High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.

Novel Frizzled-specific antibody-based Wnt mimetics and Wnt superagonists selectively activate WNT/ß-catenin signaling in target tissues.

WNTs are essential factors for stem cell biology, embryonic development, and for maintaining homeostasis and tissue repair in adults. Difficulties in purifying WNTs and their lack of receptor selectivity have hampered research and regenerative medicine development. While breakthroughs in WNT mimetic development have overcome some of these difficulties, the tools developed so far are incomplete and mimetics alone are often not sufficient. Here, we developed a complete set of WNT mimetic molecules that cover all WNT/ß-catenin-activating Frizzleds (FZDs). We show that FZD1,2,7 stimulate salivary gland expansion in vivo and salivary gland organoid expansion. We further describe the discovery of a novel WNT-modulating platform that combines WNT and RSPO mimetics' effects into one molecule. This set of molecules supports better organoid expansion in various tissues. These WNT-activating platforms can be broadly applied to organoids, pluripotent stem cells, and in vivo research, and serve as bases for future therapeutic development.

Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Development of selective bispecific Wnt mimetics for bone loss and repair.

The Wnt signaling pathway is intricately connected with bone mass regulation in humans and rodent models. We designed an antibody-based platform that generates potent and selective Wnt mimetics. Using this platform, we engineer bi-specific Wnt mimetics that target Frizzled and low-density lipoprotein receptor-related proteins and evaluate their effects on bone accrual in murine models. These synthetic Wnt agonists induce rapid and robust bone building effects, and correct bone mass deficiency and bone defects in various disease models, including osteoporosis, aging, and long bone fracture. Furthermore, when these Wnt agonists are combined with antiresorptive bisphosphonates or anti-sclerostin antibody therapies, additional bone accrual/maintenance effects are observed compared to monotherapy, which could benefit individuals with severe and/or acute bone-building deficiencies. Our data support the continued development of Wnt mimetics for the treatment of diseases of low bone mineral density, including osteoporosis.

Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis.

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis.

Glucocorticoids cause mandibular bone fragility and suppress osteocyte perilacunar-canalicular remodeling.

Osteocytes support dynamic, cell-intrinsic resorption and deposition of bone matrix through a process called perilacunar/canalicular remodeling (PLR). In long bones, PLR depends on MMP13 and is tightly regulated by PTH, sclerostin, TGFß, and glucocorticoids. However, PLR is regulated differently in the cochlea, suggesting a mechanism that is anatomically distinct. Unlike long bones, the mandible derives from neural crest and exhibits unique susceptibility to medication and radiation induced osteonecrosis. Therefore, we sought to determine if PLR in the mandible is suppressed by glucocorticoids, as it is in long bone. Hemimandibles were collected from mice subcutaneously implanted with prednisolone or vehicle containing pellets for 7, 21, or 55 days (n = 8/group) for radiographic and histological analyses. Within 21 days, micro-computed tomography revealed a glucocorticoid-dependent reduction in bone volume/total volume and trabecular thickness and a significant decrease in bone mineral density after 55 days. Within 7 days, glucocorticoids strongly and persistently repressed osteocytic expression of the key PLR enzyme MMP13 in both trabecular and cortical bone of the mandible. Cathepsin K expression was significantly reduced only after 55 days of glucocorticoid treatment, at which point histological analysis revealed a glucocorticoid-dependent reduction in the lacunocanalicular surface area. In addition to reducing bone mass and suppressing PLR, glucocorticoids also reduced the stiffness of mandibular bone in flexural tests. Thus, osteocyte PLR in the neural crest-derived mandible is susceptible to glucocorticoids, just as it is in the mesodermally-derived femur, highlighting the need to further study PLR as a target of drugs, and radiation in mandibular osteonecrosis.

Osteocyte-Intrinsic TGF-ß Signaling Regulates Bone Quality through Perilacunar/Canalicular Remodeling.

Poor bone quality contributes to bone fragility in diabetes, aging, and osteogenesis imperfecta. However, the mechanisms controlling bone quality are not well understood, contributing to the current lack of strategies to diagnose or treat bone quality deficits. Transforming growth factor beta (TGF-ß) signaling is a crucial mechanism known to regulate the material quality of bone, but its cellular target in this regulation is unknown. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix led us to hypothesize that TGF-ß controls bone quality through perilacunar/canalicular remodeling (PLR). Using inhibitors and mice with an osteocyte-intrinsic defect in TGF-ß signaling (TßRIIocy-/-), we show that TGF-ß regulates PLR in a cell-intrinsic manner to control bone quality. Altogether, this study emphasizes that osteocytes are key in executing the biological control of bone quality through PLR, thereby highlighting the fundamental role of osteocyte-mediated PLR in bone homeostasis and fragility.

Glucocorticoid suppression of osteocyte perilacunar remodeling is associated with subchondral bone degeneration in osteonecrosis.

Through a process called perilacunar remodeling, bone-embedded osteocytes dynamically resorb and replace the surrounding perilacunar bone matrix to maintain mineral homeostasis. The vital canalicular networks required for osteocyte nourishment and communication, as well as the exquisitely organized bone extracellular matrix, also depend upon perilacunar remodeling. Nonetheless, many questions remain about the regulation of perilacunar remodeling and its role in skeletal disease. Here, we find that suppression of osteocyte-driven perilacunar remodeling, a fundamental cellular mechanism, plays a critical role in the glucocorticoid-induced osteonecrosis. In glucocorticoid-treated mice, we find that glucocorticoids coordinately suppress expression of several proteases required for perilacunar remodeling while causing degeneration of the osteocyte lacunocanalicular network, collagen disorganization, and matrix hypermineralization; all of which are apparent in human osteonecrotic lesions. Thus, osteocyte-mediated perilacunar remodeling maintains bone homeostasis, is dysregulated in skeletal disease, and may represent an attractive therapeutic target for the treatment of osteonecrosis.

Activin A inhibits RANKL-mediated osteoclast formation, movement and function in murine bone marrow macrophage cultures.

The process of osteoclastic bone resorption is complex and regulated at multiple levels. The role of osteoclast (OCL) fusion and motility in bone resorption are unclear, with the movement of OCL on bone largely unexplored. RANKL (also known as TNFSF11) is a potent stimulator of murine osteoclastogenesis, and activin A (ActA) enhances that stimulation in whole bone marrow. ActA treatment does not induce osteoclastogenesis in stroma-free murine bone marrow macrophage cultures (BMM), but rather inhibits RANKL-induced osteoclastogenesis. We hypothesized that ActA and RANKL differentially regulate osteoclastogenesis by modulating OCL precursors and mature OCL migration. Time-lapse video microscopy measured ActA and RANKL effects on BMM and OCL motility and function. ActA completely inhibited RANKL-stimulated OCL motility, differentiation and bone resorption, through a mechanism mediated by ActA-dependent changes in SMAD2, AKT1 and inhibitor of nuclear factor κB (IκB) signaling. The potent and dominant inhibitory effect of ActA was associated with decreased OCL lifespan because ActA significantly increased activated caspase-3 in mature OCL and OCL precursors. Collectively, these data demonstrate a dual action for ActA on murine OCLs.

Circulating interleukin-8 levels explain breast cancer osteolysis in mice and humans.

Skeletal metastases of breast cancer and subsequent osteolysis connote a dramatic change in the prognosis for the patient and significantly increase the morbidity associated with disease. The cytokine interleukin 8 (IL-8/CXCL8) is able to directly stimulate osteoclastogenesis and bone resorption in mouse models of breast cancer bone metastasis. In this study, we determined whether circulating levels of IL-8 were associated with increased bone resorption and breast cancer bone metastasis in patients and investigated IL-8 action in vitro and in vivo in mice. Using breast cancer patient plasma (36 patients), we identified significantly elevated IL-8 levels in bone metastasis patients compared with patients lacking bone metastasis (p<0.05), as well as a correlation between plasma IL-8 and increased bone resorption (p<0.05), as measured by NTx levels. In a total of 22 ER+ and 15 ER- primary invasive ductal carcinomas, all cases examined stained positive for IL-8 expression. In vitro, human MDA-MB-231 and MDA-MET breast cancer cell lines secrete two distinct IL-8 isoforms, both of which were found to stimulate osteoclastogenesis. However, the more osteolytic MDA-MET-derived full length IL-8(1-77) had significantly higher potency than the non-osteolytic MDA-MB-231-derived IL-8(6-77), via the CXCR1 receptor. MDA-MET breast cancer cells were injected into the tibia of nude mice and 7days later treated daily with a neutralizing IL-8 monoclonal antibody. All tumor-injected mice receiving no antibody developed large osteolytic bone tumors, whereas 83% of the IL-8 antibody-treated mice had no evidence of tumor at the end of 28days and had significantly increased survival. The pro-osteoclastogenic activity of IL-8 in vivo was confirmed when transgenic mice expressing human IL-8 were examined and found to have a profound osteopenic phenotype, with elevated bone resorption and inherently low bone mass. Collectively, these data suggest that IL-8 plays an important role in breast cancer osteolysis and that anti-IL-8 therapy may be useful in the treatment of the skeletal related events associated with breast cancer.

Low bone turnover and low BMD in Down syndrome: effect of intermittent PTH treatment.

Trisomy 21 affects virtually every organ system and results in the complex clinical presentation of Down syndrome (DS). Patterns of differences are now being recognized as patients' age and these patterns bring about new opportunities for disease prevention and treatment. Low bone mineral density (BMD) has been reported in many studies of males and females with DS yet the specific effects of trisomy 21 on the skeleton remain poorly defined. Therefore we determined the bone phenotype and measured bone turnover markers in the murine DS model Ts65Dn. Male Ts65Dn DS mice are infertile and display a profound low bone mass phenotype that deteriorates with age. The low bone mass was correlated with significantly decreased osteoblast and osteoclast development, decreased bone biochemical markers, a diminished bone formation rate and reduced mechanical strength. The low bone mass observed in 3 month old Ts65Dn mice was significantly increased after 4 weeks of intermittent PTH treatment. These studies provide novel insight into the cause of the profound bone fragility in DS and identify PTH as a potential anabolic agent in the adult low bone mass DS population.

LIS1 regulates osteoclast formation and function through its interactions with dynein/dynactin and Plekhm1.

Microtubule organization and lysosomal secretion are both critical for the activation and function of osteoclasts, highly specialized polykaryons that are responsible for bone resorption and skeletal homeostasis. Here, we have identified a novel interaction between microtubule regulator LIS1 and Plekhm1, a lysosome-associated protein implicated in osteoclast secretion. Decreasing LIS1 expression by shRNA dramatically attenuated osteoclast formation and function, as shown by a decreased number of mature osteoclasts differentiated from bone marrow macrophages, diminished resorption pits formation, and reduced level of CTx-I, a bone resorption marker. The ablated osteoclast formation in LIS1-depleted macrophages was associated with a significant decrease in macrophage proliferation, osteoclast survival and differentiation, which were caused by reduced activation of ERK and AKT by M-CSF, prolonged RANKL-induced JNK activation and declined expression of NFAT-c1, a master transcription factor of osteoclast differentiation. Consistent with its critical role in microtubule organization and dynein function in other cell types, we found that LIS1 binds to and colocalizes with dynein in osteoclasts. Loss of LIS1 led to disorganized microtubules and aberrant dynein function. More importantly, the depletion of LIS1 in osteoclasts inhibited the secretion of Cathepsin K, a crucial lysosomal hydrolase for bone degradation, and reduced the motility of osteoclast precursors. These results indicate that LIS1 is a previously unrecognized regulator of osteoclast formation, microtubule organization, and lysosomal secretion by virtue of its ability to modulate dynein function and Plekhm1.

Bone turnover across the menopause transition : The role of gonadal inhibins.

Accumulating evidence demonstrates increasing bone turnover and bone loss in women prior to menopause and decreases in serum estradiol levels. Increased follicle-stimulating hormone levels have been correlated with some of these peri-menopausal changes. However, decreases in gonadal inhibins of the transforming growth factor (TGF)-beta superfamily strongly correlate with increases in bone formation and resorption markers across the menopause transition and predict lumbar bone mass in peri-menopausal women, likely as a result of direct inhibin suppression of osteoblastogenesis and osteoclastogenesis. Inhibins bind specifically to cells during osteoblastogenesis and osteoclastogenesis. They can block bone morphogenetic protein (BMP)-stimulated osteoblast and osteoclast development as well as BMP-stimulated SMAD1 phosphorylation, likely via inhibin-beta-glycan sequestration of BMP Type II receptor (BMPRII). Interestingly, continuous in vivo exposure to inhibin A is anabolic and protective against gonadectomy-induced bone loss in mice, suggesting that inhibins contribute to the endocrine regulation of bone metabolism via a bimodal mechanism of action whereby cycling inhibin exposure suppresses bone turnover and continuous exposure to inhibins is anabolic.

Reproductive hormones and bone.

Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates secretion of pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which directly regulate ovarian function. Pituitary FSH can modulate osteoclast development, and thereby influence bone turnover. Pituitary oxytocin and prolactin effects on the skeleton are not merely limited to pregnancy and lactation; oxytocin stimulates osteoblastogenesis and bone formation, whereas prolactin exerts skeletal effects in an age-dependent manner. Cyclic levels of inhibins and estrogen suppress FSH and LH, respectively, and also suppress bone turnover via their suppressive effects on osteoblast and osteoclast differentiation. However, continuous exposure to inhibins or estrogen/androgens is anabolic for the skeleton in intact animals and protects against gonadectomy-induced bone loss. Alterations of one hormone in the hypothalamic-pituitary-gonadal (HPG) axis influence other bone-active hormones in the entire feedback loop in the axis. Thus, we propose that the action of the HPG axis should be extended to include its combined effects on the skeleton, thus creating the HPG skeletal (HPGS) axis.

Characterization of a panel of six beta2-adrenergic receptor antibodies by indirect immunofluorescence microscopy.

BACKGROUND: The beta2-adrenergic receptor (beta2AR) is a primary target for medications used to treat asthma. Due to the low abundance of beta2AR, very few studies have reported its localization in tissues. However, the intracellular location of beta2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to beta-agonist medications. Thus, a method for visualizing beta2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant beta2AR in primary cell cultures. METHODS: A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat beta2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat beta2AR were identified and used to localize native beta2AR in primary cultures of rat airway smooth muscle and epithelial cells. beta2AR expression was confirmed by performing ligand binding assays using the beta-adrenergic antagonist [3H] dihydroalprenolol ([3H]DHA). RESULTS: Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human beta2AR (Ab-Bethyl) specifically recognized human but not rat beta2AR. An antibody developed against the C-terminal domain of the mouse beta2AR (Ab-sc570) specifically recognized rat but not human beta2AR. An antibody developed against 78 amino acids of the C-terminus of the human beta2AR (Ab-13989) was capable of recognizing both rat and human beta2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat beta2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface. CONCLUSION: Antibodies have been identified that recognize human beta2AR, rat beta2AR or both rat and human beta2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of beta2AR in tissues.