Antibody-mediated Cell Research Articles

Preclinical functional characterization of AGEN1181, a clinical stage Fc-engineered anti-CTLA-4 antibody for the treatment of patients with early and advanced malignancies.

e14126 Background: Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is an inhibitory receptor that modulates T cell priming and CD28-dependent T cell activation. Modulation of this pathway by CTLA-4 antibodies has demonstrated clinical responses in patients with advance disease, however the mechanism of action of these antibodies remains controversial. It is currently understood that anti-CTLA-4 antibodies mediate antitumor effects through receptor-ligand blockade, and, in preclinical models, through depletion of intratumoral regulatory T (Treg) cells via an Fc-dependent mechanism. Recently, we reported a novel Fc-mediated contribution to T cell priming underscored the importance of Fc selection for optimal CTLA-4 antibody activity. AGEN1181 is a clinical stage Fc-engineered IgG1 antibody that leverages this novel mechanism for enhanced functionality. Methods: Fab-mediated biology of AGEN1181 was confirmed by assessing binding affinity, specificity, and blockade of CTLA-4:CD80/86 interactions. Fc-mediated functions of AGEN1181 were evaluated by FcγR binding, reporter-based FcγR signaling, and antibody-mediated Treg cell cytotoxicity assays. In vitro and in vivo pharmacology of AGEN1181 (alone or in combination with anti-PD-1) were evaluated in superantigen (staphylococcal enterotoxin)-mediated T cell stimulation assays and in a non-human primate (NHP) model. Safety and tolerability of AGEN1181 were assessed in cynomolgus monkeys administered 5 once-weekly intravenous bolus infusions at doses of 5, 30, or 100 mg/kg. Results: AGEN1181 bound to CTLA-4 with high affinity and specificity and demonstrated a range of enhanced functional activities relative to first-generation CTLA-4 antibodies. This included superior CD16 (FcγRIIIA) binding, Treg depletion and T cell priming (as measured by ZAP70 phosphorylation and interleukin-2 secretion). In NHP studies, a single dose of AGEN1181, alone or in combination with anti-PD-1 antibody upregulated Ki67 and ICOS expression on peripheral T cells. Finally, AGEN1181 exhibited an acceptable toxicologic profile in multi-dose NHP studies. Conclusions: AGEN1181, an Fc-engineered anti-CTLA-4 antibody exhibits a range of enhanced functional attributes relative to first-generation anti-CTLA-4 antibodies in preclinical studies. Phase 1 clinical studies for AGEN1181 will be initiated imminently.

The Role of Histamine in the Pathophysiology of Asthma and the Clinical Efficacy of Antihistamines in Asthma Therapy.

Mast cells play a critical role in the pathogenesis of allergic asthma. Histamine is a central mediator released from mast cells through allergic reactions. Histamine plays a role in airway obstruction via smooth muscle contraction, bronchial secretion, and airway mucosal edema. However, previous clinical trials of H1 receptor antagonists (H1RAs) as a treatment for asthma were not successful. In recent years, type 2 innate immunity has been demonstrated to be involved in allergic airway inflammation. Allergic asthma is defined by IgE antibody-mediated mast cell degranulation, while group 2 innate lymphoid cells (ILC2) induce eosinophilic inflammation in nonallergic asthma without allergen-specific IgE. Anti-IgE therapy has demonstrated prominent efficacy in the treatment of severe allergic asthmatics sensitized with specific perennial allergens. Furthermore, recent trials of specific cytokine antagonists indicated that these antagonists were effective in only some subtypes of asthma. Accordingly, H1RAs may show significant clinical efficacy for some subtypes of allergic asthma in which histamine is deeply associated with the pathophysiology.

Liver-resident NK cells suppress autoimmune cholangitis and limit the proliferation of CD4+ T cells.

Liver-resident NK cells are distinct from conventional NK cells and play an important role in the maintenance of liver homeostasis. How liver-resident NK cells participate in autoimmune cholangitis remains unclear. Here, we extensively investigated the impact of NK cells in the pathogenesis of autoimmune cholangitis utilizing the well-established dnTGFβRII cholangitis model, NK cell-deficient (Nfil3-/-) mice, adoptive transfer and in vivo antibody-mediated NK cell depletion. Our data demonstrated that disease progression was associated with a significantly reduced frequency of hepatic NK cells. Depletion of NK cells resulted in exacerbated autoimmune cholangitis in dnTGFβRII mice. We further confirmed that the DX5-CD11chi liver-resident NK cell subset colocalized with CD4+ T cells and inhibited CD4+ T cell proliferation. Gene expression microarray analysis demonstrated that liver-resident NK cells had a distinct gene expression pattern consisting of the increased expression of genes involved in negative regulatory functions in the context of the inflammatory microenvironment.

Antibody-targeted chromatin enables effective intracellular delivery and functionality of CRISPR/Cas9 expression plasmids.

We report a novel system for efficient and specific targeted delivery of large nucleic acids to and into cells. Plasmid DNA and core histones were assembled to chromatin by salt gradient dialysis and subsequently connected to bispecific antibody derivatives (bsAbs) via a nucleic acid binding peptide bridge. The resulting reconstituted vehicles termed ‘plasmid-chromatin’ deliver packaged nucleic acids to and into cells expressing antigens that are recognized by the bsAb, enabling intracellular functionality without detectable cytotoxicity. High efficiency of intracellular nucleic acid delivery is revealed by intracellular expression of plasmid encoded genes in most (∼90%) target cells to which the vehicles were applied under normal growth/medium conditions in nanomolar concentrations. Specific targeting, uptake and transgene expression depends on antibody-mediated cell surface binding: plasmid chromatin of identical composition but with non-targeting bsAbs or without bsAbs is ineffective. Examples that demonstrate applicability, specificity and efficacy of antibody-targeted plasmid chromatin include reporter gene constructs as well as plasmids that enable CRISPR/Cas9 mediated genome editing of target cells.

Abstract A059: Tackling the tumor microenvironment with CD38 blockade to enhance cancer immunotherapy

Abstract The single agent or the combination of anti-PD-1, anti-PD-L1, and anti-CTLA-4 is an effective strategy that is being clinically explored to treat a variety of cancer types. Some patients display primary resistance to the treatment, while others relapse after treatment. Resistance is a major issue that needs to be addressed. Using multiple immunocompetent syngeneic and K-rasLA1/+p53R172HΔg/+ spontaneous animal models of lung cancer, we have explored the mechanisms of resistance to treatments by evaluating the molecular and cellular immune profiles of the tumor microenvironment. We observed that tumor-bearing mice treated with PD-1/PD-L1 blocking antibodies developed resistance through the up-regulation of CD38. We also observed this in the combination therapy of anti-PD-1 and anti-CTLA-4, suggesting that CD38 is a major mechanism of resistance to immune checkpoint inhibitors. In vitro and in vivo studies demonstrated that CD38 impacted CD8+ T-cell function via adenosine receptor signaling and dendritic cell-mediated B7 signaling. Antibody-mediated cell depletion assays were conducted to validate the mechanisms. To determine the applicability to patients, we analyzed 793 lung cancer patients’ specimens with immunohistochemistry staining and assessed biomarker relationships in multiple large independent patient databases (~1,900 tumors). Pathologic analysis revealed positive immunohistochemical staining for CD38 on tumor cells in 15-23% of cases, and bioinformatic analyses revealed a strong correlation between CD38 expression and the immune signature. Lastly, targeting CD38 abolished the treatment resistance by modulating the adenosine levels and thereby enhancing the effector immune cell infiltrates into the tumor microenvironment. Based on our study, CD38 blockade improves the efficacy of single-agent anti-PD-1/PD-L1, or with anti-CTLA-4 combination in lung cancer. CD38 could potentially serve as a novel biomarker of resistance for immune checkpoint inhibition. The data from this study provide a unique target, biomarker, and therapeutic strategy that can be translated into the clinical practice. Citation Format: Limo Chen, Lixia Diao, Xiaohui Yi, Bertha Leticia Rodriguez, Yanli Li, Pamela Villalobos, Tina Cascone, Xi Liu, Lin Tan, Philip Lorenzi, Jared Fradette, David Peng, Ferdinandos Skoulidis, Youhong Fan, Jaime Rodriguez-Canales, Vassiliki Papadimitrakopoulou, Ethan Dmitrovsky, Lauren A Byers, Jing Wang, Ignasio Wistuba, Jim Heymach, Don Gibbons. Tackling the tumor microenvironment with CD38 blockade to enhance cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A059.

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

Antibody-mediated pure red cell aplasia after treatment with darbepoetin-alpha postrenal transplantation

Pure red cell aplasia (PRCA) is a rare normochromic, normocytic anemia with a near-to-complete absence of erythroblasts in the bone marrow. Erythropoiesis-stimulating agents have been used widely to improve anemia in patients with chronic kidney disease. We present a case of a 38-year-old woman who was detected to have anemia (hemoglobin: 10 g/dl) and renal dysfunction (creatinine: 3 mg/dl) on a routine health check in December 2016. Her kidney biopsy showed chronic interstitial nephritis. She received intravenous (IV) iron and was started on darbepoetin-alpha. Her hemoglobin after 3 months of treatment was 12 mg/dl. She underwent preemptive one haplomatch renal transplantation on June 2017. She was discharged with serum creatinine of 0.9 mg/dl and hemoglobin of 8 g/dl. She was not on any Erthropoietin stimulating agents (ESA) at discharge. In the 4th week after transplant, her hemoglobin dropped to 7 g/dl, and she was restarted on darbepoetin-alpha 40 μg subcutaneously once a week. In the 6th week posttransplant, her hemoglobin dropped to 6 g/dl. Her peripheral smear showed normocytic normochromic anemia with no evidence of hemolysis. Transferrin saturation was 81%, serum ferritin levels were 1812 ng/ml, and reticulocyte count was 0.1%. Bone marrow biopsy showed suppression of erythroid precursors with myeloid-to-erythroid ratio of 8:1, adequate iron stores, and normal white blood cell and platelet precursors with no dysplastic cells. Paroxysmal Nocturnal hemoglobinuria (PNH) workup; antinuclear antibody; and screening for hepatitis B, hepatitis C, HIV, CMV, and human parvovirus B19 were negative. Anti-erythropoietin (EPO) antibody by enzyme-linked immunosorbent assay was positive. She was diagnosed to have PRCA secondary to anti-EPO antibodies. She was managed with pulse doses of methylprednisolone, adequate dose of tacrolimus, IV immunoglobulin of 125 g (2 g/kg) over 5 days, and two packed red cell transfusions. Darbepoetin-alpha was stopped. At present, her hemoglobin is 12.5 g/dl with serum creatinine of 1 mg/dl on triple immunosuppression with no requirement of ESA.

Assessment of Humoral Alloimmunity in Mixed Lymphocyte Reaction.

Humoral alloimmunity remains a significant and unresolved problem that constrains allograft survival. Thus, there is a need for the development of an easy, preferably non-radioactive, and inexpensive protocol for assessing the effect of various drug treatments on humoral alloimmunity. In order to satisfy this demand, we developed such a protocol in which de novo alloantibodies production is induced in one-way mixed lymphocyte reaction (MLR). The amount and capacity of the generated alloantibodies in the supernatant of each one-way MLR is assessed using an antibody-mediated cell dependent cytotoxicity (CDC) assay. The principle of the assay relies on the assessment of cellular survival of resting PBMCs isolated from the same donors, supplemented with the alloantibodies from the one-way MLR supernatant. The lesser is the cellular survival, the higher the production of alloantibodies in one-way MLR and consequently the more potent the humoral alloimmunity.

Oncogenic-Drivers Dictate Immune Responses to Control Disease Progression in Acute Myeloid Leukaemia

Immunotherapy has revolutionised therapeutic approaches to fight cancer and, in certain diseases dramatically improves survival. Clinical responses to immune checkpoint blockade have in part been attributed to high mutational burden of tumours such as melanoma. High-risk acute myeloid leukaemia (AML) is defined by molecular and cytogenetic factors. AML has a low prevalence of somatic mutations and is predicted to have low immunogenicity. We aimed to determine how AMLs driven from different classes of oncogenes interact with endogenous anti-leukemic immune responses.Methods and ResultsWe generated three oncogenically distinct models of AML: BCR-ABL+NUP98-HOXA9 (BA/NH9), MLL-AF9 (MA9), and AML1-ETO+NRASG12D (AE/NRAS), using retroviral transduced bone marrow transplanted into immune-competent, non-irradiated C57BL/6J (B6) mice or immune-deficient Rag2-/-γc-/- mice. Immunologic control of AML was dependent on the driver oncogene, as AE/NRAS AML was effectively controlled in B6, but not Rag2-/-γc-/-recipients, whereas survival of BA/NH9 AML recipients was similar between B6 and Rag2-/-γc-/-. MA9 AML had an intermediate phenotype (Figure 1A-C).To examine the mechanisms underlying immune escape in AE/NRAS, AML from immune-deficient or immune-competent hosts, was passaged through immune-competent hosts. Prior exposure to an intact immune system dramatically accelerated disease progression of AE/NRAS AML in subsequent B6 recipients, but this was not seen in passage through Rag2-/-γc-/- recipients. This demonstrates specific, functional immunoediting of AML resulting in evasion of immune control.Despite evidence of disease attenuation in immune competent hosts, functional immunoediting was not observed in MA9 AML. Antibody-mediated immune cell depletion experiments demonstrated that natural killer (NK) cells and T cells both contribute to the control AE/NRAS AML, whereas MA9 immune control was dependent on NK cells. As immunoediting was only seen in AE/NRAS model, this suggests that functional immunoediting in this model is primarily mediated by T cells.To characterise the mechanisms regulating immunoediting, we integrated proteomic and transcriptional analysis of immunoedited and non-immunoedited AE/NRAS AML. There was strong correlation between increased protein expression and transcriptional regulation. There was distinct regulation of inflammatory pathways between immunoedited and non-immunoedited AML. Immunoedited AE/NRAS cells showed increased IFN-γ-dependent response signatures, consistent with direct targeting of the leukemic cells by the immune system. Transcriptional analysis also showed modulation of expression of immune checkpoint molecules including upregulation of suppressive molecules Tim-3 and CD39 and downregulation of activating ligand CD137L. These findings were confirmed by cell-surface flow cytometry. Immunoedited AE/NRAS downregulated RAS signalling transcriptionally, with coordinate activation of MYC targets.In the murine AE/NRAS model, CD4+ and CD8+ T effector memory (TEM) cells (CD44+ CD62L-) demonstrated increased PD-1 expression compared to naïve mice. In addition, mice with high disease burden also had increased frequency of T cells co-expressing exhaustion markers PD-1, Tim-3 and LAG-3, consistent with suppression of the anti-leukemic effector immune response. To understand if these findings were relevant to AML in the clinic, we obtained single cell RNA-sequencing data from the CD45+ CD34- non-leukemic fraction of bone marrow in a patient with AML1-ETO AML at diagnosis compared to that in normal marrow. Single cell type classification and clustering using tSNE demonstrated remodelling of the immune microenvironment in AML with loss of NK cells, pre-B cells and skewing of T cell subsets. There was depletion of CD8+ TEM cells and greater proportions of CD4+ and CD8+ TEM cells expressing activation and exhaustion markers (IFN-γ, PD-1, LAG-3, TIM-3).ConclusionsThese data demonstrate that immune responses in AML are oncogene-specific and provide evidence that AE/NRAS AML cells undergo immunoediting over time in the presence of a competent immune microenvironment. Since AML is associated with alterations in T cell subsets, and changes in T cell activation and exhaustion states, these findings may inform translational strategies to use immunotherapies for patients with AML. DisclosuresSmyth:Bristol Myers Squibb: Other: Research agreement; Tizona Therapeutics: Research Funding. Lane:Janssen: Consultancy, Research Funding; Celgene: Consultancy; Novartis: Consultancy.

Rituximab-Induced Acute Thrombocytopenia in a Patient with Relapsed Follicular Lymphoma

IntroductionRituximab is a well-tolerated monoclonal antibody which is commonly used in the treatment of B cell lymphomas that bear CD20 antigen. Most of the adverse effect induced by rituximab are infusion-related symptoms. Rituximab-induced acute thrombocytopenia (RIAT) are rare than other side-effects such as serum sickness-like syndrome and most of the reports are described as case reports. Here, we reported a serious acute thrombocytopenia caused by rituximab in a refractory and relapsed (R/R) follicular lymphoma (FL) patient with massive tumor burden. To our knowledge, this is the third case that we can retrieveMethodsA 56-year-old woman was enrolled in our ward in 2011. The patient was diagnosed as FL (grade 2, stage IV, group B, IPI score 2) and received three cycles of R-CHOP without rituximab-associated serum sickness or obvious evidence of bone marrow toxicity. A partial response (PR) was achieved but the patient rejected to receive further therapy. She had experienced night sweats, fatigue, and floating during the preceding 7 months and came to the clinic in April 2018. Physical examination found multiple sites of shallow lymphadenopathy, especially in her right neck. The spleen was 7.5 cm palpable below the left costal margin and the ascites sign was positive. Re-biopsy was done in her right neck lymph node and FL (grade 3a) was confirmed again by IHC. The routine blood test showed WBC of 2,710, neut 1,100, Hb 90 g/L, and platelets count of 84. The bone marrow biopsy showed lymphocytic cells infiltration. So the diagnosis of the relapsed FL (grade 3a, stage IV, group B, FLIPI-1 Score 4) was made. We chose R-CHOP regimen again and rituximab was administrated at day 0 with a dose of 600mg. She developed shiver and fever one hour after the rituximab infusion. Blood culture showed no bacteria growth and inflammation index has no positive result. On the next day, repeat hemogram showed a platelet count of 26,000. The coagulation profile was within normal limits. The patient didn't show any sign of bleeding and did not meet the threshold criteria for platelet transfusion. On the third day post-rituximab infusion, the platelet count was 45,000 and recovered to baseline of the platelets level within one week. She had a similar episode during the following treatment contained rituximab (one cycle of R-CHOP and two cycles of R-GDP). No more intervention was given and the platelet count recovered again within one week. After two cycles of R-GDP, no obvious response was achieved and the patient died of progression of the disease.ResultsWe conducted a literature review about RIAT. It is a rare adverse event and usually occurs within 1 to 3 days after rituximab infusion. Most of the patients with RIAT share the common clinical characteristics: MCL was the predominant histological subtype; many patients present infusion-associated symptoms; they often had comorbidity of massive tumor burden, bone marrow infiltration, and splenomegaly; re-exposure to rituximab usually will not induce a repetitive RIAT but there are exceptions; RIAT is s usually transient and self-limiting. Compared to MCL, RIAT in FL is not a frequent adverse event. To the best of our knowledge, we only found two recent reports associated RIAT developed in patients with FL reported by Japanese researchers. The possible mechanisms of RIAT remain unclear and they might lie in the following ways: 1. The presence of CD20 antigen on the platelets themselves can lead to the antibody-mediated cell lysis through Fc receptors. 2. The patient had soluble CD20 antigen in the circulation, binding them to platelets causing immune-mediated cell lysis. 3. The splenomegaly also contributed to the delay and the destruction of platelets. 4. With a rapid lysis of lymphoma cells and changes in the levels of cytokines and complements, thrombocytopenia can be initiated by the induction of a disseminated intravascular coagulation. 5. P38 mitogen-activated protein kinase and Akt anti-apoptotic survival pathways might play important roles in leading to cell death.ConclusionHere we reported a patient diagnosed as R/R FL with a massive tumor burden developed repetitive and self-recovered RIAT. Massive tumor burden, bone marrow infiltration, and splenomegaly might be risk factors to develop a RIAT. Hence, rituximab should be used with caution in patients who have these factors, and routine blood count monitoring should be considered after the administration of rituximab. DisclosuresNo relevant conflicts of interest to declare.

A Role for Fc Function in Therapeutic Monoclonal Antibody-Mediated Protection against Ebola Virus

The recent Ebola virus (EBOV) epidemic highlighted the need for effective vaccines and therapeutics to limit and prevent outbreaks. Host antibodies against EBOV are critical for controlling disease, and recombinant monoclonal antibodies (mAbs) can protect from infection. However, antibodies mediate an array of antiviral functions including neutralization as well as engagement of Fc-domain receptors on immune cells, resulting in phagocytosis or NK cell-mediated killing of infected cells. Thus, to understand the antibody features mediating EBOV protection, we examined specific Fc features associated with protection using a library of EBOV-specific mAbs. Neutralization was strongly associated with therapeutic protection against EBOV. However, several neutralizing mAbs failed to protect, while several non-neutralizing or weakly neutralizing mAbs could protect. Antibody-mediated effector functions, including phagocytosis and NK cell activation, were associated with protection, particularly for antibodies with moderate neutralizing activity. This framework identifies functional correlates that can inform therapeutic and vaccine design strategies against EBOV and other pathogens.

Preventing or Eradicating Factor VIII Antibody Formation in Patients with Hemophilia A: What Can We Learn from Other Disorders?

Eradication of factor VIII (FVIII) specific neutralizing antibodies (also known as inhibitors) by the traditional method of immune tolerance induction (ITI) is costly and unsuccessful in one out of three patients. Furthermore, effective inhibitor prevention strategies are presently lacking. An overview is given in this narrative review of antidrug antibody prevention or eradication strategies that have been used in disorders beyond hemophilia A, with the aim of analyzing what we can learn from these strategies for hemophilia A. Prevention of antidrug antibody formation using rituximab, methotrexate, and intravenous immunoglobulins in patients with Pompe's disease seems effective but carries a high risk of adverse events. Based on studies in patients with rheumatoid arthritis and inflammatory bowel disease, it seems likely that treatment with methotrexate alone would also be able to prevent inhibitor formation in hemophilia A patients. Besides side effects, it is unclear whether immune tolerance to FVIII would persist after cessation of immunomodulatory therapy with methotrexate. A combination of cyclophosphamide and corticosteroids, used to treat antibody-mediated pure red cell aplasia, could be further investigated to eradicate inhibitors in hemophilia A patients who are refractory to ITI. In summary, insights gained from research on antidrug antibody formation in other diseases could be helpful in devising alternative treatment strategies for inhibitor development.

Slan+ Monocytes and Macrophages Mediate CD20-Dependent B-cell Lymphoma Elimination via ADCC and ADCP.

Terminal tissue differentiation and function of slan+ monocytes in cancer is largely unexplored. Our recent studies demonstrated that slan+ monocytes differentiate into a distinct subset of dendritic cells (DC) in human tonsils and that slan+ cells colonize metastatic carcinoma-draining lymph nodes. Herein, we report by retrospective analysis of multi-institutional cohorts that slan+ cells infiltrate various types of non-Hodgkin lymphomas (NHL), particularly the diffuse large B-cell lymphoma (DLBCL) group, including the most aggressive, nodal and extranodal, forms. Nodal slan+ cells displayed features of either immature DC or macrophages, in the latter case ingesting tumor cells and apoptotic bodies. We also found in patients with DLBCL that peripheral blood slan+ monocytes, but not CD14+ monocytes, increased in number and displayed highly efficient rituximab-mediated antibody-dependent cellular cytotoxicity, almost equivalent to that exerted by NK cells. Notably, slan+ monocytes cultured in conditioned medium from nodal DLBCL (DCM) acquired a macrophage-like phenotype, retained CD16 expression, and became very efficient in rituximab-mediated antibody-dependent cellular phagocytosis (ADCP). Macrophages derived from DCM-treated CD14+ monocytes performed very efficient rituximab-mediated ADCP, however, using different FcγRs from those used by slan+ macrophages. Our observations shed new light on the complexity of the immune microenvironment of DLBCL and demonstrate plasticity of slan+ monocytes homing to cancer tissues. Altogether, data identify slan+ monocytes and macrophages as prominent effectors of antibody-mediated tumor cell targeting in patients with DLBCL.Significance: slan+ monocytes differentiate into macrophages that function as prominent effectors of antibody-mediated tumor cell targeting in lymphoma.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3544/F1.large.jpg Cancer Res; 78(13); 3544-59. ©2018 AACR.

Development of an anti-EPO antibody detection kit based on lab-on-a-chip and bridging antibody technologies

Immunogenicity is a major concern in the use of biological drugs. In particular, antibody-mediated pure red cell aplasia (PRCA) is a rare condition that is caused by administration of recombinant erythropoietin. There are numerous assay platforms for detect EPO anti-drug antibody (ADA), and most have appropriate assay sensitivity, but in need of improvement in terms of assay turnaround time and user accessibility. Here, the new method was developed based on lab-on-a-chip technology and bridging ELISA. The FREND™ Cartridge is equipped with a microfluidic lateral flow channel, enabling easy, fast and accurate immunoassays with small sample volumes. Biotinylated EPO was immobilized on the avidin-coated solid phase of the test zone in the FREND™ cartridge. Initially, ADA in the serum sample binds to the detector conjugate (EPO-HRP-anti HRP antibody-FL bead) in the conjugation zone, and it flows into the test zone prepared with capture complex (avidin-biotinylated EPO). Unbound detector complexes are captured in the reference zone. The FREND™ system detects and quantifies the fluorescence signals in each zone and then calculates the concentration of EPO ADA in the sample. The FREND™ EPO ADA kit may be useful in local clinics as a rapid method for monitoring patients administered recombinant erythropoietin.

Contribution of NK Cell Education to both Direct and Anti-HIV-1 Antibody-Dependent NK Cell Functions.

Antibody Fc-dependent functions are linked to prevention and control of HIV-1 infection. Basic NK cell biology is likely key to understanding the contributions that anti-HIV-1 antibody-dependent NK cell activation and cytolysis make to HIV-1 susceptibility and disease progression. The importance of NK cell education through inhibitory receptors specific for self-HLA-I in determining the potency of anti-HIV-1 antibody-mediated NK cell activation and cytolysis is controversial. To address this issue more definitively, we utilized HLA-I genotyping, flow cytometry staining panels, and cytolysis assays to assess the functionality of educated and noneducated peripheral blood NK cells. We now demonstrate that educated NK cells are superior in terms of their capacity to become activated and/or mediate cytolysis following anti-HIV-1 antibody-dependent stimulation. The profiles of activation observed were similar to those observed upon direct stimulation of NK cells with target cells devoid of HLA-I. Noneducated NK cells make significantly lower contributions to total NK cell activation than would be expected from their frequency within the total NK cell population (i.e., they are hypofunctional), and educated NK cells make contributions similar to or higher than their frequency in the total NK cell population. Finally, NK cells educated through at least one killer immunoglobulin-like receptor and NKG2A exhibited the most significant difference between actual and expected contributions to the total NK cell response, based on their frequency within the total NK cell population, suggesting that summation of NK cell education through inhibitory receptors determines overall NK cell functionality. These observations have potential implications for understanding HIV-1 vaccine efficacy and disease progression.IMPORTANCE NK cells are major mediators of anti-HIV-1 antibody-dependent functions, including cytokine production and cytolysis. The mechanisms controlling the capacity of individual NK cells to mediate antibody-dependent functions remain poorly defined. We now show that NK cell education determines the capacity of NK cells to exhibit anti-HIV-1 antibody-dependent activation and mediate antibody-dependent cellular cytotoxicity. These observations suggest that the process of NK cell education could be of importance for understanding HIV-1 pathogenesis and designing immune-based prophylactics or therapeutics.

P-D8 New insights on the human anti-HIV-1 Env antibody-mediated cell cytotoxicity (ADCC) against HIV-1 virus: Allosteric regulation of FcRs binding upon antigen engagement

HIV-1 vaccine field is rapidly evolving and a deeper knowledge of the mechanisms of defense against HIV-1 infection is needed. In this regard, several studies highlighted the relevance of the antibody-mediated cell cytotoxicity (ADCC) in the context of HIV-1 infection, linking Fc-effector functions to protection against HIV-1 acquisition. The current model for ADCC activation is based on the concept that antibodies, bound to their respective antigens on the surface of HIV-1 sensitized cells, form aggregates which engage the FcRs, activating the effector cells. Our aim is to understand the molecular basis of ADCC and to identify the critical factors that lead to the triggering of the cytotoxicity against HIV-1 virus. Utilizing multiple approaches, such as ELISA, FCS (Fluorescence Correlation Spectroscopy), H/DX MS (Hydrogen/Deuterium Exchange Mass Spectroscopy) and crystallography, we studied the very first step of ADCC activation: the monovalent binding of viral antigen. Here, we demonstrate an allosteric regulation in anti-HIV gp120 Cluster A mAbs resulting from immune complex (IC) formation with a monomeric gp120-CD4 chimera antigen. We established that IC formation dramatically increases the efficiency of Ab interaction to low affinity FcRs compared to free IgG, impacting, in turn, the activation of the cytotoxicity against HIV-1 positive targets. In conclusion, we believe that monomeric antigen-antibody IC formation might be the very first step of HIV-1-specific ADCC triggering that likely precedes the IgG aggregation required for Fc receptors binding and in turn, effector cell activation. This might be a mechanism that enables the fine tuning of Fc-effector functions in vaccine regimens or HIV-1 passive treatments.

Targeting CD38 to improve anti-PD-1/CTLA-4 combination therapy in lung cancer.

144 Background: The combination of anti-PD-1 and anti-CTLA-4 is a promising strategy that is being clinically explored to treat a variety of cancer types. Some patients display primary resistance to this combination treatment, while others relapse after treatment. Although some cancer patients have long-term durable responses to the combination therapy of anti-PD-1 and anti-CTLA-4, physicians have been looking for biomarkers that predict response. Methods: Using multiple immunocompetent syngeneic and K-rasLA1/+p53R172H?g/+ spontaneous animal models of lung cancer, we have explored the mechanisms of resistance to anti-PD-1/CTLA-4 combination therapy by evaluating the molecular and cellular immune profiles of the tumor microenvironment. Antibody-mediated cell depletion assays were also conducted to validate the mechanisms. Lastly, to determine the applicability to patients, we analyzed 791 lung cancer patients’ specimens with immunohistochemistry staining and mined many immune markers in multiple large independent patient databases (~1900 tumors). Results: We observed that tumor bearing mice treated with combined PD-1 and CTLA-4 blocking antibodies developed resistance through the up-regulation of CD38, and that targeting CD38 abolished the resistance in a manner dependent on B7-costimulation. Additionally, combined PD-1 and CTLA-4 blockade eradicates CD38-deficient tumors in mice. Further study revealed that control of tumors by triple blockade of CD38, PD-1, and CTLA-4 depended on improved functionality of CD4+/CD8+ TILs, which were reactivated by enriched CD103+ dendritic cells in the tumor microenvironment. Pathologic analysis revealed positive immunohistochemical staining for CD38 on tumor cells in 15-23% of cases and bioinformatic analyses revealed a strong correlation between CD38 expression and an immune inflammatory signature. Conclusions: Targeting CD38 improves the efficacy of anti-PD-1/CTLA-4 combination therapy in lung cancer. CD38 on tumor cells could potentially serve as a novel biomarker of resistance for immune checkpoint inhibition.

Human Cytomegalovirus Infection Increases Both Antibody- and Non-Antibody-Dependent Cellular Reactivity by Natural Killer Cells.

Antibody-mediated rejection in solid organ transplantation is an important immunological barrier to successful long-term graft survival. Next to complement activation, natural killer (NK) cells have been implicated in the process. Human cytomegalovirus (CMV), independently associated with decreased graft survival, has a strong imprint on the immune response. Here, we assessed the effect of CMV status on alloreactive NK cell reactivity. We compared antibody-mediated NK cell cytolytic activity (CD107a expression) and IFNγ production between healthy CMV-seropositive (n = 8) and CMV-seronegative (n = 11) individuals, in cocultures of NK cells with anti-HLA class I or rituximab (control) antibody-coated Raji cells. First, we showed that within the NKG2C+ NK cells, it is specifically the NKG2C+/A- subset that is enriched in CMV+ individuals. We then observed that in particular the NK cell antibody-dependent cell mediated cytotoxicity (ADCC), but also non-ADCC alloreactivity toward HLA-positive target cells was increased in CMV+ individuals as compared to CMV- ones. This enhanced ADCC as well as non-ADCC NK cell reactivity in CMV+ individuals was particularly characterized by a significantly higher number of ILT2+ and NKG2C+ NK cells that possessed cytolytic activity and/or produced IFNγ in response to HLA-positive target cells. With regard to organ transplantation, these data suggest that CMV infection enhances NK cell alloreactivity, which may pose an additional adverse effect on graft survival, especially in the presence of donor specific antibodies.

In Vitro Neutralization Is Not Predictive of Prophylactic Efficacy of Broadly Neutralizing Monoclonal Antibodies CR6261 and CR9114 against Lethal H2 Influenza Virus Challenge in Mice.

Influenza viruses of the H1N1, H2N2, and H3N2 subtypes have caused previous pandemics. H2 influenza viruses represent a pandemic threat due to continued circulation in wild birds and limited immunity in the human population. In the event of a pandemic, antiviral agents are the mainstay for treatment, but broadly neutralizing antibodies (bNAbs) may be a viable alternative for short-term prophylaxis or treatment. The hemagglutinin stem binding bNAbs CR6261 and CR9114 have been shown to protect mice from severe disease following challenge with H1N1 and H5N1 and with H1N1, H3N2, and influenza B viruses, respectively. Early studies with CR6261 and CR9114 showed weak in vitro activity against human H2 influenza viruses, but the in vivo efficacy against H2 viruses is unknown. Therefore, we evaluated these antibodies against human- and animal-origin H2 viruses A/Ann Arbor/6/1960 (H2N2) (AA60) and A/swine/MO/4296424/06 (H2N3) (Sw06). In vitro, CR6261 neutralized both H2 viruses, while CR9114 only neutralized Sw06. To evaluate prophylactic efficacy, mice were given CR6261 or CR9114 and intranasally challenged 24 h later with lethal doses of AA60 or Sw06. Both antibodies reduced mortality, weight loss, airway inflammation, and pulmonary viral load. Using engineered bNAb variants, antibody-mediated cell cytotoxicity reporter assays, and Fcγ receptor-deficient (Fcer1g-/-) mice, we show that the in vivo efficacy of CR9114 against AA60 is mediated by Fcγ receptor-dependent mechanisms. Collectively, these findings demonstrate the in vivo efficacy of CR6261 and CR9114 against H2 viruses and emphasize the need for in vivo evaluation of bNAbs.IMPORTANCE bNAbs represent a strategy to prevent or treat infection by a wide range of influenza viruses. The evaluation of these antibodies against H2 viruses is important because H2 viruses caused a pandemic in 1957 and could cross into humans again. We demonstrate that CR6261 and CR9114 are effective against infection with H2 viruses of both human and animal origin in mice, despite the finding that CR9114 did not display in vitro neutralizing activity against the human H2 virus. These findings emphasize the importance of in vivo evaluation and testing of bNAbs.

Innate and adaptive type 2 immunity in lung allergic inflammation.

Allergic inflammation is a type 2 immune disorder classically characterized by high levels of immunoglobulin E (IgE) and the development of Th2 cells. Asthma is a pulmonary allergic inflammatory disease resulting in bronchial hyper-reactivity. Atopic asthma is defined by IgE antibody-mediated mast cell degranulation, while in non-atopic asthma there is no allergen-specific IgE and more involvement of innate immune cells, such as basophils, group 2 innate lymphoid cells (ILC2), and eosinophils. Recently, protease allergens were shown to cause asthmatic responses in the absence of Th2 cells, suggesting that an innate cell network (IL-33/TSLP-basophil-ILC2-IL-5/IL-13 axis) can facilitate the sensitization phase of type 2 inflammatory responses. Recent evidence also indicates that in the chronic phase, these innate immune cells directly or indirectly contribute to the adaptive Th2 cell responses. In this review, we discuss the role of Th2 cytokines (IL-4 and IL-13) and innate immune cells (mast cells, basophils, ILC2s, and dendritic cells) in the cross-talk between innate and adaptive inflammatory responses.