Antibody-based Immunotherapy Research Articles

Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Death-Ligand 1 (PD-1/PD-L1) Interaction via Transiently Induced Protein States and Dimerization of PD-L1.

Blockade of the PD-1/PD-L1 immune checkpoint pathway with monoclonal antibodies has provided significant advances in cancer treatment. The antibody-based immunotherapies carry a number of disadvantages such as the high cost of the antibodies, their limited half-life, and immunogenicity. Development of small-molecule PD-1/PD-L1 inhibitors that could overcome these drawbacks is slow because of the incomplete structural information for this pathway. The first chemical PD-1/PD-L1 inhibitors have been recently disclosed by Bristol-Myers Squibb. Here we present NMR and X-ray characterization for the two classes of these inhibitors. The X-ray structures of the PD-L1/inhibitor complexes reveal one inhibitor molecule located at the center of the PD-L1 homodimer, filling a deep hydrophobic channel-like pocket between two PD-L1 molecules. Derivatives of (2-methyl-3-biphenylyl)methanol exhibit the structures capped on one side of the channel, whereas the compounds based on [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol induce an enlarged interaction interface that results in the open "face-back" tunnel through the PD-L1 dimer.

Acquired IFN\u03b3 resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8+ T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.

Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy.

IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich) and hematological (CD20) cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

Targeting CD157 in AML using a novel, Fc-engineered antibody construct.

Antibody-based immunotherapy represents a promising strategy to eliminate chemorefractory leukemic cells in acute myeloid leukemia (AML). In this study, we evaluated a novel Fc-engineered antibody against CD157 (MEN1112) for its suitability as immunotherapy in AML. CD157 was expressed in 97% of primary AML patient samples. A significant, albeit lower expression level of CD157 was observed within the compartment of leukemia-initiating cells, which are supposed to be the major source of relapse. In healthy donor bone marrow, CD157 was expressed on CD34+ cells. In ex vivo assays, MEN1112 triggered natural killer (NK) cell-mediated cytotoxicity against AML cell lines and primary AML cells. Compared to its parental analogue, the Fc-engineered antibody exhibited higher antibody dependent cellular cytotoxicity responses. Using NK cells from AML patients, we observed heterogeneous MEN1112-mediated cytotoxicity against AML cells, most likely due to well-documented defects in AML-NK cells and corresponding inter-patient variations in NK cell function. Cytotoxicity could not be correlated to the time after completion of chemotherapy. In summary, we could demonstrate that CD157 is strongly expressed in AML. MEN1112 is a promising antibody construct that showed high cytotoxicity against AML cells and warrants further clinical testing. Due to variability in NK-cell function of AML patients, the time of application during the course of the disease as well as combinatorial strategies might influence treatment results.

SC06.04 Immunotherapy of Malignant Pleural Mesothelioma and Thymic Malignancies: The End of the Beginning?

SC06.04 Immunotherapy of Malignant Pleural Mesothelioma and Thymic Malignancies: The End of the Beginning?

Antibody-Based Immunotherapy To Treat and Prevent Infection with Hypervirulent Klebsiella pneumoniae

Hypervirulent Klebsiella pneumoniae (hvKp) strains are predicted to become a major threat in Asia if antibiotic resistance continues to spread. Anticapsular antibodies (Abs) were developed because disseminated infections caused by hvKp are associated with significant morbidity and mortality, even with antibiotic-sensitive strains. K1-serotype polysaccharide capsules (K1-CPS) are expressed by the majority of hvKp strains. In this study, K1-CPS-specific IgG Abs were generated by conjugation of K1-CPS to immunogenic anthrax protective antigen (PA) protein. Opsonophagocytic efficacy was measured in vitro and in vivo by intravital microscopy in murine livers. In vivo protection was tested in murine models, including a novel model for dissemination in hvKp-colonized mice. Protective efficacy of monoclonal antibodies (MAbs) 4C5 (IgG1) and 19A10 (IgG3) was demonstrated both in murine sepsis and pulmonary infection. In hvKp-colonized mice, MAb treatment significantly decreased dissemination of hvKp from the gut to mesenteric lymph nodes and organs. Intravital microscopy confirmed efficient opsonophagocytosis and clearance of bacteria from the liver. In vitro studies demonstrate that MAbs work predominantly by promoting FcR-mediated phagocytosis but also indicate that MAbs enhance the release of neutrophil extracellular traps (NETs). In anticipation of increasing antibiotic resistance, we propose further development of these and other Klebsiella-specific MAbs for therapeutic use.

Panobinostat Induces Upregulation of CD38 and Augments the Anti-Myeloma Efficacy of Daratumumab in Pre-Clinical Models

Background:Immunotherapy with monoclonal antibodies (mAbs) has recently entered the clinical arena in multiple myeloma, including Daratumumab that targets CD38 on malignant plasma cells. The efficacy of mAbs depends on antigen density and expression of accessory ligands on target cells to initiate cell- and complement-dependent effector mechanisms. Here, we investigate the use of the histone deacetylase inhibitor (HDACi) Panobinostat to modulate target antigen expression and ligand profile on myeloma in favor of potent mAb-mediated recognition and destruction. We show that Panobinostat augments CD38 expression specifically on myeloma cells and demonstrate powerful synergy with anti-CD38 mAb Daratumumab in pre-clinical models.Methods:The myeloma cell line MM1.S and primary myeloma cells were treated with titrated doses of Panobinostat (0, 10, 25 nM) and expression of CD38 and a panel of additional target molecules including B-cell maturation antigen (BCMA) and SLAMF7, as well as accessory ligands analyzed by flow cytometry at 24, 48 and 72 hours. Antibody-dependent cellular cytotoxicity (ADCC) against Panobinostat treated and untreated myeloma cells was analyzed at 4 and 20 hours after addition of PBMC at an effector to target ratio of 25:1 in the presence of Daratumumab (1, 10, 50 ug/mL) or an isotype control antibody.Results:We first treated the myeloma cell line MM1.S with Panobinostat and analyzed its direct cytotoxic anti-myeloma effect. Consistent with previous work, the percentage of live MM1.S myeloma cells had decreased to 85% and 50% after 48 hours of exposure to 10 and 25 nM respectively. We analyzed expression of CD38 on residual live, i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 1.5 (10 nM) and 2-fold (25 nM) increase of CD38 expression by mean fluorescence intensity (MFI) compared to baseline levels and untreated control cells. The increase in CD38 expression was already detectable after 24 hours and plateaued between 48 and 72 hours. We confirmed our observation in primary myeloma cells from multiple donors (n=4) and detected an even stronger increase to 2 (10 nM) and 4-fold (25 nM) higher CD38 expression compared to untreated cells at 48 hours. Interestingly, expression of BCMA and SLAMF7 was not increased after Panobinostat treatment at all tested concentrations and time points in both MM1.S and primary myeloma. We confirmed that Panobinostat-induced upregulation of CD38 specifically occurred in myeloma, and neither observed this phenomenon in a panel of leukemia and lymphoma cell lines including Raji (Burkitt) and JeKo-1 (mantle cell), nor on resting/activated primary CD8+ and CD4+ T cells that we isolated from peripheral blood of several donors (n=3).Next, we were interested in determining whether the increase in CD38 expression enabled superior anti-myeloma activity of the anti-CD38 mAb Daratumumab. Panobinostat pre-treatment was done for 48 hours at 10 nM as this is a clinically achievable serum level with currently approved regimens. Indeed, significantly higher ADCC was mediated by Daratumumab at all tested concentrations (1, 10 and 50 ug/mL) against MM1.S that we had exposed to Panobinostat. At 4 hours, ADCC was 45% and 25% in Panobinostat-treated and untreated MM1.S respectively, and at 20 hours, near-complete, >90% ADCC of Panobinostat-pre-treated MM1.S had occurred, whereas only 65% of MM1.S were eliminated by Daratumumab without Panobinostat pre-treatment. These data were confirmed in multiple experiments with MM1.S and PBMC from different donors, and with primary myeloma cells. Experiments to evaluate synergy of Panobinostat and Daratumumab therapy in a xenograft model (NSG/MM1.S) are ongoing.Conclusions:Our data demonstrate that the HDACi Panobinostat induces upregulation of CD38 on myeloma and a subsequent dramatic increase of Daratumumab-mediated ADCC in pre-clinical models. These data suggest that Panobinostat could be used synergistically with Daratumumab in a clinical setting to increase response rates and extend duration of responses to Daratumumab. Panobinostat has a known ability to modulate the transcriptional profile of myeloma cells and our data demonstrate for the first time that this ability can be utilized to augment the therapeutic index of antibody-based immunotherapy in multiple myeloma. DisclosuresPallaud:Novartis: Employment. Lehmann:Novartis: Employment. Hudecek:Novartis: Research Funding.

Glucose-Regulated Protein of 94kDa (Grp94)-Targeted Antibody-Based Combinatorial Immunotherapy for Intrahepatic Cholangiocarcinoma

Glucose-Regulated Protein of 94kDa (Grp94)-Targeted Antibody-Based Combinatorial Immunotherapy for Intrahepatic Cholangiocarcinoma

The antibody aducanumab reduces Aβ plaques in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by deposition of amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain, accompanied by synaptic dysfunction and neurodegeneration. Antibody-based immunotherapy against Aβ to trigger its clearance or mitigate its neurotoxicity has so far been unsuccessful. Here we report the generation of aducanumab, a human monoclonal antibody that selectively targets aggregated Aβ. In a transgenic mouse model of AD, aducanumab is shown to enter the brain, bind parenchymal Aβ, and reduce soluble and insoluble Aβ in a dose-dependent manner. In patients with prodromal or mild AD, one year of monthly intravenous infusions of aducanumab reduces brain Aβ in a dose- and time-dependent manner. This is accompanied by a slowing of clinical decline measured by Clinical Dementia Rating-Sum of Boxes and Mini Mental State Examination scores. The main safety and tolerability findings are amyloid-related imaging abnormalities. These results justify further development of aducanumab for the treatment of AD. Should the slowing of clinical decline be confirmed in ongoing phase 3 clinical trials, it would provide compelling support for the amyloid hypothesis.

An update on antibody-based immunotherapies for Clostridium difficile infection.

Clostridium difficile continues to be one of the most prevalent hospital-acquired bacterial infections in the developed world, despite the recent introduction of a novel and effective antibiotic agent (fidaxomicin). Alternative approaches under investigation to combat the anaerobic Gram-positive bacteria include fecal transplantation therapy, vaccines, and antibody-based immunotherapies. In this review, we catalog the recent advances in antibody-based approaches under development and in the clinic for the treatment of C. difficile infection. By and large, inhibitory antibodies that recognize the primary C. difficile virulence factors, toxin A and toxin B, are the most popular passive immunotherapies under investigation. We provide a detailed summary of the toxin epitopes recognized by various antitoxin antibodies and discuss general trends on toxin inhibition efficacy. In addition, antibodies to other C. difficile targets, such as surface-layer proteins, binary toxin, motility factors, and adherence and colonization factors, are introduced in this review.

Abstract 466: Therapeutic Immunization with an Antiglycosaminoglycan Antibody Reduces Atherosclerotic Lesion Progression in Apolipoprotein E-Deficient Mice

Subendothelial retention of apoB-containing lipoproteins by interaction with glycosaminoglycan-side chains of proteoglycans is considered the key initiating step of atherogenesis. Previously, we characterized the antiatherogenic properties of the chimeric mAb chP3R99, which binds sulfated GAG, inhibits LDL-chondroitin sulfate (CS) association, and abrogates LDL oxidation in vitro and in vivo. In preventive settings, rabbits and mice immunized with this mAb showed reduced atherosclerotic lesions, related with the induction of anti-chondroitin sulfate (CS) antibodies. Now we focus in define the immunization schedule which can induce in apolipoprotein E-deficient (apoE-/-) the highest anti-CS antibody response and the greater reduction of atherosclerotic lesion progression. ApoE - / - mice (6-8 wk. old) fed a chow diet received four s.c. injections of 50 or 200 μg of chP3R99 mAb. Autologous antichondroitin sulfate (CS) antibody response was evaluated in sera by ELISA. Mice who were immunized with 200 μg generated a significant higher response against CS than the ones that received 50 μg. Then, to evaluate the association of the induction of a higher anti-CS response in the atherosclerotic lesion progression, apoE-/- mice fed with a high-fat high-cholesterol diet from 4 to 18 wk. of age, received 6 doses of 50, 100 or 200 μg of the mAb starting when 5% of the aortic area was covered by lesions. Animals were sacrificed and aortas isolated to determine the presence of lesions by histologic studies. Mean aortic lesion areas of 50 and 100 μg chP3R99-treated mice were significant reduced by ~40% in comparison with mice treated with an isotype-matched control mAb. In contrast, 62% reduction in total lesion area was observed in mice treated with 200 μg of chP3R99. Again, there was an association between the level of anti-CS antibody response and the reduction of atherosclerotic lesion progression. In conclusion, this study demonstrated the dose dependence of the anti-CS antibody response and its relationship with the arresting of the atherosclerotic progression induced by chP3R99 mAb immunization. Our results also supports the potential use of this antiglycosaminoglycan antibody-based immunotherapy as a novel approach to target advanced atherosclerosis

Editorial: Biology-Driven Targeted Therapy of Pediatric Soft-Tissue and Bone Tumors: Current Opportunities and Future Challenges.

Editorial: Biology-Driven Targeted Therapy of Pediatric Soft-Tissue and Bone Tumors: Current Opportunities and Future Challenges.

Development of next generation antibody-based immunotherapy for childhood cancer neuroblastoma

BackgroundHigh risk neuroblastoma poses a significant clinical problem in paediatric oncology and new treatment strategies are needed. Antibody therapy specific for neuroblastoma surface molecule GD2 has been shown to improve outcome. Here we develop a new generation antibody-based therapeutic agent named BiTE (Bi-specific T cell Engager) that combines antibody specificity with potent T cell functions. MethodsTo identify the BiTE format with maximum activity against neuroblastoma, BiTEs were constructed with single chain variable fragments (scFv) derived from three different CD3-specific antibodies, scFv from two different GD2-specific antibodies, and three different flexible linkers between these CD3 (ie, T cell) and GD2 (ie, neuroblastoma) binders. Specific binding to CD3-positive and GD2-positive cell lines and cytolytic activity at a range of BiTE concentrations was assessed. FindingsSeven different BiTE formats have been successfully produced and purified. Whereas all seven BiTEs constructed showed binding to GD2-positive and CD3-positive cell lines and absence of binding to target antigen-negative cells, mean fluorescence intensity of CD3 binding (14·6 arbitrary units, 24·6, 255) and of GD2 binding (1366, 4258) indicated a range of affinities for CD3 and GD2. For each BiTE, lysis of GD2-target cells was only observed in the presence of T cells. Similarly, secretion of interferon γ by activated T cells only occurred in a CD3-specific and GD2-specific manner. All seven BiTEs induced GD2-specific cytolytic activity. Cytolytic activity was achieved at the lowest EC50 (ie, <1 ng/mL BiTE) for the BiTE format with highest GD2 affinity and moderate CD3 affinity. InterpretationOur findings demonstrate that the optimum BiTE design for tumour targeting can be identified by functional testing of BiTE formats incorporating CD3-specific scFvs and tumour antigen-specific scFvs with a range of binding affinities. Our optimum BiTE construct shows remarkable in-vitro activity approaching that of blinatumomab, which has proven clinical activity against leukaemia; we aim to develop this BiTE into a new therapeutic agent for neuroblastoma. FundingGreat Ormond Street Hospital Institute of Child Health Biomedical Research Centre, Wellcome Trust.

Bubble-Assisted Ultrasound: Application in Immunotherapy and Vaccination.

Bubble-assisted ultrasound is a versatile technology with great potential in immunotherapy and vaccination. This technology involves the exposure of immune cells (i.e., dendritic cells, lymphocytes) in-vitro or diseased tissues (i.e., brain, tumor) in-vivo to ultrasound treatment with gas bubbles. Bubble destruction leads to physical forces that induce the direct delivery of weakly permeant immuno-stimulatory molecules either into the cytoplasm of immune cells, or through the endothelial barrier of diseased tissues. Hence, therapeutic antibodies (i.e., antibody-based immunotherapy) and cytokine-encoding nucleic acids (i.e., cytokine gene therapy) can be successfully delivered into diseased tissues, thus improving immune responses. In addition, protein antigens, as well as antigen-encoding nucleic acids (pDNA, mRNA), can be delivered into dendritic cells (i.e., dendritic cell-based vaccines), thus leading to a long-lasting prophylactic or therapeutic immunization. This chapter focuses on the state-of-the-art of bubble-assisted ultrasound in the field of immunotherapy and vaccination.

Involvement of HMGB1 in Resistance to Tumor Vessel-Targeted, Monoclonal Antibody-Based Immunotherapy.

High mobility group box 1 (HMGB1) is a member of the “danger associated molecular patterns” (DAMPs) than can localize in various compartments of the cell (from the nucleus to the cell surface) and subserve different functions accordingly. HMGB1 is implicated in maintenance of genomic stability, autophagy, immune regulation, and tumor growth. HMGB1-induced autophagy promotes tumor resistance to chemotherapy, as shown in different models of malignancy, for example, osteosarcoma, leukemia, and gastric cancer. To the best of our knowledge, there is virtually no information on the relationships between HMGB1 and resistance to immunotherapy. A recent study from our group has shed new light on this latter issue. We have demonstrated that targeting of tumor-derived endothelial cells with an anti-human CD31 monoclonal antibody in a human neuroblastoma model was unsuccessful due to a complex chain of events involving the participation of HMGB1. These results are discussed in detail since they provide the first evidence for a role of HMGB1 in resistance of tumor cells to monoclonal antibody-based immunotherapy.

Antibody Therapies in Cancer.

Antibody-based immunotherapy has become a standard treatment for a variety of cancers. Many well-developed antibodies disrupt signaling of various growth factor receptors for the treatment of a number of cancers by targeting surface antigens expressed on tumor cells. In recent years, a new family of antibodies is currently emerging in the clinic, which target immune cells rather than cancer cells. These immune-targeted therapies strive to augment antitumor immune responses by antagonizing immunosuppressive pathways or providing exogenous immune-activating stimuli, which have achieved dramatic results in several cancers. The future of cancer therapies is likely to combine these approaches with other treatments, including conventional therapies, to generate more effective treatments.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Detection of antibody therapy-induced anti-tumor immune responses using anti-CD8 immuno-pet

Tumor heterogeneity and the dynamic tumor immune microenvironment have become important topics in the field of cancer immunotherapy. The ability to noninvasively monitor immune cells in vivo via surface markers on immune cell subsets using immuno-positron emission tomography (immuno-PET) is an attractive means of visualizing both systemic and intratumoral alterations in immune cell numbers and localization during experimental immunotherapies. Due to the critical role of tumor-infiltrating cytotoxic CD8+ T cells in anti-tumor immune responses, a radiolabeled anti-CD8 antibody fragment (cys-diabody) was developed for immuno-PET detection of cytotoxic CD8+ T cells in vivo. The anti-CD8 cys-diabody radiolabeled with 89Zr using the bifunctional chelator deferoxamine-maleimide enabled visualization of the spleen and lymph nodes in normal mice; specificity was confirmed in CD8-blocking studies. Next, anti-CD8 immuno-PET was shown to specifically detect tumor-infiltrating lymphocytes in immunotherapy models including agonistic antibody therapy (anti-CD137/4-1BB) and checkpoint blockade antibody therapy (anti-PD-L1). Balb/c mice bearing week old syngeneic CT26 colorectal tumors were treated with either anti-CD137 or anti-PD-L1 antibody therapy and imaged at day 8 post-therapy. All mice treated with anti-CD137 therapy demonstrated tumor regression and increased anti-CD8 immuno-PET targeting in the tumors of mice treated with anti-CD137 therapy compared to the tumor of untreated mice. Ex vivo biodistribution confirmed enhanced tumor uptake of anti-CD137 treated versus untreated mice (15 ± 5.5 %ID/g versus 6.0 ± 0.83 %ID/g, respectively). Anti-CD8 immuno-PET of untreated mice demonstrated a rim of activity around the tumor while anti-CD137 treated mice demonstrated enhanced intratumoral uptake. The increase in CD8+ tumor-infiltrating lymphocytes was validated by both flow cytometry and immunohistochemistry. About 30% of mice treated with anti-PD-L1 therapy demonstrated tumor regression and were grouped into non-responders (average tumor diameter > 8mm) and responders (average tumor diameter < 8mm). Anti-PD-L1 non-responders and responders had tumor anti-CD8 immuno-PET uptake of 6.4 ± 1.5 %ID/g and 11.27 ± 2.7 %ID/g as determined from ex vivo biodistribution analysis, respectively, demonstrating enhanced CD8+ T cell infiltration in responding mice that was confirmed by flow cytometry. Anti-CD8 immuno-PET was successful in the noninvasive detection of tumor-infiltrating CD8+ T cells in two differing antibody-based immunotherapy strategies that have shown promise in the clinic.

Structural mechanism of high affinity FcγRI recognition of immunoglobulin G.

Antibody-based immunotherapies are becoming powerful means of modern medicine for treating cancers and autoimmune diseases. The increasing popularity of antibody-based treatment demands a better understanding of antibody functions and in particular, their interaction with Fc receptors as effectiveness of antibodies often depends on their ability to activate or avoid effector cell functions through Fc receptors. Until recently, our understanding of antibody recognition by Fc receptors is based on the structures of low affinity Fc receptor in complex with Fc. These structural studies provided significant insights to our understanding of how an IgG antibody generally docks on Fcγ receptor and the requirement of immune complex formation for effector cell activations. They are less informative, however, to the molecular forces underlying the vast different affinities between antibodies and their Fcγ receptors. Recently, the structure of the high affinity FcγRI in complex with IgG-Fc has been determined. This review will focus on the knowledge learned from the high affinity complex structural work and a potential receptor-glycan interaction as an important contribution to the receptor affinity.

Arresting progressive atherosclerosis by immunization with an anti-glycosaminoglycan monoclonal antibody in apolipoprotein E-deficient mice

Atherogenesis is associated with the early retention of low-density lipoproteins (LDL) in the arterial intima by interaction with glycosaminoglycan (GAG)-side chains of proteoglycans. Retained LDL undergo reactive oxygen species-mediated oxidation. Oxidized LDL trigger oxidative stress (OS) and inflammation, contributing to atherosclerosis development. Recently, we reported the preventive anti-atherogenic properties of the chimeric mouse/human monoclonal antibody (mAb) chP3R99-LALA, which were related to the induction of anti-chondroitin sulfate antibody response able to inhibit chondroitin sulfate dependent LDL-enhanced oxidation. In the present work, we aimed at further investigating the impact of chP3R99-LALA mAb vaccination on progressive atherosclerosis in apolipoprotein E-deficient mice (apoE−/−) fed with a high-fat high-cholesterol diet receiving 5 doses (50µg) of the antibody subcutaneously, when ~5% of the aortic area was covered by lesions. Therapeutic immunization with chP3R99-LALA mAb halted atherosclerotic lesions progression. In addition, aortic OS was modulated, as shown by a significant (p<0.05) reduction of lipid and protein oxidation, preservation of antioxidant enzymes activity and reduced glutathione, together with a decrease of nitric oxide levels. chP3R99-LALA mAb immunization also regulated aortic NF-κB activation, diminishing the proinflammatory IL1-β and TNF-α gene expression as well as the infiltration of macrophages into the arterial wall. The therapeutic immunization of apoE−/− with progressive atheromas and persistent hypercholesterolemia using chP3R99-LALA mAb arrested further development of lesions, accompanied by a decrease of aortic OS and NF-κB-regulated pro-inflammatory cytokine gene expression. These results contribute to broaden the potential use of this anti-GAG antibody-based immunotherapy as a novel approach to target atherosclerosis at different phases of progression.