Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway.

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

Comparison of survival prediction models for bone metastases of the extremities following surgery.

Aims: This study aimed to compare the performance of survival prediction models for bone metastases of the extremities (BM-E) with pathological fractures in an Asian cohort, and investigate patient characteristics associated with survival. Methods: This retrospective cohort study included 469 patients, who underwent surgery for BM-E between January 2009 and March 2022 at a tertiary hospital in South Korea. Postoperative survival was calculated using the PATHFx3.0, SPRING13, OPTIModel, SORG, and IOR models. Model performance was assessed with area under the curve (AUC), calibration curve, Brier score, and decision curve analysis. Cox regression analyses were performed to evaluate the factors contributing to survival. Results: The SORG model demonstrated the highest discriminatory accuracy with AUC (0.80 (95% confidence interval (CI) 0.76 to 0.85)) at 12 months. In calibration analysis, the PATHfx3.0 and OPTIModel models underestimated survival, while the SPRING13 and IOR models overestimated survival. The SORG model exhibited excellent calibration with intercepts of 0.10 (95% CI -0.13 to 0.33) at 12 months. The SORG model also had lower Brier scores than the null score at three and 12 months, indicating good overall performance. Decision curve analysis showed that all five survival prediction models provided greater net benefit than the default strategy of operating on either all or no patients. Rapid growth cancer and low serum albumin levels were associated with three-, six-, and 12-month survival. Conclusion: State-of-art survival prediction models for BM-E (PATHFx3.0, SPRING13, OPTIModel, SORG, and IOR models) are useful clinical tools for orthopaedic surgeons in the decision-making process for the treatment in Asian patients, with SORG models offering the best predictive performance. Rapid growth cancer and serum albumin level are independent, statistically significant factors contributing to survival following surgery of BM-E. Further refinement of survival prediction models will bring about informed and patient-specific treatment of BM-E.

Amputation in patients with extremity soft tissue sarcoma: the experience of an East Asian referral center.

BACKGROUND: This study aimed to investigate the characteristics and clinical outcomes in a series of patients with extremity soft tissue sarcoma (STS) who underwent amputation at a large East Asian referral center. PATIENTS AND METHODS: Of the 652 patients who underwent surgery for extremity STS, data of 37 consecutive patients who underwent amputation were reviewed retrospectively. The median follow-up period was 96.0 months (range, 15-216). The patients were classified in to three cohorts. The primary localized (PL) group included patients who underwent amputation as a primary surgical procedure with curative intent. The recurrent localized (RL) group included patients who underwent amputation as a revision procedure after failure of previous limb sparing surgeries. The metastatic group included patients who underwent amputation as a palliative procedure. RESULTS: There were 22 cases of amputation in 596 STS patients and the amputation rate was 3.6% (22/596). Further, 1.8% (9/490) of patients with primary localized STS underwent amputation. Patients with localized STS who underwent amputation had a 5-year disease-specific survival (DSS) rate of 89.9% (95% Confidence Interval (CI), 87.1-92.7%), a local-recurrence-free survival (LRFS) of 84.1% (95% CI, 80.5-87.6%), and a metastasis-free survival (MFS) of 84.6%. (95% CI, 81.1-88.0%) Compared with previous studies, our results showed higher DSS and MFS rates with similar LRFS. CONCLUSIONS: The amputation rate of extremity STS in our institute in East Asia was similar but slightly lower than that reported in Western studies. The oncologic outcome of amputation reported in this study was higher than that indicated in Western studies and oncologic outcome of amputation was not statistically different from those of limb salvage surgery. However, considering the small cohort in single institute study, there is a possibility of selection bias and future multi-center study is necessary. From our results, amputation is still a feasible option for appropriately selected patients unsuitable for limb-conserving surgery.

Proximal Femur Salvage in Revision Knee Arthroplasty Due to Oncologic Indications: Long-term Results of Onlay and Overlapping Allograft in Revision Surgeries.

Background: Mechanical failures of tumor endoprosthesis in the distal femur usually require revision surgery. We investigated if the proximal femur host bone can be salvaged by onlay and overlapping allograft in revision surgeries due to aseptic loosening and stem fractures. Methods: We retrospectively reviewed 18 patients (7 men and 11 women) with osteosarcoma around the knee. The entire cohort was classified into three subgroups (no bone graft: 6, onlay allograft: 7, and overlapping allograft: 5) according to our treatment strategy. Results: The median interval from the initial surgery to the revision was 94.5 months (range, 21-219 months), and the median follow-up period from the revision surgery was 88.0 months (range, 24-179 months). At the last follow-up, 9 of the 18 patients maintained their endoprostheses, and the 5-year prosthesis survival rate was 57.9%. Limb survival was 100%. Five-year prosthesis survival rate was 66.7% in the no bone graft group, 85.7% in the onlay allograft group while 30.0% in the overlapping allograft group. In the no bone graft group and onlay allograft group, 66.7% (4/6) and 57.1% (4/7) maintained their revision prostheses while no prostheses survived in the overlapping allograft group. Recurrent stem loosening was observed in 14.2% (1/7) and 60.0% (3/5) of the onlay allograft and overlapping allograft groups, respectively, despite allograft bone union. The complication rate was 66.7% (12/18) in the entire cohort. The most common type of complication was infection (n = 6), followed by aseptic loosening (n = 4) and mechanical failure (n = 2). Conclusions: This study indicates that onlay allograft can be used as a supportive method in revising failed endoprosthesis if the extent of host bone destruction is extensive. However, applying overlapping allograft to secure bone stock showed a high rate of mechanical failures and infection in the long term. Future studies with a larger cohort are necessary to assess the prognostic factors for the higher complication rate in overlapping allograft and the need for overlapping allograft. Surveillance with consideration of the risk of anteromedial osteolysis in allograft and efforts for prevention of periprosthetic infection are essential.

Engineering bispecific T-cell engagers to deplete eosinophils for the treatment of severe eosinophilic asthma.

Severe eosinophilic asthma (SEA) is characterized by elevated eosinophil counts in the blood and airway mucosa. While monoclonal antibody therapies targeting interleukin-5 (IL-5) and its receptor (IL-5Rα) have improved treatment, some patients remain unresponsive. We propose an alternative approach to eliminate eosinophils using T cells by engineering IL-5Rα × CD3 bispecific T-cell engagers (bsTCEs) that target both IL-5Rα on eosinophils and CD3 on T cells. We designed different formats of IL-5Rα × CD3 bsTCEs, incorporating variations in valency, geometry, and affinity for the target antigen binding. We identified the single-chain variable fragment (scFv)-Fc format with the highest affinity toward the membrane-proximal domain of IL-5Rα in the IL-5Rα-binding arm showed the most potent cytotoxicity against IL-5Rα-expressing peripheral eosinophils by activating autologous primary T cells from healthy donors. This study proposes IL-5Rα × CD3 bsTCEs as potential alternatives for SEA treatment. Importantly, it demonstrates the first application of bsTCEs in eliminating disease-associated cells, including eosinophils, beyond cancer cells.

Does the Clinical Presentation of Secondary Osteosarcoma in Patients Who Survive Retinoblastoma Differ From That of Conventional Osteosarcoma and How Do We Detect Them?

BACKGROUND: Osteosarcoma is the most common secondary malignancy among survivors of retinoblastoma. Most previous reports on secondary malignancy of retinoblastoma included all types of secondary malignancies without a focus on osteosarcoma, owing to its rarity. In addition, there are few studies suggesting tools for regular surveillance for early detection. QUESTIONS/PURPOSES: (1) What are the radiologic and clinical characteristics of secondary osteosarcoma after retinoblastoma? (2) What is the clinical survivorship? (3) Is a radionuclide bone scan a reasonable imaging modality for early detection in patients with retinoblastoma? METHODS: Between February 2000 and December 2019, we treated 540 patients for retinoblastoma. Twelve patients (six male, six female) subsequently developed an osteosarcoma in the extremities; two of these patients had two sites of osteosarcoma (10 femurs, four tibiae) . A Technetium-99m bone scan image was examined annually in all patients for regular surveillance after the treatment of retinoblastoma as per our hospital's policy. All patients were treated with the same strategy as that used for primary conventional osteosarcoma, namely neoadjuvant chemotherapy, wide excision, and adjuvant chemotherapy. The median follow-up period was 12 years (range 8 to 21 years). The median age at the time of diagnosis of osteosarcoma was 9 years (range 5 to 15 years), and the median interval from retinoblastoma diagnosis to osteosarcoma diagnosis was 8 years (range 5 to 15 years). Radiologic characteristics were assessed with plain radiographs and MRI, while clinical characteristics were assessed through a retrospective review of medical records. For clinical survivorship, we evaluated overall survival, local recurrence-free survival, and metastasis-free survival. We reviewed the results of bone scans and clinical symptoms at the time of diagnosis for osteosarcoma after retinoblastoma. RESULTS: In nine of 14 patients, the tumor had a diaphyseal center, and five of the tumors were located at the metaphysis. The femur was the most common site (n = 10), followed by the tibia (n = 4). The median tumor size was 9 cm (range 5 to 13 cm). There was no local recurrence after surgical resection of the osteosarcoma, and the 5-year overall survival rate after the diagnosis of osteosarcoma was 86% (95% CI 68% to 100%). In all 14 tumors, the Technetium bone scan showed increased uptake in the lesions. Ten of 14 tumors were examined in clinic because of patient complaints of pain in the affected limb. Four patients showed no clinical symptoms detected by abnormal uptake on bone scan. CONCLUSION: For unclear reasons, secondary osteosarcomas in patients who were alive after the treatment of retinoblastoma had a slight predilection for the diaphysis of the long bone compared with patients with spontaneous osteosarcoma in other reports. The clinical survivorship of osteosarcoma as a secondary malignancy after retinoblastoma may not be inferior to that of conventional osteosarcoma. Close follow-up with at least yearly clinical assessment and bone scans or other imaging modalities appears to be helpful in detecting secondary osteosarcoma after the treatment of patients with retinoblastoma. Larger multi-institutional studies will be needed to substantiate these observations.Level of Evidenc e Level IV, therapeutic study.

An IoMT-Based Melanoma Lesion Segmentation Using Conditional Generative Adversarial Networks.

Currently, Internet of medical things-based technologies provide a foundation for remote data collection and medical assistance for various diseases. Along with developments in computer vision, the application of Artificial Intelligence and Deep Learning in IOMT devices aids in the design of effective CAD systems for various diseases such as melanoma cancer even in the absence of experts. However, accurate segmentation of melanoma skin lesions from images by CAD systems is necessary to carry out an effective diagnosis. Nevertheless, the visual similarity between normal and melanoma lesions is very high, which leads to less accuracy of various traditional, parametric, and deep learning-based methods. Hence, as a solution to the challenge of accurate segmentation, we propose an advanced generative deep learning model called the Conditional Generative Adversarial Network (cGAN) for lesion segmentation. In the suggested technique, the generation of segmented images is conditional on dermoscopic images of skin lesions to generate accurate segmentation. We assessed the proposed model using three distinct datasets including DermQuest, DermIS, and ISCI2016, and attained optimal segmentation results of 99%, 97%, and 95% performance accuracy, respectively.

Plastome-based backbone phylogeny of East Asian Phedimus (Subgenus Aizoon: Crassulaceae), with special emphasis on Korean endemics.

Although the monophyly of Phedimus has been strongly demonstrated, the species relationships among approximately 20 species of Phedimus have been difficult to determine because of the uniformity of their floral characteristics and extreme variation of their vegetative characters, often accompanied by high polyploid and aneuploid series and diverse habitats. In this study, we assembled 15 complete chloroplast genomes of Phedimus species from East Asia and generated a plastome-based backbone phylogeny of the subgenus Aizoon. As a proxy for nuclear phylogeny, we reconstructed the nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) phylogeny independently. The 15 plastomes of subg. Aizoon were highly conserved in structure and organization; hence, the complete plastome phylogeny fully resolved the species relationships with strong support. We found that P. aizoon and P. kamtschaticus were polyphyletic and morphologically distinct or ambiguous species, and they most likely evolved from the two species complex. The crown age of subg. Aizoon was estimated to be 27 Ma, suggesting its origin to be in the late Oligocene; however, the major lineages were diversified during the Miocene. The two Korean endemics, P. takesimensis and P. zokuriensis, were inferred to have originated recently during the Pleistocene, whereas the other endemic, P. latiovalifolium, originated in the late Miocene. Several mutation hotspots and seven positively selected chloroplast genes were identified in the subg. Aizoon.

Expanding the Therapeutic Window of EGFR-Targeted PE24 Immunotoxin for EGFR-Overexpressing Cancers by Tailoring the EGFR Binding Affinity.

Immunotoxins (ITs), which are toxin-fused tumor antigen-specific antibody chimeric proteins, have been developed to selectively kill targeted cancer cells. The epidermal growth factor receptor (EGFR) is an attractive target for the development of anti-EGFR ITs against solid tumors due to its overexpression on the cell surface of various solid tumors. However, the low basal level expression of EGFR in normal tissue cells can cause undesirable on-target/off-tumor toxicity and reduce the therapeutic window of anti-EGFR ITs. Here, based on an anti-EGFR monobody with cross-reactivity to both human and murine EGFR, we developed a strategy to tailor the anti-EGFR affinity of the monobody-based ITs carrying a 24-kDa fragment of Pseudomonas exotoxin A (PE24), termed ER-PE24, to distinguish tumors that overexpress EGFR from normal tissues. Five variants of ER-PE24 were generated with different EGFR affinities (KD ≈ 0.24 nM to 104 nM), showing comparable binding activity for both human and murine EGFR. ER/0.2-PE24 with the highest affinity (KD ≈ 0.24 nM) exhibited a narrow therapeutic window of 19 pM to 93 pM, whereas ER/21-PE24 with an intermediate affinity (KD ≈ 21 nM) showed a much broader therapeutic window of 73 pM to 1.5 nM in in vitro cytotoxic assays using tumor model cell lines. In EGFR-overexpressing tumor xenograft mouse models, the maximum tolerated dose (MTD) of intravenous injection of ER/21-PE24 was found to be 0.4 mg/kg, which was fourfold higher than the MTD (0.1 mg/kg) of ER/0.2-PE24. Our study provides a strategy for the development of IT targeting tumor overexpressed antigens with basal expression in broad normal tissues by tailoring tumor antigen affinities.

Improved intratumoral penetration of IL12 immunocytokine enhances the antitumor efficacy.

Tumor-targeting antibody (Ab)-fused cytokines, referred to as immunocytokines, are designed to increase antitumor efficacy and reduce toxicity through the tumor-directed delivery of cytokines. However, the poor localization and intratumoral penetration of immunocytokines, especially in solid tumors, pose a challenge to effectively stimulate antitumor immune cells to kill tumor cells within the tumor microenvironment. Here, we investigated the influence of the tumor antigen-binding kinetics of a murine interleukin 12 (mIL12)-based immunocytokine on tumor localization and diffusive intratumoral penetration, and hence the consequent antitumor activity, by activating effector T cells in immunocompetent mice bearing syngeneic colon tumors. Based on tumor-associated antigen HER2-specific Ab Herceptin (HCT)-fused mIL12 carrying one molecule of mIL12 (HCT-mono-mIL12 immunocytokine), we generated a panel of HCT-mono-mIL12 variants with different affinities (K D) mainly varying in their dissociation rates (k off) for HER2. Systemic administration of HCT-mono-mIL12 required an anti-HER2 affinity above a threshold (K D = 130 nM) for selective localization and antitumor activity to HER2-expressing tumors versus HER2-negative tumors. However, the high affinity (K D = 0.54 or 46 nM) due to the slow k off from HER2 antigen limited the depth of intratumoral penetration of HCT-mono-mIL12 and the consequent tumor infiltration of T cells, resulting in inferior antitumor activity compared with that of HCT-mono-mIL12 with moderate affinity of (K D = 130 nM) and a faster k off. The extent of intratumoral penetration of HCT-mono-mIL12 variants was strongly correlated with their tumor infiltration and intratumoral activation of CD4+ and CD8+ T cells to kill tumor cells. Collectively, our results demonstrate that when developing antitumor immunocytokines, tumor antigen-binding kinetics and affinity of the Ab moiety should be optimized to achieve maximal antitumor efficacy.

Does reverse total shoulder arthroplasty with allograft-prosthesis composite (APC) have surgical benefits over hemiarthroplasty with APC in patients with tumors of the proximal humerus?

BACKGROUND: The research on surgical outcomes of hemiarthroplasty and reverse total shoulder arthroplasty using allograft-prosthesis composites for the proximal humeral oncologic condition is still scarce. Therefore, this study aimed to compare surgical outcomes of shoulder joint reconstruction with hemiarthroplasty and reverse total shoulder arthroplasty using allograft-prosthesis composites for tumors of the proximal humerus. METHODS: Eleven patients underwent hemiarthroplasty or reverse total shoulder arthroplasty using allograft-prosthesis composites for tumors of the proximal humerus between July 2011 and April 2018 were reviewed. Radiographic analysis for bone union of allograft-host bone junction, implant loosening, stress shielding and shoulder dislocation or subluxation was performed. Functional outcomes were evaluated using visual analog scales for pain, range of motion, Simple Shoulder Test score and Musculoskeletal Tumor Society score. Furthermore, oncologic outcome and complications were also assessed, respectively. RESULTS: There were five patients with hemiarthroplasty (mean age, 23.2 years) and six patients with reverse total shoulder arthroplasty (mean age, 46.8 years, P = 0.05). Radiographically, there were no events associated with implant loosening, stress shielding and shoulder dislocation or subluxation in the two groups. There were no differences in functional outcomes between the two groups. There was no local recurrence in entire cohort. In the hemiarthroplasty group, one patient was required revision surgery to reverse total shoulder arthroplasty at postoperative 6 years due to rotator cuff dysfunction. In the reverse total shoulder arthroplasty group, one patient showed the fracture occurred at allograft-host bone junction at postoperative 6 months. CONCLUSIONS: Surgical outcomes of hemiarthroplasty with allograft-prosthesis composites were not inferior to reverse total shoulder arthroplasty when applied in properly selected patients. The authors recommended that hemiarthroplasty with allograft-prosthesis composites could be used for young age patients without glenoid metastasis involvement, and reverse total shoulder arthroplasty with allograft-prosthesis composites could be used for patients with old age or metastatic bone tumors.

Warburg-like metabolic transformation underlies neuronal degeneration in sporadic Alzheimer's disease.

The drivers of sporadic Alzheimer's disease (AD) remain incompletely understood. Utilizing directly converted induced neurons (iNs) from AD-patient-derived fibroblasts, we identified a metabolic switch to aerobic glycolysis in AD iNs. Pathological isoform switching of the glycolytic enzyme pyruvate kinase M (PKM) toward the cancer-associated PKM2 isoform conferred metabolic and transcriptional changes in AD iNs. These alterations occurred via PKM2's lack of metabolic activity and via nuclear translocation and association with STAT3 and HIF1α to promote neuronal fate loss and vulnerability. Chemical modulation of PKM2 prevented nuclear translocation, restored a mature neuronal metabolism, reversed AD-specific gene expression changes, and re-activated neuronal resilience against cell death.

Metalloendopeptidase ADAM-like Decysin 1 (ADAMDEC1) in Colonic Subepithelial PDGFRα+ Cells Is a New Marker for Inflammatory Bowel Disease.

Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRα+ cells. However, the cellular origin for this gene expression is controversial as it is also known to be expressed in intestinal macrophages. We found that Adamdec1 mRNAs were selectively expressed in colonic mucosal subepithelial PDGFRα+ cells. ADAMDEC1 protein was mainly released from PDGFRα+ cells and accumulated in the mucosal layer lamina propria space near the epithelial basement membrane. PDGFRα+ cells significantly overexpressed Adamdec1 mRNAs and protein in DSS-induced colitis mice. Adamdec1 was predominantly expressed in CD45- PDGFRα+ cells in DSS-induced colitis mice, with only minimal expression in CD45+ CD64+ macrophages. Additionally, overexpression of both ADAMDEC1 mRNA and protein was consistently observed in PDGFRα+ cells, but not in CD64+ macrophages found in human colonic mucosal tissue affected by Crohn's disease. In summary, PDGFRα+ cells selectively express ADAMDEC1, which is localized to the colon mucosa layer. ADAMDEC1 expression significantly increases in DSS-induced colitis affected mice and Crohn's disease affected human tissue, suggesting that this gene can serve as a diagnostic and/or therapeutic target for intestinal inflammation and Crohn's disease.

Yeast Mating as a Tool for Highly Effective Discovery and Engineering of Antibodies via Display Methodologies.

Yeast surface display (YSD) is a powerful methodology for discovery and engineering of antibodies, and the yeast mating has been used to overcome low transformation efficiency of yeast in antibody library generation. We developed an optimized method of yeast mating for generating a large, combinatorial antibody fragment library and heterodimeric protein library by cellular fusion between two haploid cells carrying different library each other. This method allows for increased diversity in screening of target-specific fragment antigen-binding (Fab) antibodies as well as in the development of heterodimeric Fc variants for bi-specific antibody generation and T-cell receptor (TCR). Here we describe the efficient isolation of human antibodies against the activated GTP-bound form of the oncogenic Ras mutant (KRasG12D-GTP) by sequential isolation of their heavy chains (HCs) followed by combination with light chains (LCs) via the yeast mating process. This strategy facilitates guided selection of the antigen-specific HC with either a fixed functional LC, which has cytosol penetrating ability, or an LC library to generate the Fab. It also allows for deeper exploration of a sequence space with fixed diversity, leading to a higher probability of successful isolation of human antibodies with high specificity and affinity.

Antibody-mediated delivery of a viral MHC-I epitope into the cytosol of target tumor cells repurposes virus-specific CD8+ T cells for cancer immunotherapy.

BACKGROUND: Redirecting pre-existing virus-specific cytotoxic CD8+ T lymphocytes (CTLs) to tumors by simulating a viral infection of the tumor cells has great potential for cancer immunotherapy. However, this strategy is limited by lack of amenable method for viral antigen delivery into the cytosol of target tumors. Here, we addressed the limit by developing a CD8+ T cell epitope-delivering antibody, termed a TEDbody, which was engineered to deliver a viral MHC-I epitope peptide into the cytosol of target tumor cells by fusion with a tumor-specific cytosol-penetrating antibody. METHODS: To direct human cytomegalovirus (CMV)-specific CTLs against tumors, we designed a series of TEDbodies carrying various CMV pp65 antigen-derived peptides. CMV-specific CTLs from blood of CMV-seropositive healthy donors were expanded for use in in vitro and in vivo experiments. Comprehensive cellular assays were performed to determine the presentation mechanism of TEDbody-mediated CMV peptide-MHC-I complex (CMV-pMHCI) on the surface of target tumor cells and the recognition and lysis by CMV-specific CTLs. In vivo CMV-pMHCI presentation and antitumor efficacy of TEDbody were evaluated in immunodeficient mice bearing human tumors. RESULTS: TEDbody delivered the fused epitope peptides into target tumor cells to be intracellularly processed and surface displayed in the form of CMV-pMHCI, leading to disguise target tumor cells as virally infected cells for recognition and lysis by CMV-specific CTLs. When systemically injected into tumor-bearing immunodeficient mice, TEDbody efficiently marked tumor cells with CMV-pMHCI to augment the proliferation and cytotoxic property of tumor-infiltrated CMV-specific CTLs, resulting in significant inhibition of the in vivo tumor growth by redirecting adoptively transferred CMV-specific CTLs. Further, combination of TEDbody with anti-OX40 agonistic antibody substantially enhanced the in vivo antitumor activity. CONCLUSION: Our study offers an effective technology for MHC-I antigen cytosolic delivery. TEDbody may thus have utility as a therapeutic cancer vaccine to redirect pre-existing anti-viral CTLs arising from previously exposed viral infections to attack tumors.

The clinical outcome of hemicortical resection in extremity malignant bone tumors.

INTRODUCTION: Hemicortical resection is challenging when a huge fungating tumor is covering the osteotomy site. We report the clinical outcome of hemicortical resection and reconstruction for primary bone tumors, especially with high-grade histology and extensive circumferential involvement. MATERIALS AND METHODS: We retrospectively reviewed 44 patients (males, n = 18; females, n = 26) who underwent hemicortical resection from 2005 to 2014. RESULTS: The median follow-up period was 46.0 (23-178) months. Disease-specific, local recurrence-free, and metastasis-free survival rates of patients in the malignant group at 5 years were 96.6%, 84.5%, and 93.6%, respectively. Among 42 patients, there were local recurrences (n = 6), metastasis (n = 2), and death (n = 1). Surgical margin was an independent prognostic factor for local recurrence (hazard ratio = 5.7; p = 0.038). The recycled autograft and strut allograft groups did not show statistical difference in bone union. Failure rate was 31.8% and local recurrence was the most frequent, followed by infection. CONCLUSION: Hemicortical resection can be a feasible option for extremity malignant bone tumors. Regarding reconstruction, there were no difference between autograft and allograft in bone union rate. Surgical margin was an independent prognostic factor for local recurrence.

Combination Therapy of the Active KRAS-Targeting Antibody inRas37 and a PI3K Inhibitor in Pancreatic Cancer.

KRAS activating mutations, which are present in more than 90% of pancreatic cancers, drive tumor dependency on the RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. Therefore, combined targeting of RAS/MAPK and PI3K/AKT signaling pathways may be required for optimal therapeutic effect in pancreatic cancer. However, the therapeutic efficacy of combined MAPK and PI3K/AKT signaling target inhibitors is unsatisfactory in pancreatic cancer treatment, because it is often accompanied by MAPK pathway reactivation by PI3K/AKT inhibitor. Therefore, we developed an inRas37 antibody, which directly targets the intra-cellularly activated GTP-bound form of oncogenic RAS mutation and investigated its synergistic effect in the presence of the PI3K inhibitor BEZ-235 in pancreatic cancer. In this study, inRas37 remarkably increased the drug response of BEZ-235 to pancreatic cancer cells by inhibiting MAPK reactivation. Moreover, the co-treatment synergistically inhibited cell proliferation, migration, and invasion and exhibited synergistic anticancer activity by inhibiting the MAPK and PI3K pathways. The combined administration of inRas37and BEZ-235 significantly inhibited tumor growth in mouse models. Our results demonstrated that inRas37 synergistically increased the antitumor activity of BEZ-235 by inhibiting MAPK reactivation, suggesting that inRas37 and BEZ-235 co-treatment could be a potential treatment approach for pancreatic cancer patients with KRAS mutations.

Engineering of an EpCAM-targeting cyclic peptide to improve the EpCAM-mediated cellular internalization and tumor accumulation of a peptide-fused antibody.

Fusion of a target-specific peptide to a full-length antibody (Ab) can result in a peptide-Ab fusion protein with additional specificity and enhanced activity. We recently developed an intracellular pan-RAS-targeting cytosol-penetrating antibody, RT22-ep59, in which a tumor-specific targeting ability was achieved via the fusion of an epithelial cell adhesion molecule (EpCAM) targeting cyclic peptide (ep133). Here, the aim was to enhance EpCAM-mediated endocytosis and tumor accumulation of the peptide-fused RAS-targeting Ab. Accordingly, we engineered a cyclic peptide (from ep133) that has stronger affinity for EpCAM by using yeast surface display technology and then rationally designed cyclic peptides in the Ab-fused form to enhance colloidal stability. The finally engineered EpCAM-targeting cyclic peptide (ep6)-fused Ab, ep6Ras37, has ∼10-fold stronger affinity (KD ≈ 1.9 nM) for EpCAM than that of RT22-ep59, without deterioration of biophysical properties. Compared with the parental antibody (RT22-ep59), ep6Ras37 more efficiently reached the cytosol of EpCAM-expressing cells and showed greater preferential tumor homing and accumulation in mice bearing EpCAM-expressing LoVo xenograft tumors. Thus, the high-affinity EpCAM-targeting peptide ensures efficient cellular internalization and better tumor accumulation of the peptide-fused Ab.