B-cell-directed CAR T-cell therapy activates CD8+ cytotoxic CARneg bystander T cells in patients and nonhuman primates.

ABSTRACT: Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell-derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell-targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor-independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell-targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

Implementation and evaluation of a warfarin patient self-management (PSM) program among experienced patients in a U.S. academic health system.

Warfarin patient self-management (PSM) is when a patient independently manages their warfarin therapy using a decision-support tool provided by their anticoagulation provider. Clinical trials of PSM, conducted predominantly in Europe, have consistently demonstrated superior efficacy without compromising safety. However, the evidence-based practice of PSM is rarely utilized in the United States (U.S.). We describe initiatives completed to implement a successful PSM program among experienced warfarin-taking patients in a U.S. academic health system by overcoming perceived barriers. The results showed PSM resulted in similar or improved INR control, and an estimated 68% reduction in pharmacist workload.

Wilderness Rescue of a Hiker with Multiple Trapped Limbs by a Combined Wilderness and Urban Rescue Team Using High-Pressure Airbags.

We report on the case of a 28-y-old man with both legs and left arm trapped for nearly 6 h after falling and subsequently being trapped by a boulder during a hike in the wilderness. Extrication required equipment designed for urban environments and was operated by an unconventional team of rescue professionals. The patient experienced multiple right lower-extremity orthopedic injuries, acute kidney injury secondary to rhabdomyolysis, and bilateral segmental pulmonary emboli. In this article, we detail the extrication and review the treatment guidelines for crush injuries that focus on aggressive fluid resuscitation prior to and during extrication and medication administration only if hyperkalemia presents. Wilderness rescuers should plan for the use of unconventional rescue equipment in austere prolonged rescue scenarios.

A risk-stratified approach to venous thromboembolism prophylaxis with aspirin or warfarin following total hip and knee arthroplasty: A cohort study.

INTRODUCTION: Venous thromboembolism (VTE) and bleeding events following total knee and hip arthroplasty (TKA/THA) are associated with significant morbidity. Clinical guidelines recommend administration of pharmacologic VTE prophylaxis post-operatively, although controversy exists regarding optimal prophylactic strategies. METHODS: We performed a retrospective cohort study in patients who underwent elective TKA/TKA in an academic medical center. Patients were stratified by surgery type (TKA/THA) and VTE risk determined by a novel risk stratification protocol and compared pre- and post-protocol implementation. Patients received warfarin pre-protocol and either aspirin or warfarin post-protocol for VTE prophylaxis. Natural language processing identified VTE events and ICD codes were used to identify bleeding events, with all events validated manually. RESULTS: A total of 1379 surgeries were included for analysis, 839 TKAs and 540 THAs. Post-protocol implementation, 445 (94.1%) patients following TKA and 294 (97.4%) patients following THA received aspirin for VTE prophylaxis. A significant reduction in bleeding events (hazard ratio [HR] = 0.19, p = 0.048) was observed in low-risk THA patients treated with aspirin (post-protocol) compared patients treated with warfarin (pre-protocol). Bleeding events did not differ significantly between low-risk TKA patients treated with aspirin or warfarin. No significant differences in VTE events were observed following the protocol implementation. CONCLUSIONS: The use of a novel risk stratification system to guide VTE prophylaxis selection between aspirin or warfarin following TKA and THA appears safe and effective. Among low-risk patients, aspirin use was associated with fewer bleeding events following THA, without an observed increase in VTE events.

Resistance to Pyrrolobenzodiazepine Dimers Is Associated with SLFN11 Downregulation and Can Be Reversed through Inhibition of ATR.

Resistance to antibody-drug conjugates (ADCs) has been observed in both preclinical models and clinical studies. However, mechanisms of resistance to pyrrolobenzodiazepine (PBD)-conjugated ADCs have not been well characterized and thus, this study was designed to investigate development of resistance to PBD dimer warheads and PBD-conjugated ADCs. We established a PBD-resistant cell line, 361-PBDr, by treating human breast cancer MDA-MB-361 cells with gradually increasing concentrations of SG3199, the PBD dimer released from the PBD drug-linker tesirine. 361-PBDr cells were over 20-fold less sensitive to SG3199 compared with parental cells and were cross-resistant to other PBD warhead and ADCs conjugated with PBDs. Proteomic profiling revealed that downregulation of Schlafen family member 11 (SLFN11), a putative DNA/RNA helicase, sensitizing cancer cells to DNA-damaging agents, was associated with PBD resistance. Confirmatory studies demonstrated that siRNA knockdown of SLFN11 in multiple tumor cell lines conferred reduced sensitivity to SG3199 and PBD-conjugated ADCs. Treatment with EPZ011989, an EZH2 inhibitor, derepressed SLFN11 expression in 361-PBDr and other SLFN11-deficient tumor cells, and increased sensitivity to PBD and PBD-conjugated ADCs, indicating that the suppression of SLFN11 expression is associated with histone methylation as reported. Moreover, we demonstrated that combining an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor, AZD6738, with SG3199 or PBD-based ADCs led to synergistic cytotoxicity in either resistant 361-PBDr cells or cells that SLFN11 was knocked down via siRNA. Collectively, these data provide insights into potential development of resistance to PBDs and PBD-conjugated ADCs, and more importantly, inform strategy development to overcome such resistance.

Identification and Tracking of Alloreactive T Cell Clones in Rhesus Macaques Through the RM-scTCR-Seq Platform.

T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell's transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, 'RM-scTCR-Seq', which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR's with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.

Preclinical Characterization of an Antibody-Drug Conjugate Targeting CS-1 and the Identification of Uncharacterized Populations of CS-1-Positive Cells.

Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody-drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti-CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid-erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.

ADC Analysis by Hydrophobic Interaction Chromatography.

Hydrophobic interaction chromatography (HIC) is a traditional technique used for the separation, purification, and characterization of proteins. As the number of antibody-drug conjugates (ADCs) continues to increase in clinical trials, HIC and other orthogonal methods utilizing changes in hydrophobicity are being used for ADC characterization and analysis. Unlike other techniques, HIC uniquely allows for protein analysis under mild nondenaturing conditions that preserve the native structure and activity of the molecules. Analysis of the ADC in its native form is advantageous. Herein, we describe a generic HIC protocol for the screening, analysis, and characterization of ADCs using an ammonium sulfate buffer and a high-pressure liquid chromatography system. Parameters affecting data quality and interpretation are addressed. In addition, several recommendations are included for method optimization and troubleshooting.

Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media.

Bispecific antibody (bsAb) applications have exponentially expanded with the advent of molecular engineering strategies that have addressed many of the initial challenges, including improper light chain pairing, heterodimer purity, aggregation, and pharmacokinetics. However, the lack of high-throughput methods for the generation of monovalent bsAbs has resulted in a bottleneck that has hampered their therapeutic evaluation, as current technologies can be cost-prohibitive and impractical. To address this issue, we incorporated single-matched point mutations in the CH3 domain to recapitulate the physiological process of human IgG4 Fab-arm exchange to generate monovalent bsAbs. Furthermore, we utilized the substitutions H435R and Y436F in the CH3 domain of IgG1, which incorporates residues from human IgG3, thus ablating protein A binding. By exploiting this combination of mutations and optimizing the reduction and reoxidation conditions for Fab arm exchange, highly pure monovalent bsAbs can be rapidly purified directly from combined culture media using standard protein A purification. This methodology, reported herein for the first time, allows for the high-throughput generation of monovalent bsAbs, thus increasing the capacity for evaluating monovalent bsAb iterations for therapeutic potential.

Characterization of Disulfide Bond Rebridged Fab-Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload.

We describe the characterization of antigen binding fragments (Fab)-drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolobenzodiazepine dimers, which are DNA cross-linkers, are a class of payloads used in antibody-drug conjugates (ADCs). SG3710 was designed to rebridge two adjacent cysteines, such as those that form the canonical interchain disulfide bond between the light and heavy chain in Fab fragments. The rebridging generated homogenous Fab conjugates, with a drug-to-Fab ratio of one, as demonstrated by the preparation of rebridged Fabs derived from the anti-HER2 trastuzumab antibody and from a negative control antibody both prepared using recombinant expression and papain digestion. The resulting anti-HER2 trastuzumab Fab-rebridged conjugate retained antigen binding, was stable in rat serum, and demonstrated potent and antigen-dependent cancer cell-killing ability. Disulfide rebridging with SG3710 is a generic approach to prepare Fab-pyrrolobenzodiazepine dimer conjugates, which does not require the Fabs to be engineered for conjugation. Thus, SG3710 offers a flexible and straightforward platform for the controlled assembly of pyrrolobenzodiazepine dimer conjugates from any Fab for oncology applications.

Design and characterization of homogenous antibody-drug conjugates with a drug-to-antibody ratio of one prepared using an engineered antibody and a dual-maleimide pyrrolobenzodiazepine dimer.

Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.

Antitumor Activity of MEDI3726 (ADCT-401), a Pyrrolobenzodiazepine Antibody-Drug Conjugate Targeting PSMA, in Preclinical Models of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody-drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient-derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176-86. ©2018 AACR.

Prevalence and correlates of bleeding and emotional harms in a national US sample of patients with venous thromboembolism: A cross-sectional structural equation model.

INTRODUCTION: Venous thromboembolism (VTE) including deep vein thrombosis (DVT) or pulmonary embolism (PE) is associated with reduced survival, poorer quality of life, and substantial health-care-costs. Limited research, primarily qualitative, suggests that those with VTE may have elevated fear of recurrence, and associated emotional dysfunction and distress. METHODS: A national online survey was administered to 907 patients who had experienced a VTE event in the past two years. The survey assessed for the prevalence of self-reported bleeding harms associated with VTE, the levels of anxiety, depression, cognitive dysfunction and distress experienced by patients, and a range of potential psychosocial correlates that may be associated with these bleeding or emotional harms. RESULTS: The majority (63.0%) of respondents had experienced at least one bleeding related harm following their VTE diagnosis, and 40.6% indicated they experienced fear of another clot often or almost all the time. One-in-four (24.7%) and one-in-ten (11.6%) had abnormal levels of anxiety and depression, respectively. Structural equation modeling was used to define two composite latent bleeding harm and emotional harm factors. Emotional and bleeding harms were associated with younger age, a belief that one's health is due to luck, having multiple comorbidities, having a history of prior VTE events, having multiple barriers to VTE care, and experiencing medical mistakes in diagnosis or treatment. Emotional harms were uniquely predicted by having poorer health literacy, having low self-reported medication adherence, belief others are responsible for one's health, and more recent VTE events. Bleeding harms were uniquely predicted by having a lower frequency of primary care provider contact and having a history of switching between warfarin and direct oral anticoagulants for VTE treatment. CONCLUSIONS: The findings show high levels of self-reported bleeding and emotional harms in a general population of VTE sufferers that are clearly associated with readily identifiable demographic, health status, and psychosocial characteristics. These represent targets for intervention and changes in clinical practice.

Patterns and Appropriateness of Thrombophilia Testing in an Academic Medical Center.

BACKGROUND: Clinical guidelines recommend against routine use of thrombophilia testing in patients with acute thromboembolism. Thrombophilia testing rarely changes acute management of a thrombotic event. OBJECTIVE: To determine appropriateness of thrombophilia testing in a teaching hospital. DESIGN: Retrospective cohort study. SETTING: One academic medical center in Utah. PARTICIPANTS: All patients who received thrombophilia testing between July 1, 2014, and December 31, 2014. MAIN MEASUREMENTS: Proportion of thrombophilia tests occurring in situations associated with minimal clinical utility, defined as tests meeting at least 1 of the following criteria: discharged before results available; test type not recommended; testing in situations associated with decreased accuracy; duplicate testing; and testing following a provoked thrombotic event. RESULTS: Overall, 163 patients received a total of 1451 thrombophilia tests for stroke (50% of tests; 35% of patients), venous thromboembolism (21% of tests; 21% of patients), and pregnancy-related conditions (15% of tests; 25% of patients). Of the 39 different test types performed, the most common were cardiolipin IgG and IgM antibodies (9% each), lupus anticoagulant (9%), and ß2-glycoprotein 1 IgG and IgM antibodies (8% each). In total, 911 tests (63%) were performed in situations associated with minimal clinical utility, with 126 patients (77%) receiving at least one such test. Only 2 patients (1%) had clear documentation of being offered genetic consultation. CONCLUSIONS: Thrombophilia testing in this single-center study was often associated with minimal clinical utility. Strategies to improve testing practices (eg, hematology specialty consult prior to inpatient testing, improved order panels) might help minimize inappropriate testing and promote value-driven care.

Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody-Drug Conjugate Targeting 5T4.

Antibody-drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576-87. ©2017 AACR.

Comparison of two point-of-care international normalized ratio devices and laboratory method.

: A number of factors contribute to interlaboratory variation of international normalized ratio (INR) results. This variability can lead to differences in clinical decision-making. This prospective cohort study evaluated the accuracy, precision, and clinical decision-making variability of two point-of-care (POC) devices (CoaguChek XS and the Coag-Sense) to a clinical laboratory method of INR measurement as the 'reference' and by using the laboratory's accuracy standard (±25% of the reference INR). Study subjects included adults taking warfarin undergoing a POC INR with each device and a laboratory INR on the same day and evaluated differences in clinical decision-making using a warfarin nomogram. A correction factor was derived for each POC method to estimate the corresponding laboratory INR for POC INRs more than 3.0. A total of 100 patient encounters (76 unique patients) included INR testing by all three methods. Both POC devices demonstrated excellent precision across the entire range (r = 0.98), but poor accuracy relative to the laboratory standard for POC INRs greater than 3.0 (accuracy CoaguChek 19% and Coag-Sense 35%). A correction factor applied to POC INRs greater than 3.0 improved accuracy to 99% for both devices. Applying the correction factor also significantly reduced differences in clinical decision-making (49-28% for CoaguChek and 56-40% for Coag-Sense, P < 0.001). Both POC devices demonstrated poor accuracy for INRs over 3.0. A device-specific, institution-specific correction factor applied to POC INRs greater than 3.0 can provide better accuracy of INR measurement and can significantly reduce variability in warfarin dosing decisions. More study is needed to assess impact on clinical adverse events.