An electrospun macrodevice for durable encapsulation of human cells with consistent secretion of therapeutic antibodies.

Frequent subcutaneous or intravenous administrations of therapeutic biomolecules can be costly and inconvenient for patients. Implantation of encapsulated recombinant cells represents a promising approach for the sustained delivery of biotherapeutics. However, foreign body and fibrotic response against encapsulation materials results in drastically reduced viability of encapsulated cells, presenting a major engineering challenge for biocompatibility. Here, we show that the multi-laminate electrospun retrievable macrodevice (Bio-Spun) protects genetically modified human cells after subcutaneous implant in mice. We describe here a biocompatible nanofiber device that limits fibrosis and extends implant survival. For more than 150 days, these devices supported human cells engineered to secrete the antibodies: vedolizumab, ustekinumab, and adalimumab, while eliciting minimal fibrotic response in mice. The porous electrospun cell chamber allowed secretion of the recombinant antibodies into the host bloodstream, and prevented infiltration of host cells into the chamber. High plasma levels (>50 µg/mL) of antibody were maintained in the optimized devices for more than 5 months. Our findings demonstrate that macrodevices constructed from electrospun materials are effective in protecting genetically engineered cells for the sustained administration of recombinant therapeutic antibodies.

Innate immune signaling drives late cardiac toxicity following DNA-damaging cancer therapies.

Late cardiac toxicity is a potentially lethal complication of cancer therapy, yet the pathogenic mechanism remains largely unknown, and few treatment options exist. Here we report DNA-damaging agents such as radiation and anthracycline chemotherapies inducing delayed cardiac inflammation following therapy due to activation of cGAS- and STING-dependent type I interferon signaling. Genetic ablation of cGAS-STING signaling in mice inhibits DNA damage-induced cardiac inflammation, rescues late cardiac functional decline, and prevents death from cardiac events. Treatment with a STING antagonist suppresses cardiac interferon signaling following DNA-damaging therapies and effectively mitigates cardiac toxicity. These results identify a therapeutically targetable, pathogenic mechanism for one of the most vexing treatment-related toxicities in cancer survivors.

Radioimmunotherapy Targeting Delta-like Ligand 3 in Small Cell Lung Cancer Exhibits Antitumor Efficacy with Low Toxicity.

PURPOSE: Small cell lung cancer (SCLC) is an exceptionally lethal form of lung cancer with limited treatment options. Delta-like ligand 3 (DLL3) is an attractive therapeutic target as surface expression is almost exclusive to tumor cells. EXPERIMENTAL DESIGN: We radiolabeled the anti-DLL3 mAb SC16 with the therapeutic radioisotope, Lutetium-177. [177Lu]Lu-DTPA-CHX-A"-SC16 binds to DLL3 on SCLC cells and delivers targeted radiotherapy while minimizing radiation to healthy tissue. RESULTS: [177Lu]Lu-DTPA-CHX-A"-SC16 demonstrated high tumor uptake with DLL3-target specificity in tumor xenografts. Dosimetry analyses of biodistribution studies suggested that the blood and liver were most at risk for toxicity from treatment with high doses of [177Lu]Lu-DTPA-CHX-A"-SC16. In the radioresistant NCI-H82 model, survival studies showed that 500 µCi and 750 µCi doses of [177Lu]Lu-DTPA-CHX-A"-SC16 led to prolonged survival over controls, and 3 of the 8 mice that received high doses of [177Lu]Lu-DTPA-CHX-A"-SC16 had pathologically confirmed complete responses (CR). In the patient-derived xenograft model Lu149, all doses of [177Lu]Lu-DTPA-CHX-A"-SC16 markedly prolonged survival. At the 250 µCi and 500 µCi doses, 5 of 10 and 7 of 9 mice demonstrated pathologically confirmed CRs, respectively. Four of 10 mice that received 750 µCi of [177Lu]Lu-DTPA-CHX-A"-SC16 demonstrated petechiae severe enough to warrant euthanasia, but the remaining 6 mice demonstrated pathologically confirmed CRs. IHC on residual tissues from partial responses confirmed retained DLL3 expression. Hematologic toxicity was dose-dependent and transient, with full recovery within 4 weeks. Hepatotoxicity was not observed. CONCLUSIONS: Together, the compelling antitumor efficacy, pathologic CRs, and mild and transient toxicity profile demonstrate strong potential for clinical translation of [177Lu]Lu-DTPA-CHX-A"-SC16.

Influence of Fc Modifications and IgG Subclass on Biodistribution of Humanized Antibodies Targeting L1CAM.

Immuno-PET is a powerful tool to noninvasively characterize the in vivo biodistribution of engineered antibodies. Methods: L1 cell adhesion molecule-targeting humanized (HuE71) IgG1 and IgG4 antibodies bearing identical variable heavy- and light-chain sequences but different fragment crystallizable (Fc) portions were radiolabeled with 89Zr, and the in vivo biodistribution was studied in SKOV3 ovarian cancer xenografted nude mice. Results: In addition to showing uptake in L1 cell adhesion molecule-expressing SKOV3 tumors, as does its parental counterpart HuE71 IgG1, the afucosylated variant having enhanced Fc-receptor affinity showed high nonspecific uptake in lymph nodes. On the other hand, aglycosylated HuE71 IgG1 with abrogated Fc-receptor binding did not show lymphoid uptake. The use of the IgG4 subclass showed high nonspecific uptake in the kidneys, which was prevented by mutating serine at position 228 to proline in the hinge region of the IgG4 antibody to mitigate in vivo fragment antigen-binding arm exchange. Conclusion: Our findings highlight the influence of Fc modifications and the choice of IgG subclass on the in vivo biodistribution of antibodies and the potential outcomes thereof.

ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody.

PURPOSE: Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response. EXPERIMENTAL DESIGN: The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer. RESULTS: Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors. CONCLUSIONS: The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

Exclusion of Expert Contributors From Authorship Limits the Quality of Scientific Articles.

Veterinary pathologists are key contributors to multidisciplinary biomedical research. However, they are occasionally excluded from authorship in published articles despite their substantial intellectual and data contributions. To better understand the potential origins and implications of this practice, we identified and analyzed 29 scientific publications where the contributing pathologist was excluded as an author. The amount of pathologist-generated data contributions were similar to the calculated average contributions for authors, suggesting that the amount of data contributed by the pathologist was not a valid factor for their exclusion from authorship. We then studied publications with pathologist-generated contributions to compare the effects of inclusion or exclusion of the pathologist as an author. Exclusion of the pathologist from authorship was associated with significantly lower markers of rigor and reproducibility compared to articles in which the pathologist was included as author. Although this study did not find justification for the exclusion of pathologists from authorship, potential consequences of their exclusion on data quality were readily detectable.

Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair.

After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic acute radiation syndrome (H-ARS) and higher-dose gastrointestinal acute radiation syndrome (GI-ARS) lethality. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in preclinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here, we report generation of an anti-ceramide 6B5 single-chain variable fragment (scFv) and show that s.c. 6B5 scFv delivery at 24 hours after a 90% lethal GI-ARS dose of 15 Gy mitigated mouse lethality, despite administration after DNA repair was complete. We defined an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis after radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ, and mouse survival. We posit that successful preclinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.

Ethmoidal meningoencephalocele in a C57BL/6J mouse.

An otherwise healthy two-month-old female C57BL/6J mouse presented with a left-sided head tilt. Differential diagnoses included idiopathic necrotizing arteritis, bacterial otitis media/interna (Pasteurella pneumotropica, Pseudomonas aeruginosa, Streptococcus sp., Mycoplasma pulmonis and Burkholderia gladioli), encephalitis, an abscess, neoplasia, a congenital malformation and an accidental or iatrogenic head trauma. Magnetic resonance imaging (MRI) revealed a large space-occupying right olfactory lobe intra-axial lesion with severe secondary left-sided subfalcine herniation. Following imaging, the animal was euthanized due to poor prognosis. Histopathologic examination revealed a unilateral, full-thickness bone defect at the base of the cribriform plate and nasal conchae dysplasia, resulting in the herniation of the olfactory bulb into the nasal cavity. There was also a left midline-shift of the frontal cortex and moderate catarrhal sinusitis in the left mandibular sinus. The MRI and histopathologic changes are consistent with a congenital malformation of the nasal cavity and frontal aspect of the skull known as an ethmoidal meningoencephalocele. Encephaloceles are rare abnormalities caused by herniation of contents of the brain through a defect in the skull which occur due to disruption of the neural tube closure at the level anterior neuropore or secondary to trauma, surgical complications, cleft palate or increased intracranial pressure. The etiology is incompletely understood but hypotheses include genetics, vitamin deficiency, teratogens, infectious agents and environmental factors. Ethmoidal encephaloceles have been reported in multiple species including humans but have not been reported previously in mice. There are multiple models for spontaneous and induced craniofacial malformation in mice, but none described for ethmoidal encephaloceles.

Research-Relevant Conditions and Pathology of Laboratory Mice, Rats, Gerbils, Guinea Pigs, Hamsters, Naked Mole Rats, and Rabbits.

Animals are valuable resources in biomedical research in investigations of biological processes, disease pathogenesis, therapeutic interventions, safety, toxicity, and carcinogenicity. Interpretation of data from animals requires knowledge not only of the processes or diseases (pathophysiology) under study but also recognition of spontaneous conditions and background lesions (pathology) that can influence or confound the study results. Species, strain/stock, sex, age, anatomy, physiology, spontaneous diseases (noninfectious and infectious), and neoplasia impact experimental results and interpretation as well as animal welfare. This review and the references selected aim to provide a pathology resource for researchers, pathologists, and veterinary personnel who strive to achieve research rigor and validity and must understand the spectrum of "normal" and expected conditions to accurately identify research-relevant experimental phenotypes as well as unusual illness, pathology, or other conditions that can compromise studies involving laboratory mice, rats, gerbils, guinea pigs, hamsters, naked mole rats, and rabbits.

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses.

This is the initial report of an α-based pre-targeted radioimmunotherapy (PRIT) using 225Ac and its theranostic pair, 111In. We call our novel tumor-targeting DOTA-hapten PRIT system "proteus-DOTA" or "Pr." Herein we report the first results of radiochemistry development, radiopharmacology, and stoichiometry of tumor antigen binding, including the role of specific activity, anti-tumor efficacy, and normal tissue toxicity with the Pr-PRIT approach (as α-DOTA-PRIT). A series of α-DOTA-PRIT therapy studies were performed in three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2), including evaluation of chronic toxicity at ~20 weeks of select survivors. Methods: Preliminary biodistribution experiments in SW1222 tumor-bearing mice revealed that 225Ac could not be efficiently pretargeted with current DOTA-Bn hapten utilized for 177Lu or 90Y, leading to poor tumor uptake in vivo. Therefore, we synthesized Pr consisting of an empty DOTA-chelate for 225Ac, tethered via a short polyethylene glycol linker to a lutetium-complexed DOTA for picomolar anti-DOTA chelate single-chain variable fragment (scFv) binding. Pr was radiolabeled with 225Ac and its imaging surrogate, 111In. In vitro studies verified anti-DOTA scFv recognition of [225Ac]Pr, and in vivo biodistribution and clearance studies were performed to evaluate hapten suitability and in vivo targeting efficiency. Results: Intravenously (i.v.) administered 225Ac- or 111In-radiolabeled Pr in mice showed rapid renal clearance and minimal normal tissue retention. In vivo pretargeting studies show high tumor accumulation of Pr (16.71 ± 5.11 %IA/g or 13.19 ± 3.88 %IA/g at 24 h p.i. for [225Ac]Pr and [111In]Pr, respectively) and relatively low uptake in normal tissues (all average ≤ 1.4 %IA/g at 24 h p.i.). Maximum tolerated dose (MTD) was not reached for either [225Ac]Pr alone or pretargeted [225Ac]Pr at administered activities up to 296 kBq/mouse. Single-cycle treatment consisting of α-DOTA-PRIT with either huA33-C825 bispecific anti-tumor/anti-DOTA-hapten antibody (BsAb), anti-HER2-C825 BsAb, or hu3F8-C825 BsAb for targeting GPA33, HER2, or GD2, respectively, was highly effective. In the GPA33 model, no complete responses (CRs) were observed but prolonged overall survival of treated animals was 42 d for α-DOTA-PRIT vs. 25 d for [225Ac]Pr only (P < 0.0001); for GD2, CRs (7/7, 100%) and histologic cures (4/7, 57%); and for HER2, CRs (7/19, 37%) and histologic cures (10/19, 56%) with no acute or chronic toxicity. Conclusions: [225Ac]Pr and its imaging biomarker [111In]Pr demonstrate optimal radiopharmacologic behavior for theranostic applications of α-DOTA-PRIT. For this initial evaluation of efficacy and toxicity, single-cycle treatment regimens were performed in all three systems. Histologic toxicity was not observed, so MTD was not observed. Prolonged overall survival, CRs, and histologic cures were observed in treated animals. In comparison to RIT with anti-tumor IgG antibodies, [225Ac]Pr has a much improved safety profile. Ultimately, these data will be used to guide clinical development of toxicity and efficacy studies of [225Ac]Pr, with the goal of delivering massive lethal doses of radiation to achieve a high probability of cure without toxicity.

Senolytic CAR T cells reverse senescence-associated pathologies.

Cellular senescence is characterized by stable cell-cycle arrest and a secretory program that modulates the tissue microenvironment1,2. Physiologically, senescence serves as a tumour-suppressive mechanism that prevents the expansion of premalignant cells3,4 and has a beneficial role in wound-healing responses5,6. Pathologically, the aberrant accumulation of senescent cells generates an inflammatory milieu that leads to chronic tissue damage and contributes to diseases such as liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis1,7. Accordingly, eliminating senescent cells from damaged tissues in mice ameliorates the symptoms of these pathologies and even promotes longevity1,2,8-10. Here we test the therapeutic concept that chimeric antigen receptor (CAR) T cells that target senescent cells can be effective senolytic agents. We identify the urokinase-type plasminogen activator receptor (uPAR)11 as a cell-surface protein that is broadly induced during senescence and show that uPAR-specific CAR T cells efficiently ablate senescent cells in vitro and in vivo. CAR T cells that target uPAR extend the survival of mice with lung adenocarcinoma that are treated with a senescence-inducing combination of drugs, and restore tissue homeostasis in mice in which liver fibrosis is induced chemically or by diet. These results establish the therapeutic potential of senolytic CAR T cells for senescence-associated diseases.

Radiopharmacologic screening of antibodies to the unshed ectodomain of MUC16 in ovarian cancer identifies a lead candidate for clinical translation.

INTRODUCTION: Despite its limitations, CA125 remains the most widely used biomarker for the diagnosis and treatment monitoring of ovarian cancer. Targeting the unshed portion of serum biomarkers such as CA125/MUC16 may afford more specific imaging and targeting of MUC16-positive tumors in High Grade Serous Ovarian Cancer (HGSOC) patients. METHODS: Six monoclonal antibodies raised against the 58 amino acid sequence between the extracellular cleavage site and the transmembrane region of MUC16 were radiolabeled with [89Zr]Zr4+. The radioimmunoconjugates were evaluated in vitro for molar activities, target binding affinity, cellular internalization and serum stability. In vivo characterization was performed via longitudinal positron emission tomography (PET) imaging and ex vivo biodistribution studies in mice bearing subcutaneous xenografts of SKOV3 cells transfected with the proximal 114 amino-acids of MUC16 carboxy-terminus (SKOV3+). RESULTS: In vitro screening identified 9C9 and 4H11 as the lead antibody candidates based on their comparable binding affinities, serum stability and cellular internalization profiles. Despite an identical molecular footprint for binding to MUC16, [89Zr]Zr-DFO-4H11 yielded a more favorable in vivo radiopharmacologic profile. Furthermore, a humanized variant of 4H11 capable of binding MUC16 in vitro also yielded excellent in vivo profile in subcutaneous xenograft models of SKOV3+, OVCAR3 tumors and a patient-derived xenograft model representative of HGSOC. CONCLUSION: Radiopharmacologic screening of antibodies early during their development can provide crucial information pertinent to the in vitro characterization and in vivo pharmacokinetics. The favorable in vivo profile demonstrated by humanized 4H11 combined with the use of its murine predecessor for immunohistochemical staining of biopsied tumor tissues from HGSOC patients makes a unique pair of antibodies that is poised for clinical translation.

Outbreaks of Typhlocolitis Caused by Hypervirulent Group ST1 Clostridioides difficile in Highly Immunocompromised Strains of Mice.

Clostridioides difficile is an enteric pathogen that can cause significant clinical disease in both humans and animals. However, clinical disease arises most commonly after treatment with broad-spectrum antibiotics. The organism's ability to cause naturally occurring disease in mice is rare, and little is known about its clinical significance in highly immunocompromised mice. We report on 2 outbreaks of diarrhea associated with C. difficile in mice. In outbreak 1, 182 of approximately 2, 400 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and related strains of mice became clinically ill after cessation of a 14-d course of 0.12% amoxicillin feed to control an increase in clinical signs associated with Corynebacterium bovis infection. Most mice had been engrafted with human tumors; the remainder were experimentally naïve. Affected animals exhibited 1 of 3 clinical syndromes: 1) peracute death; 2) severe diarrhea leading to euthanasia or death; or 3) mild to moderate diarrhea followed by recovery. A given cage could contain both affected and unaffected mice. Outbreak 2 involved a small breeding colony (approximately 50 mice) of NOD. CB17-Prkdcscid/NCrCrl (NOD-scid) mice that had not received antibiotics or experimental manipulations. In both outbreaks, C. difficile was isolated, and toxins A and B were detected in intestinal content or feces. Histopathologic lesions highly suggestive of C. difficile enterotoxemia included fibrinonecrotizing and neutrophilic typhlocolitis with characteristic 'volcano' erosions or pseudomembrane formation. Genomic analysis of 4 isolates (3 from outbreak 1 and 1 from outbreak 2) revealed that these isolates were closely related to a pathogenic human isolate, CD 196. To our knowledge, this report is the first to describe naturally occurring outbreaks of C. difficile-associated typhlocolitis with significant morbidity and mortality in highly immunocompromised strains of mice.

Mitochondrial arginase-2 is a cell­autonomous regulator of CD8+ T cell function and antitumor efficacy.

As sufficient extracellular arginine is crucial for T cell function, depletion of extracellular arginine by elevated arginase 1 (Arg1) activity has emerged as a hallmark immunosuppressive mechanism. However, the potential cell-autonomous roles of arginases in T cells have remained unexplored. Here, we show that the arginase isoform expressed by T cells, the mitochondrial Arg2, is a cell-intrinsic regulator of CD8+ T cell activity. Both germline Arg2 deletion and adoptive transfer of Arg2-/- CD8+ T cells significantly reduced tumor growth in preclinical cancer models by enhancing CD8+ T cell activation, effector function, and persistence. Transcriptomic, proteomic, and high-dimensional flow cytometry characterization revealed a CD8+ T cell-intrinsic role of Arg2 in modulating T cell activation, antitumor cytoxicity, and memory formation, independently of extracellular arginine availability. Furthermore, specific deletion of Arg2 in CD8+ T cells strongly synergized with PD-1 blockade for the control of tumor growth and animal survival. These observations, coupled with the finding that pharmacologic arginase inhibition accelerates activation of ex vivo human T cells, unveil Arg2 as a potentially new therapeutic target for T cell-based cancer immunotherapies.

ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease-associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10S55L mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10S55L mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10S55L-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10S55L mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.

The APMAP interactome reveals new modulators of APP processing and beta-amyloid production that are altered in Alzheimer's disease.

The adipocyte plasma membrane-associated protein APMAP is expressed in the brain where it associates with γ-secretase, a protease responsible for the generation of the amyloid-ß peptides (Aß) implicated in the pathogenesis of Alzheimer's disease (AD). In this study, behavioral investigations revealed spatial learning and memory deficiencies in our newly generated mouse line lacking the protein APMAP. In a mouse model of AD, the constitutive deletion of APMAP worsened the spatial memory phenotype and led to increased Aß production and deposition into senile plaques. To investigate at the molecular level the neurobiological functions of APMAP (memory and Aß formation) and a possible link with the pathological hallmarks of AD (memory impairment and Aß pathology), we next developed a procedure for the high-grade purification of cellular APMAP protein complexes. The biochemical characterization of these complexes revealed a series of new APMAP interactomers. Among these, the heat shock protein HSPA1A and the cation-dependent mannose-6-phosphate receptor (CD-M6PR) negatively regulated APP processing and Aß production, while clusterin, calnexin, arginase-1, PTGFRN and the cation-independent mannose-6-phosphate receptor (CI-M6PR/IGF2R) positively regulated APP and Aß production. Several of the newly identified APMAP interactomers contribute to the autophagy-lysosome system, further supporting an emergent agreement that this pathway can modulate APP metabolism and Aß generation. Importantly, we have also demonstrated increased alternative splicing of APMAP and lowered levels of the Aß controllers HSPA1A and CD-M6PR in human brains from neuropathologically verified AD cases.

Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells.

INTRODUCTION: Many human breast cancers overexpress the E3 ubiquitin ligase MDM2 and its homolog MDMX. Expression of MDM2 and MDMX occurs in estrogen receptor α-positive (ERα+) breast cancer and triple-negative breast cancer (TNBC). There are p53-independent influences of MDM2 and MDMX, and 80% of TNBC express mutant p53 (mtp53). MDM2 drives TNBC circulating tumor cells (CTCs) in mice, but the context-dependent influences of MDM2 and MDMX on different subtypes of breast cancers expressing mtp53 have not been determined. METHODS: To assess the context-dependent roles, we carried out MDM2 and MDMX knockdown in orthotopic tumors of TNBC MDA-MB-231 cells expressing mtp53 R280K and MDM2 knockdown in ERα+ T47D cells expressing mtp53 L194F. The corresponding cell proliferation was scored in vitro by growth curves and in vivo by orthotopic tumor volumes. Cell migration was assessed in vitro by wound-healing assays and cell intravasation in vivo by sorting GFP-positive CTCs by flow cytometry. The metastasis gene targets were determined by an RT-PCR array card screen and verified by qRT-PCR and Western blot analysis. RESULTS: Knocking down MDMX or MDM2 in MDA-MB-231 cells reduced cell migration and CTC detection, but only MDMX knockdown reduced tumor volumes at early time points. This is the first report of MDMX overexpression in TNBC enhancing the CTC phenotype with correlated upregulation of CXCR4. Experiments were carried out to compare MDM2-knockdown outcomes in nonmetastatic ERα+ T47D cells. The knockdown of MDM2 in ERα+ T47D orthotopic tumors decreased primary tumor volumes, supporting our previous finding that estrogen-activated MDM2 increases cell proliferation. CONCLUSIONS: This is the first report showing that the expression of MDM2 in ERα+ breast cancer and TNBC can result in different tumor-promoting outcomes. Both MDMX and MDM2 overexpression in TNBC MDA-MB-231 cells enhanced the CTC phenotype. These data indicate that both MDM2 and MDMX can promote TNBC metastasis and that it is important to consider the context-dependent roles of MDM2 family members in different subtypes of breast cancer.

Theranostic pretargeted radioimmunotherapy of internalizing solid tumor antigens in human tumor xenografts in mice: Curative treatment of HER2-positive breast carcinoma.

In recent reports, we have shown that optimized pretargeted radioimmunotherapy (PRIT) based on molecularly engineered antibody conjugates and 177Lu-DOTA chelate (DOTA-PRIT) can be used to cure mice bearing human solid tumor xenografts using antitumor antibodies to minimally internalizing membrane antigens, GPA33 (colon) and GD2 (neuroblastoma). However, many solid tumor membrane antigens are internalized after antibody binding and it is generally believed that internalizing tumor membrane antigens are not suitable targets for PRIT. In this study, we tested the hypothesis that DOTA-PRIT can be performed successfully to target HER2, an internalizing membrane antigen widely expressed in breast, ovarian, and gastroesophageal junction cancers. Methods: DOTA-PRIT was carried out in athymic nude mice bearing BT-474 xenografts, a HER2-expressing human breast cancer, using a three-step dosing regimen consisting of sequential intravenous administrations of: 1) a bispecific IgG-scFv (210 kD) format (BsAb) carrying the IgG sequence of the anti-HER2 antibody trastuzumab and the scFv "C825" with high-affinity, hapten-binding antibody for Bn-DOTA (metal) (BsAb: anti-HER2-C825), 2) a 500 kD dextran-based clearing agent, followed by 3) 177Lu-DOTA-Bn. At the time of treatment, athymic nude mice bearing established subcutaneous BT-474 tumors (medium- and smaller-sized tumors with tumor volumes of 209 ± 101 mm3 and ranging from palpable to 30 mm3, respectively), were studied along with controls. We studied single- and multi-dose regimens. For groups receiving fractionated treatment, we verified quantitative tumor targeting during each treatment cycle using non-invasive imaging with single-photon emission computed tomography/computed tomography (SPECT/CT). Results: We achieved high therapeutic indices (TI, the ratio of radiation-absorbed dose in tumor to radiation-absorbed dose to critical organs, such as bone marrow) for targeting in blood (TI = 28) and kidney (TI = 7), while delivering average radiation-absorbed doses of 39.9 cGy/MBq to tumor. Based on dosimetry estimates, we implemented a curative fractionated therapeutic regimen for medium-sized tumors that would deliver approximately 70 Gy to tumors, which required treatment with a total of 167 MBq 177Lu-DOTA-Bn/mouse (estimated absorbed tumor dose: 66 Gy). This regimen was well tolerated and achieved 100% complete responses (CRs; defined herein as tumor volume equal to or smaller than 4.2 mm3), including 62.5% histologic cure (5/8) and 37.5% microscopic residual disease (3/8) at 85 days (d). Treatment controls showed tumor progression to 207 ± 201% of pre-treatment volume at 85 d and no CRs. Finally, we show that treatment with this curative 177Lu regimen leads to a very low incidence of histopathologic abnormalities in critical organs such as bone marrow and kidney among survivors compared with non-treated controls. Conclusion: Contrary to popular belief, we demonstrate that DOTA-PRIT can be successfully adapted to an internalizing antigen-antibody system such as HER2, with sufficient TIs and absorbed tumor doses to achieve a high probability of cures of established human breast cancer xenografts while sparing critical organs of significant radiotoxicity.

CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade.

Chimeric antigen receptor (CAR) therapy targeting CD19 is an effective treatment for refractory B cell malignancies, especially acute lymphoblastic leukemia (ALL) 1 . Although a majority of patients will achieve a complete response following a single infusion of CD19-targeted CAR-modified T cells (CD19 CAR T cells)2-4, the broad applicability of this treatment is hampered by severe cytokine release syndrome (CRS), which is characterized by fever, hypotension and respiratory insufficiency associated with elevated serum cytokines, including interleukin-6 (IL-6)2,5. CRS usually occurs within days of T cell infusion at the peak of CAR T cell expansion. In ALL, it is most frequent and more severe in patients with high tumor burden2-4. CRS may respond to IL-6 receptor blockade but can require further treatment with high dose corticosteroids to curb potentially lethal severity2-9. Improved therapeutic and preventive treatments require a better understanding of CRS physiopathology, which has so far remained elusive. Here we report a murine model of CRS that develops within 2-3 d of CAR T cell infusion and that is potentially lethal and responsive to IL-6 receptor blockade. We show that its severity is mediated not by CAR T cell-derived cytokines, but by IL-6, IL-1 and nitric oxide (NO) produced by recipient macrophages, which enables new therapeutic interventions.