Highly active CAR T cells that bind to a juxtamembrane region of mesothelin and are not blocked by shed mesothelin

Mesothelin (MSLN) is a cell-surface protein that is highly expressed by many cancers. Despite many clinical trials, there is not a Food and Drug Administration (FDA)-approved antibody-based therapy targeting MSLN. Shed MSLN is present in high concentrations in tumors and in body fluids and constitutes a major barrier to antibody-based therapies. MSLN is shed from cells by the action of proteases that cut very close to the membrane. We have identified the major protease sites in MSLN and prepared a monoclonal antibody (mAb) 15B6, that binds next to the cell membrane at the protease-sensitive region and inhibits MSLN shedding. We determined the structure of a Fab-MSLN peptide complex and found the antibody binds to residues Y-V-DLSMQEL at the C terminus of MSLN. mAb 15B6 makes very active chimeric antigen receptor (CAR) T cells whose activity is not blocked by shed MSLN. The 15B6 CAR T cells have greatly superior antitumor activity in mice than CAR T cells targeting an epitope in shed MSLN.

Mesothelin (MSLN) shedding is a barrier to the activity of antibody-based therapeutics targeting MSLN. MSLN is shed from cells by the action of proteases that cut close to the cell membrane. Monoclonal antibody (mAb) 15B6 binds to the protease-sensitive region, inhibits MSLN shedding, and makes very active chimeric antigen receptor (CAR) T cells that are greatly superior in activity in mouse tumor models to CAR T cells made with mAb SS1 that binds to a distal epitope. We ascribe this enhanced activity to binding of 15B6 CAR T cells close to the cell membrane and to avoiding the inhibitory effect of shed MSLN.MSLN is a lineage-restricted cell-surface protein that is expressed by many cancers and on normal mesothelial cells (1, 2). MSLN was discovered in a search for cell-surface proteins that were expressed in cancers but not on essential organs (3, 4) and is now known to be expressed on many human cancers (1, 2). In addition, patients with high MSLN on their tumors often have a poorer prognosis (1). The specificity and pattern of MSLN expression makes it a popular target for antibody-based therapies, including CAR T cells and bispecific T cell engagers (BiTEs) (5–9). Although a few major responses have been observed with antibody-based therapies targeting MSLN (1, 10, 11), there is not yet a Food and Drug Administration (FDA)-approved drug targeting MSLN.One major impediment to antibody efficacy is MSLN shedding (12). Shed MSLN inactivates antibodies and antibody-based agents such as CAR T cells and BiTEs before they reach MSLN on the tumor cells. Shedding also releases these agents from the cell surface before they have an opportunity to kill target cells. Previous studies have indicated that MSLN shedding is due to cleavage of MSLN at several different sites close to the cell membrane (12, 13). At least five different enzymes participate in the shedding process and others must exist because for some cell lines no shedding enzymes were identified (13). Another unusual aspect of shedding is that in different cancer cell lines, different enzymes are involved. Up to now ADAM10, ADAM17, BACE1, BACE2, and MMP15 have been identified (13). MSLN levels that result from tumor shedding are much higher inside tumors than in blood (14) and are very high in ascites and pleural fluid of patients with mesothelioma, where the levels can exceed 10 μg/mL. This presents a significant barrier to therapy (15).Our goal was to acquire quantitative information on the major protease sites resulting in MSLN shedding, to use this information to make a monoclonal antibody that binds to the protease site region, and to use the antibody to make CAR T cells and other targeted therapeutics. We describe here that mAb 15B6, which blocks MSLN shedding, does not react with shed MSLN and makes a very active CAR T cell that produces complete remissions of MSLN-expressing tumors in mice. We ascribe the high activity of the CAR T cells to the fact that mAb 15B6 binds to MSLN at the site where it attaches to the cell membrane and is not inactivated by shed decoy MSLN found at high concentrations inside tumors and in ascites and pleural fluid of cancer patients (14, 15). To the best of our knowledge all other antibodies to MSLN bind to shed MSLN, although the epitopes and the distance from the cell membrane vary among them (16–20).     

Intraductal administration of transferrin receptor-targeted immunotoxin clears ductal carcinoma in situ in mouse models of breast cancer—a preclinical study

The human transferrin receptor (TFR) is overexpressed in most breast cancers, including preneoplastic ductal carcinoma in situ (DCIS). HB21(Fv)-PE40 is a single-chain immunotoxin (IT) engineered by fusing the variable region of a monoclonal antibody (HB21) against a TFR with a 40 kDa fragment of Pseudomonas exotoxin (PE). In humans, the administration of other TFR-targeted immunotoxins intrathecally led to inflammation and vascular leakage. We proposed that for treatment of DCIS, intraductal (i.duc) injection of HB21(Fv)-PE40 could avoid systemic toxicity while retaining its potent antitumor effects on visible and occult tumors in the entire ductal tree. Pharmacokinetic studies in mice showed that, in contrast to intravenous injection, IT was undetectable by enzyme-linked immunosorbent assay in blood following i.duc injection of up to 3.0 μg HB21(Fv)-PE40. We demonstrated the antitumor efficacy of HB21(Fv)-PE40 in two mammary-in-duct (MIND) models, MCF7 and SUM225, grown in NOD/SCID/gamma mice. Tumors were undetectable by In Vivo Imaging System (IVIS) imaging in intraductally treated mice within 1 wk of initiation of the regimen (IT once weekly/3 wk, 1.5 μg/teat). MCF7 tumor–bearing mice remained tumor free for up to 60 d of observation with i.duc IT, whereas the HB21 antibody alone or intraperitoneal IT treatment had minimal/no antitumor effects. These and similar findings in the SUM225 MIND model were substantiated by analysis of mammary gland whole mounts, histology, and immunohistochemistry for the proteins Ki67, CD31, CD71 (TFR), and Ku80. This study provides a strong preclinical foundation for conducting feasibility and safety trials in patients with stage 0 breast cancer.

With the advent of monoclonal antibodies (mAb) in the 1980s, the vision that cell-specific targeted antibodies or antibodies linked to potent plant toxins could achieve tumor-specific killing has become a reality (1, 2). Since then, many immunotoxins (ITs) have been engineered to specifically target cancer cells (3–5).One of these, HB21(Fv)-PE40, consists of the variable regions of HB21, a monoclonal antibody to the human transferrin receptor (TFR), fused to PE40, a truncated form of Pseudomonas exotoxin (PE) A that is devoid of its binding domain and, once internalized, leads to protein translation arrest and cell death (6, 7). Clinical success was achieved with moxetumomab pasudotox (an Fv fragment of an anti-CD22 antibody fused to a truncated 38 kDa PE) for the treatment of hairy cell leukemia (8). In a pivotal trial evaluating 80 patients with relapsed or refractory hairy cell leukemia, the complete response rate was 41% and the overall response rate was 75%. This trial led to FDA approval of moxetumomab pasudotox in 2018 (8) and was followed by additional reports of its clinical effectiveness (4, 9, 10).The TFR is expressed at markedly higher levels in all subtypes of breast cancer compared to normal breast epithelium (11–14). The most comprehensive study of TFR expression in various breast pathologies was conducted by Singh et al. (12), who reported low-level expression in benign breast lesions (n = 172), abundant expression in ductal carcinoma in situ (DCIS) (n = 65; > 50% cells) and in invasive breast cancer (n = 38; > 80% cells), but negligible expression in normal breast tissue (n = 127). This widespread, tumor-specific expression renders TFR a potent target for antibody–toxin conjugates.A barrier to the systemic use of TFR-directed ITs in the clinic is the known widespread distribution of TFR in many important tissues like the liver and blood vessels that would lead to adverse events. Previously, a conjugate of a monoclonal antibody to the TFR and recombinant ricin A chain (454A12-rRA) was investigated for local treatment of glioblastoma (15). A dose finding toxicity with 454A12-rRA showed that administered intraventricularly, half of the eight patients exhibited temporary tumor responses. No acute or chronic drug toxicity was seen in patients who received up to 38 μ⩽g IT. Doses more than or equal to 120 μg caused a cerebrospinal fluid (CSF) inflammatory response that was associated with transient headache, vomiting, and altered mental status that was responsive to steroids and CSF drainage. No systemic toxicity was detected (15). Although promising, many hurdles remain in realizing the potential of local IT treatment in the brain, including inconsistent drug delivery and local toxicity (16).Based on the high levels of expression of TFR in invasive breast cancer and DCIS, we hypothesized that we can circumvent the problem of systemic toxicity while at the same time control local breast cancer by administering HB21(Fv)-PE40 through the intraductal (i.duc) route. DCIS is a preneoplastic lesion in which the cancer cells are confined within the myoepithelial lining of the mammary ducts (17). Currently, there is concern that many patients with DCIS are overtreated by ablative surgery and radiation (18–20). The consensus opinion is that treatment with minimally invasive procedures should receive serious consideration to improve the quality of life of patients with DCIS (18–20). It is unlikely that surgery will be replaced as a treatment for DCIS, unless a safe and equally effective alternative is provided.That i.duc therapy shows promise for treating DCIS has been demonstrated in several preclinical studies (21–28). As shown by our group, the superior effects of the i.duc route, compared to the systemic route, for the delivery of chemotherapy (21, 22), radioactive compounds (24), and hormonal agents (18, 21) stems from the ability to confine the anticancer effects of the agent to the tumor site in the duct while sparing the remainder of the body from its toxic effects. We demonstrated the feasibility of and ready acceptance by women of the i.duc route for delivering drugs in a clinical study performed using i.duc Doxil in women prior to surgery (22).While i.duc chemotherapy (21, 22), radiotherapy (24), and endocrine therapy (21, 26) approaches significantly reduced tumor burden, none of these approaches eradicated tumor cells. As a result, tumors regrew in the animals upon long-term follow-up. In this study, we tested the anticancer efficacy of i.duc administration of HB21(Fv)-PE40 in mammary-in-duct (MIND) models of DCIS using MCF7 and SUM225 (24, 29–31). We show that the i.duc administration of IT rendered 100% of the mice bearing i.duc MCF7 and SUM225 mammary outgrowths tumor free. Unlike intravenous (i.v.) injected IT, no toxin was measurable in the blood of mice by enzyme-linked immunosorbent assay (ELISA) 5 to 30 min after i.duc injection. Compared to current treatments, this approach addresses the ablation of DCIS, both visible and occult, in the entire diseased ductal tree. In addition, unlike i.duc chemotherapy and radiotherapy there is no risk of causing mutations in DNA using ITs. Our findings provide a strong foundation for the potential use of i.duc ITs as a therapeutic approach for DCIS.     

The role of coronary artery calcium in allocating pharmacotherapy for primary prevention of cardiovascular disease: The ABCs of CAC

Determining optimal candidates for the numerous potential pharmacotherapies for primary prevention of atherosclerotic cardiovascular disease remains challenging. Selective use of coronary artery calcium (CAC) scoring is recommended by the 2018 and 2019 American Heart Association/American College of Cardiology Cholesterol and Primary Prevention Guidelines as a tool for refining cardiovascular disease risk assessment. A growing body of research shows that CAC has potential value in allocation of primary prevention aspirin, determining blood pressure targets and treatment intensity, the intensity of cholesterol management, and use of the more expensive medications for type 2 diabetes. We also review the literature regarding very elevated CAC scores greater than 400 or 1000 and how these scores appear to confer a risk for cardiovascular disease on par with secondary prevention cohorts.     

Small cell lung cancer in young patients: trends in sociodemographic factors,diagnosis, treatment,and survival

BackgroundSmall cell lung cancer (SCLC) in patients <50 years old has unique socioeconomic and clinical implications. We aimed to examine the demographics, treatment patterns, and survival of young patients with SCLC and compared them to older adults.MethodsThe National Cancer Database (NCDB) was queried to identify SCLC cases diagnosed from 2004 to 2016. Patients were divided into three age groups: ≥18–<50, ≥50–<70, and ≥70 years. Patient characteristics were evaluated for survival within each age group. Kaplan-Meier and Cox regression analyses were used to assess survival.ResultsOf the 172,453 evaluated SCLC patients (median age 66 years), 8,792 were ≥18–<50 years old. Compared to the older groups, patients under 50 were more likely to be Black, uninsured or on Medicaid, have household income <$30,000, and present with stage III or IV disease (P<0.0001 for all). While young patients were more likely to receive guideline-concordant care (GCC), the hazard of death increased to 1.96 (95% CI: 1.80–2.14; P<0.0001) with receipt of nonstandard therapy. Private insurance, female gender, non-White race, Hispanic ethnicity, and higher income were associated with better survival. The youngest cohort had significantly better survival overall when compared to the older patients (P<0.0001), but the survival advantage was reduced with the advancing stage.ConclusionsSCLC patients under 50 years old represent a socioeconomically disadvantaged group with advanced disease at presentation. Despite having fewer comorbidities and being offered guideline-concordant treatment, younger patients with SCLC have only marginally better survival than older patients in advanced stages.     

Therapeutic implications of crocin in Parkinson's disease: A review of preclinical research

Parkinson's disease is among the most common forms of neurodegenerative illness, with present treatment being primarily symptomatic and frequently coming with substantial adverse effects. Neuronal degeneration may arise due to a variety of pathological events, like inflammatory responses, neurotransmitter dysregulation, oxidative damage, mitochondrial malfunction, apoptosis, and genetic factors. The health issue and financial burden brought on by Parkinson's disease can worsen as the population ages. In the search for new and secure therapeutic agents for Parkinson's disease, several natural compounds have been shown to exert considerable neuroprotective benefits. Crocin, a naturally occurring carotenoid molecule, was found to have neuroprotective potential in the therapy of this disorder. Taking into account, the outcomes of various studies and the restorative actions of crocin, the present study emphasized the protective ability of crocin in this disease. Given the strong evidence supporting the neuroprotective ability of crocin, it is inferred that crocin inhibits inflammatory, apoptotic, and antioxidant processes through multiple mechanisms. Therefore, this compound is considered a safe and effective therapeutic choice for neurodegenerative illnesses like Parkinson's disease. However, more research on its efficacy as a treatment of Parkinson's disease is needed, specifically examining its mechanisms and the results obtained in clinical trials.     

Plasma cell-free DNA methylome profiling in pre- and post-surgery oral cavity squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC), a highly heterogeneous disease that involves multiple anatomic sites, is a leading cause of cancer-related mortality worldwide. Although the utility of noninvasive biomarkers based on circulating cell-free DNA (cfDNA) methylation profiling has been widely recognized, limited studies have been reported so far regarding the dynamics of cfDNA methylome in oral cavity squamous cell carcinoma (OCSCC). It is hypothesized in this study that comparison of methylation profiles in pre- and postsurgery plasma samples will reveal OCSCC-specific prognostic and diagnostic biomarkers. As a strategy to further prioritize tumor-specific targets, top differential methylated regions (DMRs) were called by reanalyzing methylation data from paired tumor and normal tissue collected in the the cancer genome atlas head-neck squamous cell carcinoma (TCGA) head and neck cancer cohort. Matched plasma samples from eight patients with OCSCC were collected at Moffitt Cancer Center before and after surgical resection. Plasma-derived cfDNA was analyzed by cfMBD-seq, which is a high-sensitive methylation profiling assay. Differential methylation analysis was then performed based on the matched samples profiled. In the top 200 HNSCC-specific DMRs detected based on the TCGA data set, a total of 23 regions reached significance in the plasma-based DMR test. The top five validated DMR regions (ranked by the significance in the plasma study) are located in the promoter regions of genes PENK, NXPH1, ZIK1, TBXT, and CDO1, respectively. The genome-wide cfDNA DMR analysis further highlighted candidate biomarkers located in genes SFRP4, SOX1, IRF4, and PCDH17. The prognostic relevance of candidate genes was confirmed by survival analysis using the TCGA data. This study supports the utility of cfDNA-based methylome profiling as a promising noninvasive biomarker source for OCSCC and HNSCC.