Preclinical efficacy and pharmacokinetics of an RNA-encoded T cell-engaging bispecific antibody targeting human claudin 6.

We present the preclinical pharmacology of BNT142, a lipid nanoparticle (LNP)-formulated RNA (RNA-LNP) encoding a T cell-engaging bispecific antibody that monovalently binds the T cell marker CD3 and bivalently binds claudin 6 (CLDN6), an oncofetal antigen that is absent from normal adult tissue but expressed on various solid tumors. Upon BNT142 RNA-LNP delivery in cell culture, mice, and cynomolgus monkeys, RNA is translated, followed by self-assembly into and secretion of the functional bispecific antibody RiboMab02.1. In vitro, RiboMab02.1 mediated CLDN6 target cell-specific activation and proliferation of T cells, and potent target cell killing. In mice and cynomolgus monkeys, intravenously administered BNT142 RNA-LNP maintained therapeutic serum concentrations of the encoded antibody. Concentrations of RNA-encoded RiboMab02.1 were maintained longer in circulation in mice than concentrations of directly injected, sequence-identical protein. Weekly injections of mice with BNT142 RNA-LNP in the 0.1- to 1-µg dose range were sufficient to eliminate CLDN6-positive subcutaneous human xenograft tumors and increase survival over controls. Tumor regression was associated with an influx of T cells and depletion of CLDN6-positive cells. BNT142 induced only transient and low cytokine production in CLDN6-positive tumor-bearing mice humanized with peripheral blood mononuclear cells (PBMCs). No signs of adverse effects from BNT142 RNA-LNP administration were observed in mice or cynomolgus monkeys. On the basis of these and other findings, a phase 1/2 first-in-human clinical trial has been initiated to assess the safety and preliminary efficacy of BNT142 RNA-LNP in patients with CLDN6-positive advanced solid tumors (NCT05262530).

Osteoclasts and Macrophages-Their Role in Bone Marrow Cavity Formation During Mouse Embryonic Development.

The formation of the bone marrow cavity is a prerequisite for endochondral ossification. In reviews and textbooks, it is occasionally reported that osteoclasts are essential for bone marrow cavity formation removing hypertrophic chondrocytes. Mice lacking osteoclasts or having functionally defective osteoclasts have osteopetrotic bones, yet they still form a bone marrow cavity. Here, we investigated the role of osteoclasts and macrophages in bone marrow cavity formation during embryogenesis. Macrophages can assist osteoclasts in matrix removal by phagocytosing resorption byproducts. Rank-deficient mice, lacking osteoclasts, and Pu.1-deficient mice, lacking monocytes, macrophages, and osteoclasts, displayed a delay in bone marrow cavity formation and a lengthening of the zone of hypertrophic chondrocytes. F4/80-positive monocyte/macrophage numbers increased by about fourfold in the bone marrow cavity of E18.5 Rank-deficient mice. Based on lineage-tracing experiments, the majority of the excess F4/80 cells were derived from definitive hematopoietic precursors of the fetal liver. In long bones of both Rank-/- and Pu.1-/- specimens, Mmp9-positive cells were still present. In addition to monocytes, macrophages, and osteoclasts, Ctsb-positive septoclasts were lost in Pu.1-/- specimens. The mineralization pattern was altered in Rank-/- and Pu.1-/- specimens, revealing a significant rise in transverse-oriented mineralized structures. Taken together, our findings imply that early on during bone marrow cavity formation, osteoclasts facilitate the entry of blood vessels and later the turnover of hypertrophic chondrocytes, whereas macrophages appear to play no major role. Furthermore, the absence of septoclasts in Pu.1-/- specimens suggests that septoclasts are either derived from Pu.1-dependent precursors or require PU.1 activity for their differentiation. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Only the Co-Transcriptional Activity of ß-Catenin Is Required for the Local Regulatory Effects in Hypertrophic Chondrocytes on Developmental Bone Modeling.

In hypertrophic chondrocytes, ß-catenin has two roles. First, it locally suppresses the differentiation of osteoclasts at the chondro-osseous junction by maintaining the pro-osteoclastic factor receptor activator of NF-κB ligand (RANKL) at low levels. Second, it promotes the differentiation of osteoblast-precursors from chondrocytes. Yet, ß-catenin is a dual-function protein, which can either participate in cell-cell adherens junctions or serve as a transcriptional co-activator in canonical Wnt signaling interacting with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors. Hence, whenever studying tissue-specific requirements of ß-catenin using a conventional conditional knockout approach, the functional mechanisms underlying the defects in the conditional mutants remain ambiguous. To decipher mechanistically which of the two molecular functions of ß-catenin is required in hypertrophic chondrocytes, we used different approaches. We analyzed the long bones of newborn mice carrying either the null-alleles of Lef1 or Tcf7, or mice in which Tcf7l2 was conditionally deleted in the hypertrophic chondrocytes, as well as double mutants for Lef1 and Tcf7l2, and Tcf7 and Tcf7l2. Furthermore, we analyzed Ctnnb1 mutant newborns expressing a signaling-defective allele that retains the cell adhesion function in hypertrophic chondrocytes. None of the analyzed Tcf/Lef single or double mutants recapitulated the previously published phenotype upon loss of ß-catenin in hypertrophic chondrocytes. However, using this particular Ctnnb1 allele, maintaining cell adhesion function, we show that it is the co-transcriptional activity of ß-catenin, which is required in hypertrophic chondrocytes to suppress osteoclastogenesis and to promote chondrocyte-derived osteoblast differentiation. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

CaMKII Signaling Stimulates Mef2c Activity In Vitro but Only Minimally Affects Murine Long Bone Development in vivo.

The long bones of vertebrate limbs form by endochondral ossification, whereby mesenchymal cells differentiate into chondrogenic progenitors, which then differentiate into chondrocytes. Chondrocytes undergo further differentiation from proliferating to prehypertrophic, and finally to hypertrophic chondrocytes. Several signaling pathways and transcription factors regulate this process. Previously, we and others have shown in chicken that overexpression of an activated form of Calcium/calmodulin-dependent kinase II (CaMKII) results in ectopic chondrocyte maturation. Here, we show that this is not the case in the mouse. Although, in vitro Mef2c activity was upregulated by about 55-fold in response to expression of an activated form of CaMKII (DACaMKII), transgenic mice that expressed a dominant-active form of CaMKII under the control of the Col2a1 regulatory elements display only a very transient and mild phenotype. Here, only the onset of chondrocyte hypertrophy at E12.5 is accelerated. It is also this early step in chondrocyte differentiation that is temporarily delayed around E13.5 in transgenic mice expressing the peptide inhibitor CaM-KIIN from rat (rKIIN) under the control of the Col2a1 regulatory elements. Yet, ultimately DACaMKII, as well as rKIIN transgenic mice are born with completely normal skeletal elements with regard to their length and growth plate organization. Hence, our in vivo analysis suggests that CaMKII signaling plays a minor role in chondrocyte maturation in mice.

ß-catenin activity in late hypertrophic chondrocytes locally orchestrates osteoblastogenesis and osteoclastogenesis.

Trabecular bone formation is the last step in endochondral ossification. This remodeling process of cartilage into bone involves blood vessel invasion and removal of hypertrophic chondrocytes (HTCs) by chondroclasts and osteoclasts. Periosteal- and chondrocyte-derived osteoprogenitors utilize the leftover mineralized HTC matrix as a scaffold for primary spongiosa formation. Here, we show genetically that ß-catenin (encoded by Ctnnb1), a key component of the canonical Wnt pathway, orchestrates this remodeling process at multiple levels. Conditional inactivation or stabilization of ß-catenin in HTCs by a Col10a1-Cre line locally modulated osteoclastogenesis by altering the Rankl:Opg ratio in HTCs. Lack of ß-catenin resulted in a severe decrease of trabecular bone in the embryonic long bones. Gain of ß-catenin activity interfered with removal of late HTCs and bone marrow formation, leading to a continuous mineralized hypertrophic core in the embryo and resulting in an osteopetrotic-like phenotype in adult mice. Furthermore, ß-catenin activity in late HTCs is required for chondrocyte-derived osteoblastogenesis at the chondro-osseous junction. The latter contributes to the severe trabecular bone phenotype in mutants lacking ß-catenin activity in HTCs.

Mechanical forces induce changes in VEGF and VEGFR-1/sFlt-1 expression in human chondrocytes.

Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1). The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.

Role of platelet-released growth factors in detoxification of reactive oxygen species in osteoblasts.

INTRODUCTION: Oxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2-ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2). METHODS: Platelets and PRGF were obtained from healthy human donors. HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors. RESULTS: The activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells. CONCLUSIONS: These results provide a new insight into PRGF's mode of action in osteoblasts. PRGF not only leads to increase the endogenous VEGF, but also it may be involved in preventing oxidative damage through the Nrf2-ARE signalling. Nrf2 activation via PRGF may have great potential as an effective therapeutic drug target in fracture healing.

Platelets display potent antimicrobial activity and release human beta-defensin 2.

Platelet-rich plasma (PRP) is a potent agent that improves soft tissue and bone healing. By the release of growth factors and cytokines, PRP is believed to locally boost physiologic healing processes. Recently, antimicrobial activity of PRP has been demonstrated against S. aureus strains. Major scientific effort is being put into the understanding and prevention of infections i.e. by delivery of antimicrobial substances. In previous studies we showed the ideal antibacterial activity-profile of the human beta-defensin 2 (hBD-2) for orthopaedic infections and therefore hypothesized that hBD-2 may be the effector of antimicrobial platelet action. Platelet concentrates were produced from human platelet phresis obtained from a hospital blood bank. They were screened by immunohistochemistry, Western Blot and ELISA for the human beta defensin-2. In vitro susceptibility to PRP was investigated by a standard disc diffusion test with or without pre-incubation of PRP with anti-hBD-2 antibody. SPSS statistical software was used for statistical analysis. PRP contains hBD-2 470 pg/10(9) platelets or 1786 pg/ml, respectively, (ELISA), which was confirmed by immunohistochemistry and Western Blot. In antimicrobial testing, PRP demonstrates effective inhibition of E. coli, B. megaterium, P. aeruginosa, E. faecalis and P. mirabilis. With this study we confirm the previously reported antimicrobial action of platelet concentrates i.e. PRP. In opposition to previously reported effects against gram positive bacteria our study focuses on gram negative and less common gram positive bacteria that do frequently cause clinical complications. We provide a possible molecular mechanism at least for E. coli and P. mirabilis for this effect by the detection of an antimicrobial peptide (hBD-2). This study may advocate the clinical use of PRP by highlighting a new aspect of platelet action.