectively. An in vivo efficacy study demonstrated that B7-H4 antibodies significantly inhibited tumor growth in B-hB7-H4 mice bearing tumors derived from the B16-F10 murine melanoma line. These data confirm that the B-hB7-H4 humanized mouse model is a powerful tool for evaluating the preclinical potential of B7-H4-targeting immune-therapeutics. Keywords: Humanized mice, B7-H4, Immunotherapy Citation Format: Ruili Lyu, Chonghui Liu, John Charpentier, Suman Zhao, Luke (Zhaoxue) Yu. A humanized mouse model of the promising immune checkpoint molecule B7-H4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1652.

Characterization of a splice variant of perilipin 5

Lipid storage provides energy stores for the body in times of increased oxidative requirements, such as fasting or exercise, but when the balance of lipid storage and mobilization is disrupted human health suffers. Implications for increased lipid storage include risk factors for developing type II diabetes mellitus and non‐alcoholic fatty liver disease. The perilipin family of proteins associate with the surface of lipid droplets and are essential to regulation of stored triacylglycerols. Perilipin 5 functions to maintain a balance between the storage of triacylglycerols in lipid droplets or their mobilization when energetically required. Several members of the perilipin gene family exhibit differential splicing, most notably perilipin 1 but reports exist of alternative messages for shortened forms of perilipins 2 and 3. We have identified a shortened form of perilipin 5 we have termed perilipin 5b. Western blots indicate that the protein, of approximately 35 kDa, is present in C2C12 cultured myoblasts and in heart and liver tissue from fed and fasted mice. Analysis of murine genomic DNA sequences reveals stop codons found in the introns between exons 6 and 7 and 7 and 8, either of which could give rise to a protein of approximately 35 kDa, indicating the likelihood of the observed protein being a splice variant, rather than a post‐translationally modified protein. Using RT‐PCR we have identified multiple amplicons suggesting that perilipin‐5b contains a read through of intron 6 of the murine perilipin 5 gene. Mining transcriptome databases reveals several transcripts with retained introns. Analysis of the amino acid sequence of perilipin 5b indicates that this truncation would lack the reported C‐terminal domain essential for interactions with ATGL and CGI‐58 but would retain the PKA site found at Ser155. The function of perilipin 5b is yet unknown, but the fact that it is found primarily in heart tissue seems to suggest that it plays a role in the oxidation of the lipid droplet. These data are consistent with what is currently known about Perilipin‐5 and the other PAT family proteins. Determination of perilipin 5b function will give a more accurate picture of the overall mechanism of lipid storage and metabolism.

Spironolactone alleviates myocardial fibrosis via inhibition of Ets-1 in mice with experimental autoimmune myocarditis.

Spironolactone improves cardiac structure, function and prognosis in patients with heart failure and delays the progression of cardiac fibrosis. However, the exact underlying mechanism of this process remains to be elucidated. The present study therefore aimed to explore the protective effect and underlying mechanism of the aldosterone receptor antagonist, spironolactone, on myocardial fibrosis in mice with experimental autoimmune myocarditis (EAM). The EAM model was induced in BALB/c mice via immunization with murine cardiac α-myosin heavy chain sequence polypeptides. The cardiac function of the mice was assessed using echocardiography and the levels of inflammatory cytokines were quantified using ELISA. E26 transformation-specific sequence-1 (Ets-1) expression was knocked down using lentivirus-mediated small interference RNA. Total collagen deposition was assessed using Masson's trichrome and Ets-1, TGF-β1, Smad2/3, collagen I and III protein expression levels were detected using immunohistochemistry and western blotting. MMP-2 and MMP-9 mRNA expression levels and activity was determined using reverse transcription-quantitative PCR and gelatin zymography, respectively. The results of the present study demonstrated that spironolactone significantly improved myocardium hypertrophy, diastolic cardiac function and decreased myocardial inflammation and collagen deposition induced by EAM. Spironolactone treatment significantly inhibited Ets-1 and smad2/3 phosphorylation. In addition, inhibition of Ets-1 reduced the expression and activity of MMP-2 and MMP-9 and decreased cardiac fibrosis in EAM mice. The results indicated that the improvement of myocardial fibrosis by spironolactone may be associated with the TGF-β1/Smad-2/3/Ets-1 signaling pathway in EAM mice.

Identification and profiling of Trichinella spiralis circulating antigens and proteins in sera of mice with trichinellosis.

Trichinellosis is a zoonotic disease caused by the ingestion of the Trichinella nematode. With a worldwide incidence of approximately 10,000 cases per year, Trichinella spiralis is responsible for most human infections. There are no specific signs or symptoms of this parasitic infection. Muscle biopsy is the gold diagnostic standard for trichinellosis, but the technique is invasive and unable to detect the early stage of infection. Although immunodiagnostics are also available, antibody detection usually occurs after 3 weeks and prolonged up to 19 years after the acute phase. Therefore, additional diagnostic biomarkers must be identified to improve trichinellosis diagnosis. This study aimed to measure concentration changes in mouse serum proteins prior to T. spiralis infection and 2, 4 and 8 weeks after infection, and to identify T. spiralis circulating proteins and antigens using mass spectrometry-based proteomics. Mouse muscle-related proteins including inter-alpha-trypsin inhibitor heavy chain H2, a protein involved in the response to muscle tissue damage, were up-regulated in mouse sera during the T. spiralis larvae invasion. Additionally, 33 circulatory parasite proteins were identified in infected mouse sera. Notably, T. spiralis long-chain fatty acid transport protein 1 could be detected in the early stage of infection and peroxidasin-like protein was identified 2, 4 and 8 weeks after infection. Seventeen T. spiralis circulating antigens were detected in mouse immune complexes, with PX domain protein being found 2, 4 and 8 weeks after infection. Because peroxidasin-like protein and PX domain protein were detected at all post-infection time points, sequence alignments of these proteins were performed, which showed they are conserved among Trichinella spp. and have less similarity to the human and murine sequences. Integrative analysis of T. spiralis biomarkers throughout the course of infection may reveal additional diagnostic targets to improve early diagnosis of trichinellosis.