B7-H3-Targeting Chimeric Antigen Receptors Epstein-Barr Virus-specific T Cells Provides a Tumor Agnostic Off-The-Shelf Therapy Against B7-H3-positive Solid Tumors.

Encouraged by the observations of significant B7-H3 protein overexpression in many human solid tumors compared to healthy tissues, we directed our focus towards targeting B7-H3 using chimeric antigen receptor (CAR) T cells. We utilized a nanobody as the B7-H3-targeting domain in our CAR construct to circumvent the stability issues associated with single-chain variable fragment-based domains. In efforts to expand patient access to CAR T-cell therapy, we engineered our nanobody-based CAR into human Epstein-Barr virus-specific T cells (EBVST), offering a readily available off-the-shelf treatment. B7H3.CAR-armored EBVSTs demonstrated potent in vitro and in vivo activities against multiple B7-H3-positive human tumor cell lines and patient-derived xenograft models. Murine T cells expressing a murine equivalent of our B7H3.CAR exhibited no life-threatening toxicities in immunocompetent mice bearing syngeneic tumors. Further in vitro evaluation revealed that while human T, B, and natural killer cells were unaffected by B7H3.CAR EBVSTs, monocytes were targeted because of upregulation of B7-H3. Such targeting of myeloid cells, which are key mediators of cytokine release syndrome (CRS), contributed to a low incidence of CRS in humanized mice after B7H3.CAR EBVST treatment. Notably, we showed that B7H3.CAR EBVSTs can target B7-H3-expressing myeloid-derived suppressor cells (MDSC), thereby mitigating MDSC-driven immune suppression. In summary, our data demonstrate that our nanobody-based B7H3.CAR EBVSTs are effective as an off-the-shelf therapy for B7-H3-positive solid tumors. These cells also offer an avenue to modulate the immunosuppressive tumor microenvironment, highlighting their promising clinical potential in targeting solid tumors. SIGNIFICANCE: Clinical application of EBVSTs armored with B7-H3-targeting CARs offer an attractive solution to translate off-the-shelf CAR T cells as therapy for solid tumors.

Breastfeeding Duration and Development of Dysglycemia in Women Who Had Gestational Diabetes Mellitus: Evidence from the GUSTO Cohort Study.

(1) Background: Breastfeeding has been shown to support glucose homeostasis in women after a pregnancy complicated by gestational diabetes mellitus (GDM) and is potentially effective at reducing long-term diabetes risk. (2) Methods: Data from the Growing Up in Singapore Towards healthy Outcomes (GUSTO) study were analyzed to understand the influence of breastfeeding duration on long-term dysglycemia (prediabetes and diabetes) risk in women who had GDM in the index pregnancy. GDM and dysglycemia four to seven years postpartum were determined by the oral glucose tolerance test (OGTT). A Poisson regression model with a robust error variance was used to estimate incidence rate ratios (IRRs) for dysglycemia four to seven years post-delivery according to groupings of the duration of any breastfeeding (<1, ≥1 to <6, and ≥6 months). (3) Results: Women who had GDM during the index pregnancy and complete breastfeeding information and OGTT four to seven years postpartum were included in this study (n = 116). Fifty-one women (44%) had postpartum dysglycemia. Unadjusted IRRs showed an inverse association between dysglycemia risk and ≥1 month to <6 months (IRR 0.91; 95% confidence interval [CI] 0.57, 1.43; p = 0.68) and ≥6 months (IRR 0.50; 95% CI 0.27, 0.91; p = 0.02) breastfeeding compared to <1 month of any breastfeeding. After adjusting for key confounders, the IRR for the ≥6 months group remained significant (IRR 0.42; 95% CI 0.22, 0.80; p = 0.008). (4) Conclusions: Our results suggest that any breastfeeding of six months or longer may reduce long-term dysglycemia risk in women with a history of GDM in an Asian setting. Breastfeeding has benefits for mothers beyond weight loss, particularly for those with GDM.

Direct Admission from the Emergency Department to a Subacute Care Ward: An Alternative to Acute Hospitalization.

In recent years, subacute care units (SCUs) have emerged as alternatives to acute hospitalization for selected emergency department (ED) patients who might benefit from a short period of inpatient stay within a less acute setting. We developed a new protocol to directly admit selected older patients from our acute hospital's (AH) ED to the SCU of a partner community hospital, making use of our ED's short-stay ward as a transit area to overcome administrative, financial, and clinical barriers. The new protocol has removed the need for intervening stays of longer than 24 hours at our AH, reduced overall length of stay across both institutions, decreased hospital admissions, and reduced the number of patient hand-offs.

Therapeutic anti-cancer activity of antibodies targeting sulfhydryl bond constrained epitopes on unglycosylated RON receptor tyrosine kinase.

Recepteur d'origine nantais (RON) receptor tyrosine kinase (RTK) and its ligand, serum macrophage-stimulating protein (MSP), are well-established oncogenic drivers for tumorigenesis and metastasis. RON is often found to be alternatively spliced resulting in various isoforms that are constitutively active. RON is therefore an attractive target for cancer therapeutics, including small molecular inhibitors and monoclonal antibodies. While small molecule inhibitors of RON may inhibit other protein kinases including the highly similar MET kinase, monoclonal antibodies targeting RON are more specific, potentially inducing fewer side effects. Although anti-RON monoclonal antibody therapies have been developed and tested in clinical trials, they were met with limited success. Cancer cells have been associated with aberrant glycosylation mechanisms. Notably for RON, the loss of N-bisected glycosylation is a direct cause for tumorigenesis and poorer prognosis in cancer patients. Particularly in gastric cancer, aberrant RON glycosylation augments RON activation. Here, we present a novel panel of monoclonal antibodies which potentially widens the specific targeting of not only the glycosylated RON, but also unglycosylated and aberrantly glycosylated RON. Our antibodies can bind strongly to deglycosylated RON from tunicamycin treated cells, recognise RON in IHC/IF and possess superior therapeutic efficacy in RON expressing xenograft tumours. Our most potent antibody in xenograft assays, is directed to the RON alpha chain and targets a sulfhydryl bond constrained epitope that appears to be cryptic in the crystal structure. This establishes the paradigm that such constrained and cryptic epitopes represent good targets for therapeutic antibodies.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

Monoclonal Antibodies against Specific p53 Hotspot Mutants as Potential Tools for Precision Medicine.

The large number of mutations identified across all cancers represents an untapped reservoir of targets that can be useful for therapeutic targeting if highly selective, mutation-specific reagents are available. We report here our attempt to generate such reagents: monoclonal antibodies against the most common R175H, R248Q, and R273H hotspot mutants of the tumor suppressor p53. These antibodies recognize their intended specific alterations without any cross-reactivity against wild-type (WT) p53 or other p53 mutants, including at the same position (as exemplified by anti-R248Q antibody, which does not recognize the R248W mutation), evaluated by direct immunoblotting, immunoprecipitation, and immunofluorescence methods on transfected and endogenous proteins. Moreover, their clinical utility to diagnose the presence of specific p53 mutants in human tumor microarrays by immunohistochemistry is also shown. Together, the data demonstrate that antibodies against specific single-amino-acid alterations can be generated reproducibly and highlight their utility, which could potentially be extended to therapeutic settings.

Inhibiting p53 Acetylation Reduces Cancer Chemotoxicity.

Chemotoxicity due to unwanted p53 activation in the bone marrow remains an unmet clinical challenge. Doxorubicin, a first-line chemotherapy drug, often causes myelosuppression in patients, thus limiting its effectiveness. In this study, we discovered that C646, a reversible p300 inhibitor, downregulates p53 transcription and selectively protects noncancerous cells from p53-dependent apoptosis. C646 treatment blocked acetylation of specific lysine residues that regulate p53 activity. Exploitation of differential p53 genetic backgrounds between human hematopoietic and colorectal cancer cells improved the therapeutic index of doxorubicin with C646 cotreatment. C646 administration in mice afflicted with p53-mutant tumors protected them from doxorubicin-induced neutropenia and anemia while retaining antitumor efficacy. We deduce that temporary and reversible inhibition of p53 acetylation in cancer subjects, especially those with p53-mutant tumors, may protect them from severe chemotoxicity while allowing treatment regimens to effectively proceed. Cancer Res; 77(16); 4342-54. ©2017 AACR.

A His6-SUMO-eXact tag for producing human prepro-urocortin 2 in Escherichia coli for raising monoclonal antibodies.

This is a first report of recombinant production of human prepro-Urocortin 2 in Escherichia coli by N-terminal fusion with a triple His6-SUMO-eXact tag and its subsequent use as an antigen for the production and screening of very high affinity monoclonal antibodies. The rationale for this combinatorial construct is that the His tag allows first step protein purification of insoluble and soluble proteins, the SUMO tag enhances protein expression level and solubility, while the eXact tag facilitates anion-triggered on-column cleavage of the triple tag to recover pure native proteins in a simple two-step protein purification procedure. Compared with an eXact fusion alone, the presence of the SUMO moiety enhanced overall expression levels by 4 to 10 fold but not the solubility of the highly basic prepro-Urocortin 2. Insoluble SUMO-eXact-preproUCN2 was purified in milligram quantities by denaturing IMAC and solubilized in native phosphate buffer by on-column refolding or step-wise dialysis. Only a small fraction of this solubilized protein was able to bind onto the eXact™ affinity column and cleaved by NaF treatment. To test whether binding and cleavage failure was due to improperly refolded SUMO-eXact-preproUCN2 or to the presence of N- and C-terminal sequences flanking the eXact moiety, we created a SUMO-eXact-thioredoxin construct which was overexpressed mainly in the soluble form. This protein bound to and was cleaved efficiently on the eXact™ column to yield native thioredoxin. Solubilized SUMO-eXact-preproUCN2 was used successfully to generate two high affinity mouse monoclonal antibodies (KD~10⁻¹° and 10⁻¹¹ M) specific to the pro-region of Urocortin 2.