Administration Of mAb Research Articles

Passive Immunization with Anti-Pertussis Toxin Humanized Monoclonal Antibody Mitigates Clinical Signs of Pertussis Infection in Newborn Baboons

BackgroundPertussis is a significant mortality risk in the developing world, killing up to 200,000 infants annually. Maternal vaccination as a strategy to protect newborns has shown some success, but is unlikely to capture all eligible mothers. Hu1B7 is a monoclonal antibody (mAb) that potently neutralizes pertussis toxin. Hu1B7, when administered as part of a binary mAb cocktail, demonstrated therapeutic efficacy in pertussis-infected weanling baboons. Here, the prophylactic potential of hu1B7 to protect infants during their first few months, when mortality risk is highest, was evaluated.MethodsNeonatal baboons of normal gestational age (180 days ± 10), normal birth weight (~1.0kg), and anti-Fha titer < 5 IU/ml (verifying no prior exposure to Bordetella species) were recruited into the study. At 2 days of age, treated baboons received hu1B7 (40mg/kg, IV), while control animals were untreated. Serum levels of hu1B7 were followed for 5 weeks, at which time the animals were infected with 108 cfu of B. pertussis strain D420. Animals were monitored for clinical signs of disease.ResultsSix controls and seven treated animals were evaluated. All animals were heavily colonized with B. pertussis during the first week after infection. Controls developed significant leukocytosis, most coughed, and three required euthanasia. In contrast, white blood cell counts for all treated animals remained within the normal range, coughing was virtually absent, and all animals maintained normal activity. As expected for a humanized mAb in a nonhuman primate, hu1B7 had an elimination half-life of 11.8±3.4 days.ConclusionProtection of newborn baboons from pertussis was achieved by mAb administration 5 weeks prior to pertussis infection. Hu1B7, when systemically present, mitigated the clinical signs of pertussis, including leukocytosis and coughing, but did not prevent bacterial colonization. Assuming a half-life in humans of 3 weeks, mAb administration at birth could potentially provide 4 months of prophylaxis and is a viable strategy to complement maternal vaccination. Moreover, strategies that extend the mAb half-life and lower the dose could further support developing world application.Disclosures J. Maynard, Synthetic Biologics, Inc.: Collaborator, Research support; A. Nguyen, Synthetic Biologics, Inc.: Collaborator, Research support; R. Wolf, Synthetic Biologics, Inc.: Collaborator, Research support;; J. Papin, Synthetic Biologics, Inc.: Collaborator, Research support; S. Connelly, Synthetic Biologics, Inc.: Employee, Salary; M. Kaleko, Synthetic Biologics, Inc.: Employee and Shareholder, Salary and stock options

Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.

Protective capacity of neutralizing and non-neutralizing antibodies against glycoprotein B of cytomegalovirus.

Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in immunodeficient hosts. When present before infection, both neutralizing and non-neutralizing anti-gB exhibited protective capacity.

Anti-CD25 monoclonal antibody enhances the protective efficacy of Schistosoma japonicum GST vaccine via inhibition of CD4+CD25+Foxp3+ regulatory T cells.

The effect of anti-CD25 monoclonal antibody (anti-CD25 mAb) on the protection efficacy of Schistosoma japonicum 26kDa GST (glutathione-S-transferase) vaccine was evaluated. Mice were immunized with GST before infection with S. japonicum cercariae and then injected with anti-CD25 mAb. The worm reduction rate was promoted from 24.18% in mice with GST immunization to 47.09% in mice with GST plus anti-CD25 mAb. Compared with the control group, the percentages of splenic CD4+CD25+Foxp3+ regulatory T cells (Tregs) were significantly lower after administration of anti-CD25 mAb; meanwhile, elevated levels of IFN-γ and IL-2 were secreted by splenocytes. These results indicate that the poor protective efficacy of the GST vaccine against S. japonicum results from the presence of CD4+CD25+Foxp3+ Tregs, while anti-CD25 mAb can partially block CD4+CD25+Foxp3+ Tregs and thus enhance the protective efficacy of the GST vaccine.

Innate IFN-γ-Producing Cells Developing in the Absence of IL-2 Receptor Common γ-Chain.

IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2-/-γc-/-), indicating their innate nature and their γc cytokine independence. Rag2-/-γc-/- mice are as resistant to Mycobacterium avium as Rag2-/- mice, whereas Rag2-/- mice lacking IFN-γ are more susceptible than either Rag2-/- or Rag2-/-γc-/- These lineage-negative CD45+/Thy1.2+ cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2-/-γc-/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2-/-γc-/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2+ non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice.

Systemic and topical use of monoclonal antibodies to prevent the sexual transmission of HIV.

: Passive immunization, the transfer of antibodies to a nonimmune individual to provide immunological protection, has been used for over 100 years to prevent and treat human infectious diseases. The introduction of techniques to produce human mAbs has revolutionized the field, and a large number of human mAbs have been licensed for the treatment of cancer, autoimmune and inflammatory diseases. With the recent discovery and production of highly potent broadly neutralizing and other multifunctional antibodies to HIV, mAbs are now being considered for HIV therapy and prophylaxis. In this review, we briefly present recent advances in the anti-HIV mAb field and outline strategies for the selection, engineering and production of human mAbs, including the modification of their structure for optimized stability and function. We also describe results from nonhuman primate studies and phase 1 clinical trials that have tested the safety, tolerability, pharmacokinetics, and efficacy of mAb-based HIV prevention strategies, and discuss the future of parenteral and topical mAb administration for the prevention of HIV transmission.

Abstract 3663: An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice

Abstract Administration of an anti-CD4 depleting antibody induces tumor-specific CD8+ T cell responses in tumor-bearing mice, although the mechanisms underlying this phenomenon remain unclear. CD4+ Foxp3+ regulatory T cells (Treg) impair antigen presentation by DCs through CTLA4-mediated down-regulation of CD80/CD86. In the present study, we investigated the effects of an anti-CD4 depleting monoclonal antibody (mAb) on DC responses in the B16F10 and LLC subcutaneous tumor models, and compared these effects with those of Treg targeting therapy. Administration of anti-CD4 mAb up-regulated CD80/CD86 in tumor-infiltrating DCs, and in migrating-type but not resident-type DC subsets in the tumor-draining lymph node. The upregulation of CD80/CD86 on these DC populations was correlated with increased proliferation of tumor-specific CD8+ T cells in the tumor-draining lymph node. Anti-CD4 mAb-induced CD80/CD86 up-regulation and tumor-specific CD8+ T cell responses were remarkably higher than that induced by an anti-CD25 depleting mAb or an anti-CTLA4 blocking mAb, and were equivalent to that induced by diphtheria-toxin-induced selective depletion of Tregs in Foxp3-DTR mice. Importantly, the activation of CD4+ conventional T cells and B cells in the peripheral lymph nodes that was observed following Treg depletion was not observed following anti-CD4 mAb administration. In conclusion, anti-CD4 depleting antibody therapy is an effective way to evoke anti-tumor CD8+ T cell responses by reversing regulatory T cell-induced suppression of dendritic cells, and to reduce the risk of severe autoimmunity in cancer patients. Citation Format: Satoshi Ueha, Haru Ogiwara, Shoji Yokochi, Yoshiro Ishiwata, Francis Shand, Shohei Hori, Kazuhiro Kakimi, Satoru Ito, Kouji Matsushima. An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3663. doi:10.1158/1538-7445.AM2017-3663

Abstract LB-188: Selective targeting of SIRP alpha induces potent memory antitumor immune responses without presenting hematological toxicity

Abstract Targeting immune checkpoints of the adaptive immunity has shown great therapeutic efficacy to fight cancers, but in a limited proportion of patients. Myeloid cells represent the most abundant immune cell type in many solid tumors, and are often associated with a poor outcome. Interaction of SIRPalpha (SIRPα), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response, involved in the regulation of macrophages, dendritic cells and neutrophils function (e.g. phagocytosis). Thus, combining immunotherapies targeting both adaptive and innate immune cells represents a promising therapeutic strategy against cancer. Since agents targeting CD47 (anti-CD47 or SIRPa-Fc) recently experienced hematological toxicity (anemia or thrombocytopenia), we evaluated the preclinical safety and efficacy of an antagonistic anti-SIRPa monoclonal antibody (mAb). Here, we found that administration of anti-SIRPa mAb at high dose (10 mg/kg) in healthy or tumor-bearing mice did not modify red blood cells and platelets numbers, hematocrit and hemoglobin levels, while in parallel anti-CD47 mAbs rapidly induced anemia within three days, owing to the high expression of CD47 on red blood cells. Next, we evaluated the therapeutic potential of antagonist anti-SIRPa mAbs in combination with adaptive immune checkpoint inhibitors (anti-PD-L1 mAb) or an adaptive immune checkpoint agonist (anti-4-1BB mAb) using an orthotopic hepatocellular carcinoma (HCC) preclinical model in immunocompetent mice. Monotherapy with antagonist anti-SIRPa mAbs prolonged survival in 25% of mice but did not induce remission (p&amp;lt;0.01). While PD-1 blockade alone was also poorly effective in this model (MST: 22 days, n=8), combination with SIRPα blockade induced durable remission in 60% of mice (p&amp;lt;0.05). Association of SIRPα blockade with 4-1BB agonist was highly efficient with 80% of long-term remission (p&amp;lt;0.01) while monotherapy with 4-1BB mAb led to tumor elimination in only 26% of mice (ns). Mice presenting a remission after selective SIRPα blockade in combination with PD-L1 or 4-1BB agents acquired a robust memory immune responses mediated, at least, by T lymphocytes since a second tumor challenge (performed up to one month after treatment withdrawal) was always rejected (p&amp;lt;0.01). In addition, adoptive transfer of T lymphocytes from cured mice in naïve untreated mice protected them from orthotopic HCC development. In conclusion, we showed that targeting selectively SIRPa is devoid of hematological toxicity and that combination of immunotherapies targeting both adaptive and innate immune cells is promising to generate robust antitumor memory immunity. Citation Format: Vanessa Gauttier, Sabrina Pengam, Sophie Conchon, Bernard Vanhove, Nicolas Poirier. Selective targeting of SIRP alpha induces potent memory antitumor immune responses without presenting hematological toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-188. doi:10.1158/1538-7445.AM2017-LB-188

Targeting the Human T-Cell Inducible COStimulator Molecule with a Monoclonal Antibody Prevents Graft-vs-Host Disease and Preserves Graft vs Leukemia in a Xenograft Murine Model.

Graft-vs-host disease (GVHD) is a major complication of allogenic bone marrow transplantation (BMT). Targeting costimulatory molecules with antagonist antibodies could dampen the excessive immune response that occurs, while preserving the beneficial graft vs leukemia (GVL) of the allogeneic response. Previous studies using a mouse model of GVHD have shown that targeting the T-cell Inducible COStimulator (ICOS, CD278) molecule is beneficial, but it is unclear whether the same applies to human cells. Here, we assessed whether a monoclonal antibody (mAb) to human ICOS was able to antagonize the costimulatory signal delivered in vivo to human T cells. To test this hypothesis, we used a xenogeneic model of GVHD where human peripheral blood mononuclear cells were adoptively transferred in immunocompromised NOD.SCID.gc-null mice (NSG). In this model, control mice invariably lost weight and died by day 50. In contrast, 65% of the mice receiving a single injection of the anti-hICOS mAb survived beyond 100 days. Moreover, a significant improvement in survival was obtained in a curative xeno-GVHD setting. Mechanistically, administration of the anti-hICOS mAb was associated with a strong reduction in perivascular infiltrates in liver and lungs and reduction in frequencies and numbers of human T cells in the spleen. In addition, the mAb prevented T-cell expansion in the blood during xeno-GVHD. Importantly, GVHD-protected mice retained the ability to control the P815 mastocytoma cell line, mimicking GVL in humans. A mAb-targeting human ICOS alleviated GVHD without impairing GVL in a xenograft murine model. Thus, ICOS represents a promising target in the management of BMT, preventing GVHD while preserving GVL.

Improving theranostics in pancreatic cancer.

Pancreatic cancer is the fourth most deadly cancer in the United States, and is expected to be the second most deadly by 2030. The major difficulty in treating pancreatic cancer is the late onset of symptoms. Generally, patients show metastatic disease by the time of diagnosis, with a survival rate of 5% beyond 5 years. In patients without metastatic disease, surgical resection increases 5 year survival rate to 25%. The remaining 75% succumb to undetected metastases. Clearly, improvements to both detection, surgical intervention, and therapeutic strategies will be needed to improve patient outcome in pancreatic cancer. Recent literature has been surveyed and atomic models of new therapeutic approaches were generated. Here, we focus on the recent progress employing monoclonal antibodies (mAbs) to target pancreatic cancer associated markers, and more specifically on recent chemical and protein engineering efforts to improve the homogeneity, stability, and administration of mAbs to precisely deliver imaging agents and cytotoxins to sites of disease.

In vivo imaging reveals a tumor-associated macrophage-mediated resistance pathway in anti-PD-1 therapy.

Monoclonal antibodies (mAbs) targeting the immune checkpoint anti-programmed cell death protein 1 (aPD-1) have demonstrated impressive benefits for the treatment of some cancers; however, these drugs are not always effective, and we still have a limited understanding of the mechanisms that contribute to their efficacy or lack thereof. We used in vivo imaging to uncover the fate and activity of aPD-1 mAbs in real time and at subcellular resolution in mice. We show that aPD-1 mAbs effectively bind PD-1+ tumor-infiltrating CD8+ T cells at early time points after administration. However, this engagement is transient, and aPD-1 mAbs are captured within minutes from the T cell surface by PD-1- tumor-associated macrophages. We further show that macrophage accrual of aPD-1 mAbs depends both on the drug's Fc domain glycan and on Fcγ receptors (FcγRs) expressed by host myeloid cells and extend these findings to the human setting. Finally, we demonstrate that in vivo blockade of FcγRs before aPD-1 mAb administration substantially prolongs aPD-1 mAb binding to tumor-infiltrating CD8+ T cells and enhances immunotherapy-induced tumor regression in mice. These investigations yield insight into aPD-1 target engagement in vivo and identify specific Fc/FcγR interactions that can be modulated to improve checkpoint blockade therapy.

Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection.

Dengue virus (DENV) co-circulates as four serotypes (DENV1-4). Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody dependent enhancement (ADE). Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM) of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2) and DENV-2 prM monoclonal antibody (prM mAb) could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs) specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo, interferon-α and γ receptor-deficient mice (AG6) were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10) and alaninea minotransferase (ALT) in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo, suggested that anti-idiotypic antibodies might be a new choice to be considered to treat DENV infection.

Polymorphisms in FcγRII (CD32) mediate abnormal antibody half-life in NOD SCID mice and reduce antibody-mediated tumour cell deletion.

Abstract NOD SCID (NS) mice are commonly used for the assessment of monoclonal antibody (mAb) therapeutics and form the genetic background of NSG mice widely used in human patient derived xenograft models. However, relatively little is known regarding how the NS background effects monoclonal antibody (mAb) pharmacokinetics and how this might influence therapeutic response. To address this we examined mAb half-life and clearance of hCD20 transgenic EμTCL-1 tumours in NS mice following administration of anti-hCD20 hIgG1 mAb and showed mAb half-life to be significantly shorter in NS versus SCID mice, correlating with reduced therapy. Reduced half-life was also seen with mIgG2a but not mIgG1 or hIgG2 isotypes. Additionally, NS mice lacking CD32 or the FcR γ-chain demonstrated this rapid loss was dependent on the expression of CD32 and not activatory FcγRs. Sequencing confirmed the NOD strains carry the rare ly17.1 variant of CD32. Recombinant ly17.1 displayed increased affinity for hIgG1 compared to the more common ly17.2 variant, providing a potential mechanism for its more rapid removal from serum. Importantly, the reduced half-life was dependent on the presence of both SCID and NOD mutations. SCID mice lack endogenous IgG and so we reasoned this might accentuate the effects of the NOD CD32 polymorphic variant. Accordingly, reconstitution of NS mice with physiological levels of mIgG restored normal half-life. These findings highlight the importance of examining mAb pharmacokinetics in complex mouse strains and demonstrate that therapeutic studies in mice on the NS background should be performed in the presence of exogenous IgG.

FCγ Chimeric Receptor-Engineered T Cells: Methodology, Advantages, Limitations, and Clinical Relevance.

For many years, disappointing results have been generated by many investigations, which have utilized a variety of immunologic strategies to enhance the ability of a patient’s immune system to recognize and eliminate malignant cells. However, in recent years, immunotherapy has been used successfully for the treatment of hematologic and solid malignancies. The impressive clinical responses observed in many types of cancer have convinced even the most skeptical clinical oncologists that a patient’s immune system can recognize and reject his tumor if appropriate strategies are implemented. The success immunotherapy is due to the development of at least three therapeutic strategies. They include tumor-associated antigen (TAA)-specific monoclonal antibodies (mAbs), T cell checkpoint blockade, and TAA-specific chimeric antigen receptors (CARs) T cell-based immunotherapy. However, the full realization of the therapeutic potential of these approaches requires the development of strategies to counteract and overcome some limitations. They include off-target toxicity and mechanisms of cancer immune evasion, which obstacle the successful clinical application of mAbs and CAR T cell-based immunotherapies. Thus, we and others have developed the Fc gamma chimeric receptors (Fcγ-CRs)-based strategy. Like CARs, Fcγ-CRs are composed of an intracellular tail resulting from the fusion of a co-stimulatory molecule with the T cell receptor ζ chain. In contrast, the extracellular CAR single-chain variable fragment (scFv), which recognizes the targeted TAA, has been replaced with the extracellular portion of the FcγRIIIA (CD16). Fcγ-CR T cells have a few intriguing features. First, given in combination with mAbs, Fcγ-CR T cells mediate anticancer activity in vitro and in vivo by an antibody-mediated cellular cytotoxicity mechanism. Second, CD16-CR T cells can target multiple cancer types provided that TAA-specific mAbs with the appropriate specificity are available. Third, the off-target effect of CD16-CR T cells may be controlled by withdrawing the mAb administration. The goal of this manuscript was threefold. First, we review the current state-of-the-art of preclinical CD16-CR T cell technology. Second, we describe its in vitro and in vivo antitumor activity. Finally, we compare the advantages and limitations of the CD16-CR T cell technology with those of CAR T cell methodology.

Anti-high mobility group box-1 (HMGB1) antibody inhibits hemorrhage-induced brain injury and improved neurological deficits in rats

As one of the most lethal stroke subtypes, intracerebral hemorrhage (ICH) is acknowledged as a serious clinical problem lacking effective treatment. Available evidence from preclinical and clinical studies suggests that inflammatory mechanisms are involved in the progression of ICH-induced secondary brain injury. High mobility group box-1 (HMGB1) is a ubiquitous and abundant nonhistone DNA-binding protein, and is also an important proinflammatory molecule once released into the extracellular space from the nuclei. Here, we show that treatment with neutralizing anti-HMGB1 mAb (1 mg/kg, i.v. twice) remarkably ameliorated ICH-injury induced by local injection of collagenase IV in the striatum of rats. Administration of anti-HMGB1 mAb inhibited the release of HMGB1 into the extracellular space in the peri-hematomal region, reduced serum HMGB1 levels and decreased brain edema by protecting blood-brain barrier integrity, in association with decreased activated microglia and the expression of inflammation-related factors at 24 h after ICH. Consequently, anti-HMGB1 mAb reduced the oxidative stress and improved the behavioral performance of rats. These results strongly indicate that HMGB1 plays a critical role in the development of ICH-induced secondary injury through the amplification of plural inflammatory responses. Intravenous injection of neutralizing anti-HMGB1 mAb has potential as a novel therapeutic strategy for ICH.

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

A New Approach to PCSK9 Therapeutics.

PCSK9 inhibition is an effective therapy to reduce LDL cholesterol (LDL-C) and cardiovascular events. A recent study shows that one or two doses of inclisiran, a long-acting synthetic small-interfering RNA (siRNA) that selectively targets hepatic PCSK9, causes a sustained reduction of plasma LDL-C for up to 6 months. Pending further studies of safety and efficacy, this may represent an important addition to the armamentarium for inhibiting PCSK9.

Immunosuppressive tumor-infiltrating myeloid cells mediate adaptive immune resistance via a PD-1/PD-L1 mechanism in glioblastoma.

Adaptive immune resistance in the tumor microenvironment appears to attenuate the immunotherapeutic targeting of glioblastoma (GBM). In this study, we identified a tumor-infiltrating myeloid cell (TIM) population that expands in response to dendritic cell (DC) vaccine treatment. The aim of this study was to understand how this programmed death ligand 1 (PD-L1)-expressing population restricts activation and tumor-cytolytic function of vaccine-induced tumor-infiltrating lymphocytes (TILs). To test this hypothesis in our in vivo preclinical model, we treated mice bearing intracranial gliomas with DC vaccination ± murine anti-PD-1 monoclonal antibody (mAb) blockade or a colony stimulating factor 1 receptor inhibitor (CSF-1Ri) (PLX3397) and measured overall survival. We then harvested and characterized the PD-L1+ TIM population and its role in TIL activation and tumor cytolysis in vitro. Our data indicated that the majority of PD-L1 expression in the GBM environment is contributed by TIMs rather than by tumor cells themselves. While PD-1 blockade partially reversed the TIL dysfunction, targeting TIMs directly with CSF-1Ri altered TIM expression of key chemotactic factors associated with promoting increased TIL infiltration after vaccination. Neither PD-1 mAb nor CSF-1Ri had a demonstrable therapeutic benefit alone, but when combined with DC vaccination, a significant survival benefit was observed. When the tripartite regimen was given (DC vaccine, PD-1 mAb, PLX3397), long-term survival was noted together with an increase in the number of TILs and TIL activation. Together, these studies elucidate the role that TIMs play in mediating adaptive immune resistance in the GBM microenvironment and provide evidence that they can be manipulated pharmacologically with agents that are clinically available. Development of immune resistance in response to active vaccination in GBM can be reversed with dual administration of CSF-1Ri and PD-1 mAb.

Factor VIIa Interaction with Endothelial Cell Protein C Receptor: Its Role in Hemostatic Effect of rFVIIa in Treating Hemophilia

Recent studies from our laboratory and others established that clotting factor FVII/FVIIa, which binds tissue factor and triggers the coagulation cascade, also binds endothelial cell protein C receptor (EPCR), a receptor that plays a critical role in protein C/activated protein C (APC)-mediated anticoagulation pathway. We postulated that FVIIa binding to EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions by down-regulating the EPCR-mediated anticoagulation pathway. Our recent studies showing the blockade of endogenous protein C binding to EPCR by administration of EPCR blocking antibodies augmented the hemostatic effect of rFVIIa in a mouse hemophilia model provided an indirect support to this hypothesis. However, studies from other investigators raised a possibility that FVIIa interaction with EPCR may influence the hemostatic effect of rFVIIa in vivo through EPCR-FVIIa directly activating FX or EPCR tethering FVIIa to provide an extended locale of procoagulant reactions on the endothelium. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of FVIIa in hemophilia therapy. The study employed wild-type, transgenic mice expressing no EPCR (EPCR-/-) or overexpressing EPCR (Tie2-EPCR). FVIII mAb (1 mg/kg) were given to mice to induce hemophilia. The saphenous vein incision bleeding model was used to evaluate the hemostatic effect of human rFVIIa. In this model, bleeding is induced by a sharp incision to the saphenous vein and the bleeding from the cut was monitored for 30 min. After each hemostasis incident, the clot was disrupted gently to reinitiate a new bleeding episode and average time to achieve hemostasis (ATH) was calculated.First, we investigated the role of EPCR in hemostasis by comparing the bleeding episodes in unchallenged wild-type, EPCR-/- and Tie2-EPCR mice following the saphenous vein incision. In this model, control wild-type mice had a median ATH of 55 sec (mean 63 ± 7 sec; n=9) and EPCR-/- mice had a median ATH of 67 sec (mean 74 ± 6 sec; n =12). The difference between them is not statistically significant. Surprisingly, the median ATH in EPCR overexpressing Tie2-EPCR mice was significantly lower (a median ATH of 43 sec; mean 46 ± 2 sec; n=10) compared to the ATH in wild-type or EPCR-/- mice. The reason for a shorter ATH observed in EPCR overexpressing mice is unknown at present. Analysis of blood count showed no significant differences among wild-type, EPCR-/- and Tie2-EPCR in their platelet count, RBC, and other blood cell count.Next, we induced acquired hemophilia condition in wild-type, EPCR-/- and Tie2-EPCR by injecting FVIII mAb (1 mg/kg) i.v. 2 h before inducing the bleeding. Administration of FVIII mAb markedly prolonged the bleeding time in wild-type and Tie2-EPCR mice (between 300 to 1800 sec) with a median ATH of 600 sec (mean 730 ± 277 sec) in wild-type mice and 750 sec (mean 939 ± 200 sec) in Tie2-EPCR mice. Interestingly, EPCR-/- mice were protected from the antibody-induced acquired hemophilia. Administration of FVIII mAb prolonged the bleeding time only marginally in this group of mice to a median ATH of 120 sec (mean 163 ± 38 sec). Administration of a low dose of rFVIIa (0.25 mg/kg) fully corrected the mild bleeding defect in EPCR-/- acquired hemophilia mice (median ATH of 67 sec; mean 86 ± 17 sec). Administration of 0.25 mg/kg rFVIIa is corrected the bleeding disorder only partially in wild-type acquired hemophilia mice (median ATH 164 sec; mean 303 ± 94 sec; n=5). Administration of either 1 or 4 mg/kg of rFVIIa was required to correct the bleeding in these mice (1 mg/kg dose: median ATH of 75 sec, mean 74 ± 1 sec; 4 mg/kg dose: median ATH of 37 sec; mean 40 ± 2 sec, n=5). In Tie2-EPCR hemophilia mice, administration of 1 mg/kg rFVIIa was not sufficient to fully achieve the hemostatic effect (median ATH 164 sec; mean 521 ± 237 sec; n=6). Administration of 4 mg/kg rFVIIa was required to restore the hemostasis in these mice (median ATH of 90 sec; mean 94 ± 7 sec, n= 6). In summary, the present data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating antibody-induced hemophilia. The present study rules out the possibility of direct activation of FX by FVIIa-EPCR complexes or EPCR tethering of FVIIa as probable mechanisms by which EPCR-FVIIa influences the hemostatic effect of rFVIIa. DisclosuresNo relevant conflicts of interest to declare.

Anti-high mobility group box-1 (HMGB1) antibody attenuates delayed cerebral vasospasm and brain injury after subarachnoid hemorrhage in rats.

Although delayed cerebral vasospasm (DCV) following subarachnoid hemorrhage (SAH) is closely related to the progression of brain damage, little is known about the molecular mechanism underlying its development. High mobility group box-1 (HMGB1) plays an important role as an initial inflammatory mediator in SAH. In this study, an SAH rat model was employed to evaluate the effects of anti-HMGB1 monoclonal antibody (mAb) on DCV after SAH. A vasoconstriction of the basilar artery (BA) associated with a reduction of nuclear HMGB1 and its translocation in vascular smooth muscle cells were observed in SAH rats, and anti-HMGB1 mAb administration significantly suppressed these effects. Up-regulations of inflammation-related molecules and vasoconstriction-mediating receptors in the BA of SAH rats were inhibited by anti-HMGB1 mAb treatment. Anti-HMGB1 mAb attenuated the enhanced vasocontractile response to thrombin of the isolated BA from SAH rats and prevented activation of cerebrocortical microglia. Moreover, locomotor activity and weight loss recovery were also enhanced by anti-HMGB1 mAb administration. The vasocontractile response of the BA under SAH may be induced by events that are downstream of responses to HMGB1-induced inflammation and inhibited by anti-HMGB1 mAb. Anti-HMGB1 mAb treatment may provide a novel therapeutic strategy for DCV and early brain injury after SAH.