Addressing Domain Specificity in the Development of a Cell-Based Binding Assay for the Detection of Neutralizing Antibodies Against a CD47xPD-L1 Bispecific Antibody.

PF-07257876 is a bispecific antibody being developed for the treatment of certain advanced or metastatic solid tumors. To support clinical development of PF-07257876, neutralizing antibody (NAb) assays were developed as part of a tiered immunogenicity testing approach. Because PF-07257876 targets both CD47 and PD-L1, determination of domain specificity of a NAb response may provide additional insight relating to PK, efficacy, and safety. Due to limitations of functional cell systems, two cell-based binding assays were developed using electrochemiluminescence to detect domain-specific NAb. While both NAb assays utilized a cell-based binding approach and shared certain requirements, such as sensitivity and tolerance to potentially interfering substances, the development of each assay faced unique challenges. Among the hurdles encountered, achieving drug tolerance while preserving domain specificity for CD47 proved particularly challenging. Consequently, a sample pretreatment procedure to isolate NAb from potentially interfering substances was necessary. The sample pretreatment procedure developed was based on a bead-extraction and acid dissociation (BEAD) approach. However, the use of the standard BEAD approach with whole drug to capture NAb resulted in loss of NAb detection under certain circumstances. Specifically, mock samples containing a mixture of NAb positive controls against both binding domains of the bispecific antibody produced false-negative results in the cell-based binding assay. An adaptation made to the standard BEAD approach restored domain-specific NAb detection, while also contributing to an assay sensitivity of 1 µg/mL in the presence of a clinically relevant drug tolerance level of up to 400 µg/mL.

Spontaneous Development of Immune-Mediated Necrotizing Myopathy (IMNM) After Chronic Use of Statins: A Case Report.

Statin medications, in addition to lifestyle modifications, have been the regimen of choice for addressing dyslipidemia in the general population. Its widespread use has been justified by increasing evidence that hyperlipidemia is a strong risk factor for the development of atherosclerotic disease resulting in myocardial infarction and other cardiovascular events. Unfortunately, this medication is not tolerated by some patients as it causes uncomfortable side effects such as myalgias, arthralgias, and headaches to name a few.  On rare occasions, some patients may develop immunity against the medication itself resulting in a condition known as immune-mediated necrotizing myopathy (IMNM). In such instances, patients carry antibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) or signal recognition particle (SRP). A small subset of patients may develop IMNM even in the absence of these two antibodies and they are termed seronegative. In this care report, we review the case of a 55-year-old Hispanic male with a history of Hashimoto's thyroiditis and hyperlipidemia who presented to an outpatient rheumatology office for severe proximal muscle weakness after being asymptomatic on rosuvastatin for over 20 years. The patient was stabilized with high-dose steroids and was subsequently given a regimen of mycophenolate and intravenous immunoglobulin (IVIG). He was able to regain approximately 75%-80% of his baseline muscle strength.

Development of a Cell-Based Assay for the Detection of Neutralizing Antibodies to PF-06730512 Using Homogenous Time-Resolved Fluorescence.

The administration of biotherapeutics has the potential to induce potent immune responses. Among these responses, the production of anti-drug antibodies (ADA), including a subset of ADA referred to as neutralizing antibodies (NAb), is of heightened concern. Aside from their capacity to alter the pharmacological profile of a given biotherapeutic, NAb can also pose significant safety risks, especially in instances where an endogenous counterpart to the drug exists. As such, the inclusion of an assay to detect NAb in clinical samples is critical to the effectiveness of a tiered approach to immunogenicity assessment. PF-06730512 is a biotherapeutic protein being developed for the treatment of primary Focal Segmental Glomerulosclerosis (FSGS). To support the immunogenicity assessment of PF-06730512, a cell-based assay was developed for the detection of NAb in FSGS serum samples. Herein, we describe the development of the assay with a focus on the challenges faced, including drug and blood collection tube interferences in NAb detection. The outcome of our efforts was a robust assay capable of detecting 1 µg/mL of a NAb positive control in the presence of clinically relevant drug concentrations up to 30 µg/mL.

Addressing soluble target interference in the development of a functional assay for the detection of neutralizing antibodies against a BCMA-CD3 bispecific antibody.

Proper evaluation of immunogenicity during clinical development of biotherapeutics is a major challenge to bioanalytical scientists, in part due to matrix interference in anti-drug antibody (ADA) and neutralizing antibody (NAB) assays. If not addressed, matrix interference could confound the immunogenicity assessment of a given biotherapeutic in clinical development. To support clinical development of a B cell maturation antigen (BCMA)-CD3 bispecific antibody, a cell-based NAB assay was developed as part of a tiered approach to evaluating the immunogenicity of the drug. The assay endpoint (T cell activation) was chosen based on its strong association with the mechanism of action of the drug. The BCMA-CD3 bispecific antibody activates T cells through simultaneous binding of CD3 on T cells and BCMA on target cells. In this system, T cell activation was assessed through the measurement of luciferase activity in an engineered Jurkat cell line. In the presence of NAB, the degree of T cell activation measured by the amount of luciferase activity can be reduced. During method development, soluble BCMA (sBCMA) interference in the NAB assay was apparent. The binding of sBCMA to the anti-BCMA domain of the bispecific drug led to reduced T cell activation, which caused false positive results in NAB testing. To mitigate this interference, several strategies to eliminate sBCMA were investigated. Among the procedures tested, a bead-based approach proved most effective in depleting sBCMA, while maintaining robust assay performance and achieving fit-for-purpose sensitivity. Using this sample pretreatment procedure, the NAB assay tolerated sBCMA up to 2 µg/mL, or approximately four times the estimated median sBCMA concentration in serum samples from patients with active multiple myeloma.

Interdisciplinary Approach to an Unusual Case of Myocarditis in Pregnancy.

We present a case of myocarditis in a 26-year-old pregnant woman at 29 weeks gestation. Despite optimal medical therapy, she experienced a cardiac arrest 10 days postadmission. An interdisciplinary team facilitated emergency delivery of her baby by perimortem (ie, during maternal cardiac arrest) Caesarean section and initiation of emergency mechanical circulatory support. A cardiac biopsy revealed a mixed eosinophilic and histiocytic infiltrate. After a course of steroid therapy, she experienced full recovery. Both the patient and the infant are alive and well. The case highlights the success of modern interdisciplinary care, as well as ongoing gaps in our knowledge of myocarditis.

Nous présentons un cas de myocardite chez une femme de 26 ans enceinte de 29 semaines, qui a subi un arrêt cardiaque 10 jours après son admission. Une équipe interdisciplinaire a favorisé l'accouchement d'urgence par césarienne perimortem (c.-à-d. durant l'arrêt cardiaque de la mère), et la mise en place en urgence d'une assistance mécanique. Une biopsie cardiaque a révélé un infiltrat mixte d'éosinophiles et d'histiocytes. Il y a eu récupération complète de la fonction ventriculaire après un traitement aux stéroïdes. La patiente et l'enfant sont en vie et se portent bien. Le cas témoigne de la réussite de la pratique moderne des soins interdisciplinaires, et met en lumière les lacunes actuelles de nos connaissances sur la myocardite.

Detecting Underlying Cardiovascular Disease in Young Competitive Athletes.

BACKGROUND: Sudden cardiac death (SCD) is frequently the first manifestation of underlying cardiovascular disease in young competitive athletes (YCAs), yet there are no Canadian guidelines for preparticipation screening in this population. The goal of this study was to determine the prevalence of potentially lethal cardiovascular disease in a sample of Canadian YCAs by comparing 2 screening strategies. METHODS: We prospectively screened 1419 YCAs in British Columbia, Canada (age 12-35 years). We initially screened 714 YCAs using the American Heart Association 12-element recommendations, physical examination, and electrocardiogram (ECG) examination (phase 1). This strategy yielded a high number of false positive results; 705 YCAs were subsequently screened using a novel SportsCardiologyBC (SCBC) questionnaire and ECG examination in the absence of a physical examination (phase 2). RESULTS: Overall, 7 YCAs (0.52%) were found to have clinically significant diagnoses associated with SCD (4 pre-excitation, 1 long QT syndrome, 1 mitral valve prolapse, 1 hypertrophic cardiomyopathy). Six of the 7 athletes (85.7%) with disease possessed an abnormal ECG. Conversely, only 2 had a positive personal or family history (1 athlete had an abnormal ECG and family history). The SCBC questionnaire and protocol (phase 2) was associated with fewer false positive screens; 3.7% (25 of 679) compared with 8.1% (55 of 680) in phase 1 (P = 0.0012). CONCLUSIONS: The prevalence of conditions associated with SCD in a cohort of Canadian YCAs was comparable with American and European populations. The SCBC questionnaire and protocol were associated with fewer false positive screens. The ECG identified most of the positive cases irrespective of screening strategy used.

Implementation of a quality improvement initiative to reduce daily chest radiographs in the intensive care unit.

OBJECTIVE: To reduce the number of routine chest radiographs (CXRs) done in a tertiary care intensive care unit (ICU). METHODS: Using a quality improvement approach, we measured the number of CXRs done per patient-day before (15 June 2010-15 June 2011) and after (15 June 2011-15 June 2012) a multipronged intervention in a 15-bed medical-surgical ICU in a 350-bed tertiary care teaching hospital. We studied a total of 1492 patients who were admitted to this ICU-738 patients during the preintervention period and 754 patients during the postintervention period. Interventions were education for the ICU house staff, developing indications for routine CXRs on the computer order-entry system, and visual posters/signage to remind ICU staff that there were no indications for routine, daily CXRs. The primary outcome was the number of CXRs per patient-day, but we also measured CTs of the chest, mechanical ventilator days, length of ICU stay and ICU and hospital mortality. RESULTS: There were 0.73 CXRs per patient-day done during the preintervention period and 0.54 CXRs per patient-day done during the postintervention period, a 26% reduction. There were no differences between the periods in age, sex or severity of illness (Acute Physiology and Chronic Health Evaluation (APACHE) II score) of the patients, number of chest CTs, mechanical ventilator days, length of ICU stay and ICU or hospital mortality. CONCLUSIONS: A quality improvement that includes education, reminders of appropriate indications and computerised decision support can decrease the number of routine CXRs in an ICU.

Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself.

BACKGROUND: Toll-like receptor 4 (TLR4) is activated by bacterial endotoxin, a prototypical pathogen-associated molecular pattern (PAMP). It has been suggested that TLR4 can also be activated by damage-associated molecular pattern (DAMP) proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification. RESULTS: Here we report that the activation of TLR4 on primary human macrophage cultures by recombinant HSP70 is not solely due to contaminating endotoxin. Polymyxin B pretreatment of HSP70 preparations to neutralize contaminating endotoxin caused significant reductions in the amount of TNF-α induced by the recombinant protein as determined by ELISA. However, digestion of HSP70 with Proteinase K-agarose beads also dramatically reduced the TNF-α response of macrophages to HSP70, while leaving levels of contaminating endotoxin largely unchanged relative to controls. CONCLUSIONS: These results indicate that the stimulatory effect of recombinant HSP70 requires both the presence of endotoxin and structural integrity of the heat shock protein itself.

Identification and SAR of a new series of thieno[3,2-d]pyrimidines as Tpl2 kinase inhibitors.

We report here the synthesis and SAR of a new series of thieno[3,2-d]pyrimidines as potent Tpl2 kinase inhibitors. The proposed binding mode suggests the potential flipped binding mode depending on the substitution. Biacore studies show evidence of binding of these molecules to the protein kinase. The kinome inhibition profile of these molecules suggests good selectivity.

Discovery of indazoles as inhibitors of Tpl2 kinase.

Synthesis, modeling and structure-activity relationship of indazoles as inhibitors of Tpl2 kinase are described. From a high throughput screening effort, we identified an indazole hit compound 5 that has a single digit micromolar Tpl2 activity. Through SAR modifications at the C3 and C5 positions of the indazole, we discovered compound 31 with good potency in LANCE assay and cell-based p-Erk assay.

In vivo characterization of rabbit anti-mouse thymocyte globulin: a surrogate for rabbit anti-human thymocyte globulin.

BACKGROUND: Polyclonal rabbit anti-human thymocyte globulin (Thymoglobulin) is used clinically for immunosuppression in solid organ transplantation; however, it is difficult to fully characterize the effects of this agent in humans. METHODS: A surrogate rabbit anti-murine thymocyte globulin (mATG) was generated analogously to the commercial product Thymoglobulin and in vivo activities were evaluated, including pharmacokinetics, T-cell depletion, dose response and kinetics, depletion/sparing of T-cell subsets or other leukocyte populations, and depletion in different lymphoid organs. RESULTS: Within 1 day, T cells are depleted by mATG in the blood, spleen, lymph node, and bone marrow down to doses of 1 mg/kg. Although mATG binds and depletes thymocytes in vitro, there is no thymocyte depletion in vivo at any dose level, suggesting decreased antibody accessibility to the thymus. After two doses of mATG given 3 days apart, T-cell reconstitution begins as early as day 9 and returns to basal levels by day 21 and 29 for CD4 and CD8 T cells, respectively. There is also preferential depletion of naïve T cells that results in increased ratios of regulatory and memory T cells within 1 day after mATG administration. Depletion of natural killer-T cells, natural killer cells, plasma cells, and plasmablasts occurs, but is modest and more transient compared with T cells. B cells, macrophages, dendritic cells, hematopoetic stem cells, and bone marrow stromal cells seem resistant to mATG depletion. CONCLUSIONS: These studies characterize the depletive effects of mATG in normal mice and provide insight into mechanisms of action of Thymoglobulin.

P38 MAP kinase in valve interstitial cells is activated by angiotensin II or nitric oxide/peroxynitrite, but reduced by Toll-like receptor-2 stimulation.

BACKGROUND AND AIM OF THE STUDY: The involvement of p38 MAPK in mediating factors that may produce aortic valve disease is unknown. Angiotensin II (Ang II) has been implicated in the development of aortic stenosis through either the generation of free radicals and/or the modulation of inflammatory responses. A variety of proinflammatory factors utilize Toll-like receptors, and these may also play a role in the development of aortic valve disease. METHODS: Valve interstitial cells (VICs) were cultured from porcine aortic valves. Cells were treated with Ang II, 3-morpholinosydnonimine (SIN-1), which liberates NO and superoxide anion generating peroxynitrite, or the lipopetide Toll-like receptor-2 (TLR-2) agonist Pam3CSK4. RESULTS: In response to Ang II (1 microM), MAPK phosphorylation levels were increased by 3.5-fold after 15 min, peaked at 4.6-fold after 60 min, and decreased to 1.9-fold greater than control after 120 min of treatment. In response to SIN-1, phosphorylation levels were increased progressively throughout the 90 min of treatment and were significantly (p < 0.05) twofold (1.9 +/- 0.3) greater than control or native p38 MAPK (2.3 +/- 0.4) after 90 min. SB202190, a relatively selective inhibitor of the p38a MAPK isoform, reduced SIN-1-induced p38 MAPK phosphorylation. In contrast, there was a rapid and marked decline in phosphorylated p38 MAPK, in response to Pam3CSK4 that was evident at 30 min; after 90 min, the p38 MAPK level was 85% lower than baseline. CONCLUSION: p38 MAPK is present in VICs, and is activated by Ang II. Peroxynitrite similarly increased p38 MAPK phosphorylation, which suggests that these two factors involve similar pathways in their effect on VICs. Alternatively, peroxynitrite may be involved in the pathway by which Ang II activates p38 MAPK. The dramatic reduction in p38 MAPK phosphorylation by TLR-2 stimulation excludes a role for this receptor type in mediating Ang II or peroxynitrite effects, and suggests that inflammatory factors that act through TLR-2 to dephosphorylate p38 MAPK utilize pathways different from Ang II or peroxynitrite, to produce their effect on the aortic valve.

Effects of fungal N- and O-linked mannosylation on the immunogenicity of model vaccines.

Targeting dendritic cell mannose receptors by mannosylating antigens represents a promising vaccination strategy. Using the model antigen ovalbumin (OVA) expressed recombinantly in bacterial and yeast vectors, we have previously demonstrated fungal mannosylation enhances antigen immunogenicity in the context of CD4(+) T cell responses. However, because protection against many tumors and pathogens is thought to require MHC class I-restricted T cell responses, the capacity of differentially mannosylated OVA antigens to induce antigen-specific CD8(+) T cell proliferation was determined. We found that mannosylated yeast-derived OVA antigens were more potent than their unmannosylated counterparts at inducing antigen-specific T cell proliferation. However, the type of mannosylation was critical as addition of extensive O-linked mannosylation increased lymphoproliferative responses while the presence of N-linked mannosylation was associated with decreased responses. Mannosylated OVA failed to stimulate TNF-alpha and IL-12 production from dendritic cells. These data suggest that vaccines incorporating mannosylation must take into account how the mannose groups are linked to the core antigen and may need to include an adjuvant to stimulate cytokine production.

Tumor suppressor pRB functions as a co-repressor of the CCAAT displacement protein (CDP/cut) to regulate cell cycle controlled histone H4 transcription.

The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key proliferation-related function as the DNA binding subunit of the cell cycle controlled HiNF-D complex. HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell-cycle dependent histone genes, which are transcriptionally and coordinately activated at the G(1)/S phase transition independent of E2F. The tumor suppressor pRB/p105 is an intrinsic component of the HiNF-D complex. However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined. Using transient transfections, we show that CDP represses the H4 gene promoter and that pRB functions with CDP as a co-repressor. Direct physical interaction between CDP and pRB was observed in glutathione-S-transferase (GST) pull-down assays. Furthermore, interactions between these proteins were established by yeast and mammalian two-hybrid experiments and co-immunoprecipitation assays. Confocal microscopy shows that subsets of each protein are co-localized in situ. Using a series of pRB mutants, we find that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB. Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters.

Gene conversion-like sequence transfers between transgenic antibody V genes are independent of RAD54.

Homology-based Ig gene conversion is a major mechanism for Ab diversification in chickens and the Rad54 DNA repair protein plays an important role in this process. In mice, although gene conversion appears to be rare among endogenous Ig genes, Ab H chain transgenes undergo isotype switching and gene conversion-like sequence transfer processes that also appear to involve homologous recombination or gene conversion. Furthermore, homology-based DNA repair has been suggested to be important for somatic mutation of endogenous mouse Ig genes. To assess the role of Rad54 in these mouse B cell processes, we have analyzed H chain transgene isotype switching, sequence transfer, and somatic hypermutation in mice that lack RAD54. We find that Rad54 is not required for either transgene switching or transgene hypermutation. Furthermore, even transgene sequence transfers that are known to require homology-based recombinations are Rad54 independent. These results indicate that mouse B cells must use factors for promoting homologous recombination that are distinct from the Rad54 proteins important in homology-based chicken Ab gene recombinations. Our findings also suggest that mouse H chain transgene sequence transfers might be more closely related to an error-prone homology-based somatic hypermutational mechanism than to the hyperconversion mechanism that operates in chicken B cells.