A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo.

The CEACAM5 gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)-based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02-expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.

Chimeric Antigen Receptors Directed at Mutant KRAS Exhibit an Inverse Relationship Between Functional Potency and Neoantigen Selectivity.

Neoantigens are among the most intriguing potential immuno-oncology targets because, unlike many cancer targets that are expressed on normal tissues, they are by definition restricted to cancer cells. Medicines directed at common neoantigens such as mutant KRAS are especially interesting because they may offer the convenience and cost of an off-the-shelf therapy. However, all common KRAS mutations produce proteins that differ from the wild type at a single amino acid, creating challenges for molecular discrimination. We have undertaken an effort to optimize single-chain variable fragments (scFv) against peptide/major histocompatibility antigen complexes composed of HLA-A*11 and either G12V- or G12D-mutant KRAS peptides. These scFvs could in principle be used in chimeric antigen receptor (CAR) T-cell therapies for selected patients whose tumors bear either of these mutations. Here we show that optimization of such CARs involves a trade-off between potency and selectivity. We further show that targeting this family without high selectivity engenders risks of cross-reactivity against other members of the G-protein family to which KRAS belongs. Significance: We report an effort to generate high potency, selective CARs directed at mutant KRAS peptides. Although the heavily optimized CARs maintain high selectivity against wild-type KRAS, they lose selectivity against other KRAS-related peptides derived from human proteins. To our knowledge, this work is the first to examine the trade-off between potency and selectivity with regard to KRAS pMHC-directed CARs, illustrating the challenge to achieve both sufficient potency and high selectivity.

Extensive functional comparisons between chimeric antigen receptors and T cell receptors highlight fundamental similarities.

Though TCRs have been subject to limited engineering in the context of therapeutic design and optimization, they are used largely as found in nature. On the other hand, CARs are artificial, composed of different segments of proteins that function in the immune system. This characteristic raises the possibility of altered response to immune regulatory stimuli. Here we describe a large-scale, systematic comparison of CARs and TCRs across 5 different pMHC targets, with a total of 19 constructs examined in vitro. These functional measurements include CAR- and TCR-mediated activation, proliferation, and cytotoxicity in both acute and chronic settings. Surprisingly, we find no consistent difference between CARs and TCRs as receptor classes with respect to their relative sensitivity to major regulators of T cell activation: PD-L1, CD80/86 and IL-2. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved and compared head to head in functional tests, CARs appear remarkably similar to TCRs with respect to immune modulation.

Potent, Selective CARs as Potential T-Cell Therapeutics for HPV-positive Cancers.

Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients' T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629-38 complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.

Re-examination of MAGE-A3 as a T-cell Therapeutic Target.

In 2013, an innovative MAGE-A3-directed cancer therapeutic of great potential value was terminated in the clinic because of neurotoxicity. The safety problems were hypothesized to originate from off-target T-cell receptor activity against a closely related MAGE-A12 peptide. A combination of published and new data led us to test this hypothesis with current technology. Our results call into question MAGE-A12 as the source of the neurotoxicity. Rather, the data imply that an alternative related peptide from EPS8L2 may be responsible. Given the qualities of MAGE-A3 as an onco-testis antigen widely expressed in tumors and largely absent from normal adult tissues, these findings suggest that MAGE-A3 may deserve further consideration as a cancer target. As a step in this direction, the authors isolated 2 MAGE-A3 peptide-major histocompatibility complex-directed chimeric antigen receptors, 1 targeting the same peptide as the clinical T-cell receptor. Both chimeric antigen receptors have improved selectivity over the EPS8L2 peptide that represents a significant risk for MAGE-A3-targeted therapeutics, showing that there may be other options for MAGE-A3 cell therapy.

Structure-function relationships of chimeric antigen receptors in acute T cell responses to antigen.

Chimeric antigen receptors (CARs) and their parent signaling molecule, the T cell receptor (TCR), are fascinating proteins of increasing relevance to disease therapy. Here we use a collection of 1221 pMHC-directed CAR constructs representing 10 pMHC targets to study aspects of CAR structure-activity relationships (SAR), with particular focus on the extracellular and transmembrane structural components. These experiments that involve pMHC targets whose number/cell can be manipulated by peptide dosing in vitro enable systematic analysis of the SAR of CARs in carefully controlled experimental situations (Harris and Kranz, 2016). We find that CARs tolerate a wide range of structural variation, with the ligand-binding domains (LBDs) dominating the SAR of CAR antigen sensitivity. Notwithstanding the critical role of the LBD, CAR antigen-binding on the cell surface, measured by pMHC tetramer staining, is not an effective predictor of functional sensitivity. These results have important implications for the design and testing of CARs aimed toward the clinic.

Short PlGF-derived peptides bind VEGFR-1 and VEGFR-2 in vitro and on the surface of endothelial cells.

The placental growth factor (PlGF), a member of VEGF family, plays a crucial role in pathological angiogenesis, especially ischemia, inflammation, and cancer. This activity is mediated by its selective binding to VEGF receptor 1 (VEGFR-1), which occurs predominantly through receptor domains 2 and 3. The PlGF ß-hairpin region spanning residues Q87 to V100 is one of the key binding elements on the protein side. We have undertaken a study on the design, preparation, and functional characterization of the peptide reproducing this region and of a set of analogues where glycine 94, occurring at the corner of the hairpin in the native protein, is replaced by charged as well as hydrophobic residues. Also, some peptides with arginine 96 replaced by other residues have been studied. We find that the parent peptide weakly binds VEGFR-1, but replacement of G94 with residues bearing H-bond donating residues significantly improves the affinity. Replacement of R96 instead blocks the interaction between the peptide and the domain. The strongest affinity is observed with the G94H (peptide PlGF-2) and G94W (peptide PlGF-10) mutants, while the peptide PlGF-8, bearing the R96G mutation, is totally inactive. The PlGF-1 and PlGF-2 peptides also bind the VEGFR-2 receptors, though with a reduced affinity, and are able to interfere with the VEGF-induced receptor signaling on endothelial cells. The peptides also bind VEGFR-2 on the surface of cells, while PlGF-8 is inactive. Data suggest that these peptides have potential applications as PlGF/VEGF mimic in various experimental settings.

Immunodominant West Nile Virus T Cell Epitopes Are Fewer in Number and Fashionably Late.

Class I HLA molecules mark infected cells for immune targeting by presenting pathogen-encoded peptides on the cell surface. Characterization of viral peptides unique to infected cells is important for understanding CD8(+) T cell responses and for the development of T cell-based immunotherapies. Having previously reported a series of West Nile virus (WNV) epitopes that are naturally presented by HLA-A*02:01, in this study we generated TCR mimic (TCRm) mAbs to three of these peptide/HLA complexes-the immunodominant SVG9 (E protein), the subdominant SLF9 (NS4B protein), and the immunorecessive YTM9 (NS3 protein)-and used these TCRm mAbs to stain WNV-infected cell lines and primary APCs. TCRm staining of WNV-infected cells demonstrated that the immunorecessive YTM9 appeared several hours earlier and at 5- to 10-fold greater density than the more immunogenic SLF9 and SVG9 ligands, respectively. Moreover, staining following inhibition of the TAP demonstrated that all three viral ligands were presented in a TAP-dependent manner despite originating from different cellular compartments. To our knowledge, this study represents the first use of TCRm mAbs to define the kinetics and magnitude of HLA presentation for a series of epitopes encoded by one virus, and the results depict a pattern whereby individual epitopes differ considerably in abundance and availability. The observations that immunodominant ligands can be found at lower levels and at later time points after infection suggest that a reevaluation of the factors that combine to shape T cell reactivity may be warranted.

Quantitative Ultrasound for Measuring Obstructive Severity in Children with Hydronephrosis.

PURPOSE: We define sonographic biomarkers for hydronephrotic renal units that can predict the necessity of diuretic nuclear renography. MATERIALS AND METHODS: We selected a cohort of 50 consecutive patients with hydronephrosis of varying severity in whom 2-dimensional sonography and diuretic mercaptoacetyltriglycine renography had been performed. A total of 131 morphological parameters were computed using quantitative image analysis algorithms. Machine learning techniques were then applied to identify ultrasound based safety thresholds that agreed with the t½ for washout. A best fit model was then derived for each threshold level of t½ that would be clinically relevant at 20, 30 and 40 minutes. Receiver operating characteristic curve analysis was performed. Sensitivity, specificity and area under the receiver operating characteristic curve were determined. Improvement obtained by the quantitative imaging method compared to the Society for Fetal Urology grading system and the hydronephrosis index was statistically verified. RESULTS: For the 3 thresholds considered and at 100% sensitivity the specificities of the quantitative imaging method were 94%, 70% and 74%, respectively. Corresponding area under the receiver operating characteristic curve values were 0.98, 0.94 and 0.94, respectively. Improvement obtained by the quantitative imaging method over the Society for Fetal Urology grade and hydronephrosis index was statistically significant (p <0.05 in all cases). CONCLUSIONS: Quantitative imaging analysis of renal sonograms in children with hydronephrosis can identify thresholds of clinically significant washout times with 100% sensitivity to decrease the number of diuretic renograms in up to 62% of children.

Management of Bladder Diverticula in Menkes Syndrome: A Case Report and Review of the Literature.

Menkes syndrome is a genetic disorder of copper metabolism, often with urologic complications, including bladder diverticula and vesicoureteral reflux. A 1-year-old boy with Menkes syndrome presented with recurrent urinary tract infections and incomplete bladder emptying secondary to 2 large bladder diverticula. He underwent robot-assisted excision of both diverticula with subsequent improved emptying and resolution of urinary tract infections. There is no consensus on management of bladder diverticula in Menkes syndrome. Because the life span of these patients is significantly shortened, one must select an intervention based on their clinical condition, potential morbidities, and informed expectations of the family.

Trends in Toilet Training and Voiding Habits among Children with Down Syndrome.

PURPOSE: Children with Down syndrome are at risk for lower urinary tract dysfunction and delayed toilet training. Comparative studies regarding voiding function in the Down syndrome population are lacking. We assessed urinary continence and voiding function in patients with Down syndrome and a control group. MATERIALS AND METHODS: A questionnaire designed to assess toilet training, continence status, symptoms of lower urinary tract dysfunction and prior evaluation of urological complaints was sent to parents of 326 children with Down syndrome who had been seen at our institution previously. The same survey was administered to parents of patients without Down syndrome. Data were compiled, and descriptive and comparative statistical analyses were performed. RESULTS: A total of 77 patients comprised the Down syndrome group and 78 patients without Down syndrome comprised the control group. Average age of reported toilet training completion was 5.5 years in children with Down syndrome and 2.2 years in controls. Of children 5 years or older 79% with Down syndrome were toilet trained, compared to 100% of those without Down syndrome. Incontinence was reported in 46% of previously toilet trained children with Down syndrome and 24.5% of controls. These findings were statistically significant. No significant difference was observed in the rate of urinary tract infection, symptoms of lower urinary tract dysfunction or evaluation for urological complaints. CONCLUSIONS: Children with Down syndrome can experience marked delay in toilet training and are more likely to suffer incontinence afterward. This study was ineffective in determining whether symptoms of lower urinary tract dysfunction could be related to decreased continence rates.

The "flowerpot" sign: inference of poor renal function in high grade vesicoureteral reflux by calyceal orientation.

INTRODUCTION/OBJECTIVE: Modern radiographic advances have allowed for detailed and accurate imaging of not only urologic anatomy but also urologic function. The art of observational inference of subtle anatomic features and function from a static radiograph is being traded for new, more precise, and more expensive modalities. While the superiority of these methods cannot be denied, the total information provided in simpler tests should not be ignored. The relationship between high grade vesicoureteral reflux with the dilated calyces arranged cephalad to a dilated funnel-shaped renal pelvis on VCUG and reduced differential renal function has not been previously described, but has been anecdotally designated a "flowerpot" sign by our clinicians. We hypothesize that the appearance of a "flowerpot" kidney as described herein is an indicator of poor renal function in the setting of high grade VUR. STUDY DESIGN: IRB approval was obtained and 315 patients were identified from system-wide VCUG reports from 2004-2012 with diagnosed "high grade" or "severe" vesicoureteral reflux. Inclusion into the study required grade IV or V VUR on initial VCUG and an initial radionuclide study for determination of differential function. Patients with a solitary kidney, posterior urethral valve, multicystic dysplastic kidney, renal ectopia, or duplex collecting systems were excluded. Grade of reflux, angle of the inferior-superior calyceal axis relative to the lumbar spine, and differential uptake were recorded along with presence of the new "flowerpot" sign. Variables were analyzed using the Mann-Whitney U test to determine statistical significance. RESULTS: Fifty seven patients met inclusion criteria with 11 being designated as "flowerpot" kidneys. These "flowerpot" kidneys could be objectively differentiated from other kidneys with grade IV and/or grade V VUR both by inferior-superior calyceal axis (median angle, 52° [37-66] vs. 13° [2-37], respectively p < 0.001) and by differential renal uptake (median, 23% [5-49] vs. 45% [15-81], respectively p < 0.001). Likewise, there was no difference between either calyceal axis (median angle, 13° [3-20] vs. 13° [2-37]) or differential function (median, 48% [24-81] vs. 40% [15-66], p = 0.129) when comparing kidneys with grade IV and grade V VUR, respectively, that did not demonstrate the "flowerpot" sign. DISCUSSION/CONCLUSION: Grading of VUR is used to provide a common language for scientific discussion and determine prognosis for children with similar attributes. The dysmorphic calyceal system in the "flowerpot" kidneys supports the theory of abnormal renal blastema induction associated with abnormal differentiation of the ureteral bud. Even in the absence of urinary tract infections and/or pyelonephritis, renal abnormalities and decreased differential function can be observed on renal scintigraphy. This study also confirms the male predominance and functional similarities between grade 4 and 5 refluxing renal units. Recognizing this is a limited observational study based on imaging alone, the "flowerpot" sign is an indicator of the most severe form of grade 5 VUR but is only one factor in predicting long term overall renal prognosis. However, 14% (8/57) of our cohort had a relative uptake of less than 20% with 5 of these exhibiting the "flowerpot" sign. The "flowerpot" sign on VCUG can be used as indirect evidence of poor differential renal function and, therefore, useful in guiding parental expectations prior to formal functional imaging.

The prevalence of bell clapper anomaly in the solitary testis in cases of prior perinatal torsion.

PURPOSE: Bell clapper anomaly is associated with an increased risk of intravaginal testicular torsion. However, perinatal torsion is thought to be secondary to an extravaginal process. We quantified the contralateral prevalence of bell clapper anomaly in children found to have atrophic testicular nubbins secondary to presumed torsion during gestation to better define the subsequent risk of metachronous testicular torsion. MATERIALS AND METHODS: Inspection results for the presence of contralateral bell clapper anomaly was recorded by a single surgeon in 50 consecutive cases in which exploration for nonpalpable testes revealed a testicular nubbin. For comparison data were collected in 27 consecutive cases of acute testicular torsion. Anatomy of the normal contralateral testis was compared between the 2 groups. RESULTS: Average age at surgery in the perinatal torsion group was 15 months vs 12.7 years in the acute torsion group. One case of partial contralateral bell clapper anomaly was discovered in the perinatal torsion group but no complete anomaly was found. In contrast, in older boys with acute testicular torsion complete bell clapper anomaly was found in 21 of the 27 contralateral testes (78%). CONCLUSIONS: In older boys with acute testicular torsion contralateral bell clapper anomaly is highly prevalent, supporting the standard practice of contralateral testicular fixation in this clinical situation. However, the prevalence of contralateral bell clapper anomaly is exceedingly small in cases of monorchism after perinatal torsion, substantiating an insufficient risk of subsequent torsion to justify routine fixation of the solitary testis.

Most infants with dilating vesicoureteral reflux can be treated nonoperatively.

PURPOSE: Dilating vesicoureteral reflux provokes concern for physicians and parents that often leads to corrective surgery in young children. Since there are limited data describing the natural history of dilating vesicoureteral reflux in infants, we identified factors predictive of resolution/improvement in infants initially treated nonoperatively. MATERIALS AND METHODS: We reviewed the medical records of 90 infants 6 months old or younger from 2004 to 2010 who were referred for prenatal hydronephrosis or initial febrile urinary tract infection and found to have dilating vesicoureteral reflux (grade 3 or greater). Variables of interest included presentation, dimercapto-succinic acid results, sex, breakthrough febrile urinary tract infections, reflux grade and bilateral reflux. Cox regression analysis was performed to determine predictors of spontaneous resolution and/or improvement to reflux grade less than 3 as well as predictors of surgical intervention. RESULTS: Included in final analysis were 80 infants (113 renal units). Of the patients 51 (64%) experienced spontaneous resolution/improvement with a mean followup of 29 months before resolution, discharge home and/or end of followup. Only 20 patients (25%) underwent surgery. Cox regression analysis revealed that a normal initial dimercapto-succinic acid scan, initial reflux grade less than 5 and absent breakthrough febrile urinary tract infections were predictive of reflux resolution/improvement (p <0.05). Dimercapto-succinic acid scan abnormalities, prenatal hydronephrosis and breakthrough febrile urinary tract infections were significant predictors of surgery (p <0.05). CONCLUSIONS: Dilating vesicoureteral reflux in infancy often resolves/improves spontaneously. Therefore, surgery should be directed toward patients unlikely to experience resolution, ie those with an abnormal initial dimercapto-succinic acid scan, grade 5 vesicoureteral reflux and breakthrough febrile urinary tract infections.

Human ß-defensin HBD3 binds to immobilized Bla g2 from the German cockroach (Blattella germanica).

Human ß-defensin 3 (HBD3) is a small, well-characterized peptide in mucosal secretions with broad antimicrobial activities and diverse innate immune functions. Among these functions is the ability of HBD3 to bind to antigens. In this study, we hypothesize that HBD3 binds to the allergen Bla g2 from the German cockroach (Blattella germanica). The ability of HBD1 (used as a control ß-defensin) and HBD3 to bind to Bla g2 and human serum albumin (HSA, used as a control ligand) was assessed using the SensíQ Pioneer surface plasmon resonance (SPR) spectroscopy biosensor system. HBD1 was observed to bind weakly to Bla g2, while HBD3 demonstrated a stronger affinity for the allergen. HBD3 was assessed under two buffer conditions using 0.15 M and 0.3 M NaCl to control the electrostatic attraction of the peptide to the chip surface. The apparent K(D) of HBD3 binding Bla g2 was 5.9±2.1 µM and for binding HSA was 4.2±0.7 µM, respectively. Thus, HBD3, found in mucosal secretions has the ability to bind to allergens like Bla g2 possibly by electrostatic interaction, and may alter the ability of Bla g2 to induce localized allergic and/or inflammatory mucosal responses.

Stereoscopic augmented reality for laparoscopic surgery.

BACKGROUND: Conventional laparoscopes provide a flat representation of the three-dimensional (3D) operating field and are incapable of visualizing internal structures located beneath visible organ surfaces. Computed tomography (CT) and magnetic resonance (MR) images are difficult to fuse in real time with laparoscopic views due to the deformable nature of soft-tissue organs. Utilizing emerging camera technology, we have developed a real-time stereoscopic augmented-reality (AR) system for laparoscopic surgery by merging live laparoscopic ultrasound (LUS) with stereoscopic video. The system creates two new visual cues: (1) perception of true depth with improved understanding of 3D spatial relationships among anatomical structures, and (2) visualization of critical internal structures along with a more comprehensive visualization of the operating field. METHODS: The stereoscopic AR system has been designed for near-term clinical translation with seamless integration into the existing surgical workflow. It is composed of a stereoscopic vision system, a LUS system, and an optical tracker. Specialized software processes streams of imaging data from the tracked devices and registers those in real time. The resulting two ultrasound-augmented video streams (one for the left and one for the right eye) give a live stereoscopic AR view of the operating field. The team conducted a series of stereoscopic AR interrogations of the liver, gallbladder, biliary tree, and kidneys in two swine. RESULTS: The preclinical studies demonstrated the feasibility of the stereoscopic AR system during in vivo procedures. Major internal structures could be easily identified. The system exhibited unobservable latency with acceptable image-to-video registration accuracy. CONCLUSIONS: We presented the first in vivo use of a complete system with stereoscopic AR visualization capability. This new capability introduces new visual cues and enhances visualization of the surgical anatomy. The system shows promise to improve the precision and expand the capacity of minimally invasive laparoscopic surgeries.

Histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and alters HagB-induced chemokine responses.

Histatins are human salivary gland peptides with anti-microbial and anti-inflammatory activities. In this study, we hypothesized that histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates HagB-induced chemokine responses in human myeloid dendritic cells. Histatin 5 bound to immobilized HagB in a surface plasmon resonance (SPR) spectroscopy-based biosensor system. SPR spectroscopy kinetic and equilibrium analyses, protein microarray studies, and I-TASSER structural modeling studies all demonstrated two histatin 5 binding sites on HagB. One site had a stronger affinity with a KD1 of 1.9 µM and one site had a weaker affinity with a KD2 of 60.0 µM. Binding has biological implications and predictive modeling studies and exposure of dendritic cells both demonstrated that 20.0 µM histatin 5 attenuated (p < 0.05) 0.02 µM HagB-induced CCL3/MIP-1α, CCL4/MIP-1ß, and TNFα responses. Thus histatin 5 is capable of attenuating chemokine responses, which may help control oral inflammation.

Do Not Judge a Kidney by Its Pelvis: A Rare Case of an Unobstructed Dilated Renal Pelvis.

We present a rare case of an unobstructed dilated renal pelvis in a newborn female. Prenatal imaging documented a large abdominal cyst in a stable fetus. Postnatal imaging confirmed persistence of this large cyst but with an unclear etiology. The child was taken to surgery by the pediatric surgeons with concern for a possible harmful nonurologic diagnosis. Intraoperative findings were that of a severely dilated renal pelvis; however, in the absence of an expected ureteropelvic junction obstruction. Reduction pyeloplasty without interference of the ureteropelvic junction proved successful.