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Chimeric antigen receptors (CARs) can effectively redirect cytotoxic T cells toward highly expressed surface antigens on tumor cells. The low expression of several tumor-associated antigens (TAAs) on normal tissues, however, hinders their safe targeting by CAR T cells due to on-target/off-tumor effects. Using the multiple myeloma (MM)-associated CD38 antigen as a model system, here, we present a rational approach for effective and tumor-selective targeting of such TAAs. Using “light-chain exchange” technology, we combined the heavy chains of two high-affinity CD38 antibodies with 176 germline light chains and generated ∼124 new antibodies with 10- to >1,000-fold lower affinities to CD38. After categorizing them into three distinct affinity classes, we incorporated the single-chain variable fragments of eight antibodies from each class into new CARs. T cells carrying these CD38-CARs were extensively evaluated for their on-tumor/off-tumor cytotoxicity as well as CD38-dependent proliferation and cytokine production. We identified CD38-CAR T cells of ∼1,000- fold reduced affinity, which optimally proliferated, produced Th1-like cytokines, and effectively lysed CD382+ MM cells, but spared CD38+ healthy hematopoietic cells in vitro and in vivo. Thus, this systematic approach is highly suitable for the generation of optimal CARs for effective and selective targeting of TAAs. Drent et al. used the light-chain exchange method to identify CD38-CAR T cells of reduced affinity, which effectively lysed multiple myeloma cells but spared healthy hematopoietic cells, in vitro and in vivo. This paper proposes a rational strategy for the selective targeting of tumor-associated antigens by CAR T cells.

Biomaterials can be endowed with biologically instructive properties by changing basic parameters such as elasticity and surface texture. However, translation from in vitro proof of concept to clinical application is largely missing. Porous calcium phosphate ceramics are used to treat small bone defects but in general do not induce stem cell differentiation, which is essential for regenerating large bone defects. Here, we prepared calcium phosphate ceramics with varying physicochemical and structural characteristics. Microporosity correlated to their propensity to stimulate osteogenic differentiation of stem cells in vitro and bone induction in vivo. Implantation in a large bone defect in sheep unequivocally demonstrated that osteoinductive ceramics are equally efficient in bone repair as autologous bone grafts. Our results provide proof of concept for the clinical application of \"smart\" biomaterials.

Heterotopic ossification (HO) is a double‐edged sword. Pathological HO presents as an undesired clinical complication, whereas controlled heterotopic bone formation by synthetic osteoinductive materials shows promising therapeutic potentials for bone regeneration. However, the mechanism of material‐induced heterotopic bone formation remains largely unknown. Early acquired HO being usually accompanied by severe tissue hypoxia prompts the hypothesis that hypoxia caused by the implantation coordinates serial cellular events and ultimately induces heterotopic bone formation in osteoinductive materials. The data presented herein shows a link between hypoxia, macrophage polarization to M2, osteoclastogenesis, and material‐induced bone formation. Hypoxia inducible factor‐1α (HIF‐1α), a crucial mediator of cellular responses to hypoxia, is highly expressed in an osteoinductive calcium phosphate ceramic (CaP) during the early phase of implantation, while pharmacological inhibition of HIF‐1α significantly inhibits M2 macrophage, subsequent osteoclast, and material‐induced bone formation. Similarly, in vitro, hypoxia enhances M2 macrophage and osteoclast formation. Osteoclast‐conditioned medium enhances osteogenic differentiation of mesenchymal stem cells, such enhancement disappears with the presence of HIF‐1α inhibitor. Furthermore, metabolomics analysis reveals that hypoxia enhances osteoclastogenesis via the axis of M2/lipid‐loaded macrophages. The current findings shed new light on the mechanism of HO and favor the design of more potent osteoinductive materials for bone regeneration. Hypoxia enhances macrophage polarization to M2, stimulates lipid loading into M2 macrophage to facilitate osteoclastogenesis, factors generated in osteoclastogenesis initiate heterotopic bone formation. On the contrary, via the axis of M2/lipid‐loaded macrophages, HIF‐1α inhibitors inhibit osteoclastogenesis, material‐induced heterotopic bone formation is blocked once osteoclasts are deleted.

Critical-size bone defects (CSDs), which are those that do not self-repair in a given period, are essential for evaluating bone-regeneration strategies. We established CSDs models in the rabbit cranium and ulna, and the bone-regeneration capacities of porous calcium phosphate (CaP) ceramics were assessed. A 12.6-mm cranial defect was confirmed as a CSDs after 12 weeks, with submicron surface-structured biphasic calcium-phosphate (BCP) implants [consisting of 20% hydroxyapatite and 80% tricalcium phosphate (TCP)] demonstrating significantly higher bone formation (32.2% ± 10.6%) than micron surface-structured TCP (TCP-B) implants (17.8% ± 4.6%, p = 0.0121). Ulna defects (15.0 mm in length) failed to heal spontaneously within 24 weeks when the periosteum was removed from both the ulna and radius, and the radius was covered with an expanded polytetrafluoroethylene (ePTFE) membrane. No bone bridging (i.e., union) was observed in the BCP implants at 12 weeks, whereas 80% of BCP implants (four out of five) achieved union by 24 weeks. Furthermore, the bone area within the available space of BCP implants increased significantly from 19.3% ± 7.3% at 12 weeks to 37.7% ± 8.5% at 24 weeks ( p = 0.0063), accompanied by significant BCP resorption (14.8% at 12 weeks and 30.2% at 24 weeks). This study offers two rabbit CSDs models for evaluating bone-regeneration strategies (including bone substitution), and the overall data obtained in the current study indicate the possibility of repairing CSDs with CaP ceramics demonstrating improved bone-forming ability given adequate implantation time.

The osteoinductive potential, or bone induction potency, of two calcium phosphate ceramics was evaluated after intramuscular implantation in goats. The ceramics were comprised of hydroxyapatite (HA) and biphasic calcium phosphate (BCP), the later of which contained a 85/15 mixture of hydroxyapatite and tricalcium phosphate (TCP). Both ceramics had a similar macroporosity of around 55% and a pore distribution between 100 and 800 microm. Besides the difference in chemistry, BCP was also microporous and hence had a different surface microstructure. After implantation in the back muscles of four goats for 12 weeks, all 8 BCP samples (7x7x7 mm(3)) showed the presence of bone formation in the macropores (1+/-1%), while no bone was found in any of the HA samples. The used BCP can therefore be characterized as an osteoinductive material. Having the ability to induce bone formation in soft tissues, the BCP presented herein may be a useful biomaterial for bone repair when combined with cultured osteogenic cells, growth factors or both.

Since the application of the autologous bone graft, the need for an alternative has been recognized. Tissue engineering (TE) of bone by combining bone marrow stromal cells (BMSCs) with a porous scaffold, is considered a promising technique. In this study we investigated the potential of tissue engineered bone to heal a critical sized defect in the goat. Orthotopic bone formation was compared to ectopic bone formation in comparable constructs. TE constructs were prepared from goat BMSCs and porous biphasic calcium phosphate ceramic scaffolds. These constructs and scaffolds without cells were implanted paired in critical sized iliac wing defects. Comparable samples were implanted intramuscularly. After 9 ( n=7) and 12 ( n=8) weeks implantation, the samples were analyzed histomorphometrically. After 9-weeks implantation in the iliac wing defect, significantly more bone apposition was found in the TE condition. After 12 weeks, the defects were almost completely filled with bone, but no significant advantage of TE was determined anymore. This contrasted with the intramuscular samples where TE implants showed significantly more bone at both time points. In conclusion, bone TE is feasible in critical sized defects. However, when appropriate osteoconductive/inductive materials are applied the effect of cell seeding may be temporary.

ABSTRACT Multiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow. Most patients suffer from an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic protein 4 (BMP4) is important for both pre‐ and postnatal bone formation and induces growth arrest and apoptosis of myeloma cells. BMP4‐treatment of myeloma patients could have the potential to reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human‐mouse model of multiple myeloma where humanized bone scaffolds were implanted subcutaneously in RAG2−/− γC−/−mice. Mice were treated with adeno‐associated virus serotype 8 BMP4 vectors (AAV8‐BMP4) to express BMP4 in the liver. When mature BMP4 was detectable in the circulation, myeloma cells were injected into the scaffolds and tumor growth was examined by weekly imaging. Strikingly, the tumor burden was reduced in AAV8‐BMP4 mice compared with the AAV8‐CTRL mice, suggesting that increased circulating BMP4 reduced tumor growth. BMP4‐treatment also prevented bone loss in the scaffolds, most likely due to reduced tumor load. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from cancer cells, we examined the unaffected, non‐myeloma femurs by μCT. Surprisingly, the AAV8‐BMP4 mice had significantly reduced trabecular bone volume, trabecular numbers, as well as significantly increased trabecular separation compared with the AAV8‐CTRL mice. There was no difference in cortical bone parameters between the two groups. Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced the amount of trabecular bone in mice. Our data suggest that care should be taken when considering using BMP4 as a therapeutic agent. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The most common application of bone grafts is spinal fusion surgery, in which the use of iliac crest autograft is the gold standard. Harvesting of autograft, however, requires an extra surgical procedure, which is associated with additional morbidity. Allograft is the well-known alternative, but it is generally considered less effective in posterior fusions. Therefore, the need for an effective alternative remains. Recently, it was shown that ceramics can be endowed with biologically instructive properties by changing the basic parameters of the material. In this study, we compared a novel tricalcium phosphate ceramic (TCP) to iliac crest autograft and allograft, in instrumented posterolateral fusions in a goat model. A total of nine goats were included, who underwent a two-level lumbar fusion. Each side of the spine was randomized into one type of graft: iliac crest autograft; fresh-frozen allograft; TCP alone; or TCP combined with local autograft (50:50). The fusion rates after 16 weeks were comparable between the groups (autograft 3/8, allograft 4/8, TCP 4/8, and TCP/local autograft 5/8). Calculation of the fusion volume on computed tomography images, showed significantly greater volume in the control groups (autograft 7.8 mL and allograft 8.9 mL) compared with the groups with TCP (TCP 6.1 mL and TCP/local autograft 6.0 mL). No adverse tissue response was seen on histological analysis and TCP was almost completely resorbed. The results demonstrate that TCP is capable of achieving fusion at a similar rate to iliac crest autograft in posterolateral fusions, while almost completely resorbing within 16 weeks. Despite the lower fusion volume, the TCP is a promising alternative circumventing the disadvantages of autograft and allograft.

Different materials were implanted in muscles of dogs to study the osteoinduction of calcium phosphate biomaterials. Bone formation was only seen in calcium phosphate biomaterials with micropores, and could be found in hydroxyapatite (HA) ceramic, tricalcium phosphate/hydroxyapatite ceramic (BCP), beta-TCP ceramic and calcium phosphate cement. The osteoinductive potential was different in different materials. The results indicate that osteoinduction can be a property of calcium phosphate biomaterials when they exhibit specific chemical and structural characteristics.

Material-induced bone formation reported in canine, bovid, suid, and primate species does not often occur in lagomorph or rodent models. In this study, we test biphasic calcium phosphate and hydroxyapatite- induced bone formation in subcutaneous pockets of mice and intramuscular pockets in rats, rabbits, and dogs. All scaffolds are of similar size, and all animals were sacrificed at 90 days post-implantation. In dogs (N = 8), all implants showed bone formation with significantly more bone formed in biphasic calcium phosphates (30 ± 6%, N = 8) as compared to hydroxyapatite (14 ± 5%, N = 8) ( p = 0.003). Hydroxyapatite implants did not induce bone formation in mice, rats, or rabbits. Biphasic calcium phosphate induced bone in 6 of 8 scaffolds implanted in 4 rabbits and 3 of 16 scaffolds implanted in 16 mice, whereas it did not induce bone formation in any of the 8 rats. The results presented herein suggest that the incidence of material-induced bone formation varies with animal species and is related to the implant material used.