Human Isotype Research Articles

Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity.

SUMMARYKinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor. We show that the KLC inhibits the kinesin-microtubule interaction independently from the proposed intramolecular interaction within KHC. Cargo-adaptor proteins that bind the KLC stimulated processive movement, but the landing rate of activated kinesin complexes remained low. Microtubule-associated protein 7 (MAP7) enhanced motility by increasing the landing rate and run length of the activated kinesin motors. Our results support a model whereby the motor activity of the kinesin is regulated by synergistic inhibition mechanisms and that cargo-adaptor binding to the KLC releases both mechanisms. However, a non-motor MAP is required for robust microtubule association of the activated motor. Thus, human kinesin is regulated by synergistic autoinhibition and activation mechanisms.

Computational study of interactions of anti-cancer drug eribulin with human tubulin isotypes.

Microtubules (MTs) are widely targeted for the treatment of various types of cancer due to their essential role in cell division. MTs are polymers made of αβ-tubulin heterodimers. These α- and β-tubulins have 8 and 10 different isotypes, respectively. It is known that a few tubulin isotypes have anti-cancer drug resistance properties, especially βIII, which shows poor sensitivity to many potent anti-cancer drugs such as eribulin. However, the molecular-level understanding of drug-resistance due to tubulin isotype variation is poorly understood. This paper presents the study of differential binding affinities of different tubulin isotypes with the potent anti-cancer drug eribulin. Eribulin (MT destabilizer) binds at the inter-dimer interface of MTs near the vinca site and induces a lattice deformation, which results in catastrophic events in MT dynamics. In this study, sequence analysis has been done throughway and the binding sites and based on that 2α-tubulin isotypes (αI and αVIII) and 7β tubulin isotypes (βI, βIIa, βIII, βIVa, βVI, βVII and βVIII) were selected. In total, 14 combinations were prepared after building homology models of these selected isotypes. Molecular docking and molecular dynamics simulations were performed to deeply understand the binding mode of eribulin at different MT compositions. RMSD, RMSF, radius of gyration, SASA, ligand-protein interactions, and calculations of binding free energy were performed to investigate the eribulin binding variations to tubulin isotypes and it was found that αIβII showed the maximum binding affinity among all 14 systems to eribulin. The βIII-tubulin isotype, which shows low sensitivity to eribulin in experimental results, had the least binding affinity in the system αVIIIβIII complex and the average binding affinity in the system αIβIII among all 14 systems. Additionally, we performed steered MD simulations and DynDom analysis of the systems with the lowest binding energy (αIβII) and the highest binding energy (αVIIIβIII) and extracted force, displacement, and H-bonding profiles during the pulling simulations to get a better insight.

AO-176, a Differentiated Clinical-Stage Anti-CD47 Antibody, Demonstrates Potent Anti-Tumor Activity across Multiple Preclinical Models of B Cell Neoplasms

Upregulation of CD47, the “don't eat me” signal, on the surface of tumors to evade immune surveillance is a common escape mechanism utilized during hematological malignancy and solid tumor development, progression, and relapse. We recently reported that AO-176, a clinical stage humanized anti-CD47 IgG2 antibody, possesses differentiated characteristics such as preferential binding of tumor cells compared to normal cells, negligible binding to red blood cells, non-ADCC direct tumor killing and elicits immunogenic cell death and DAMP induction, all in addition to single-agent phagocytosis. In vivo, AO-176 has exhibited broad anti-tumor activity in preclinical xenograft models of multiple myeloma (MM), acute myeloid leukemia, T cell acute lymphoblastic leukemia, and Burkitt lymphoma. In this study, the anti-tumor activity of AO-176 in an expanded set of preclinical models of B cell neoplasms was evaluated. We assessed In vivo anti-tumor activity in a diffuse large B cell lymphoma (DLBCL) preclinical xenograft model by inoculating Toledo cells into NSG mice and treating once weekly with either 25 mg/kg AO-176 or human IgG2 isotype control. Treatment with AO-176 resulted in profound tumor shrinkage, achieved complete responses in 8/10 mice, and extended survival for all treated mice through the 46 day dosing period, compared to all isotype control treated tumors reaching endpoint by day 21.Having previously observed significant tumor shrinkage and extension of survival in subcutaneous xenograft models of MM, we sought to evaluate anti-tumor activity of AO-176 in an orthotopic model of MM. Luciferase expressing RPMI-8226 cells were inoculated via intratibial injection into NOD-SCID mice and treated with either 25 mg/kg of AO-176 or human IgG2 isotype control once weekly. AO-176-treated mice showed significant reductions of bioluminescence on study days 7, 21, 35, and 41, and serum paraprotein at study end. Evaluation of bone lesions by x-ray showed significantly reduced average bone lysis score in the AO-176 treatment group at study day 41. We then compared the anti-tumor activity of AO-176 against the second generation proteosome inhibitor carfilzomib in a myeloma xenograft model. AO-176 dosed at 25 mg/kg once weekly achieved 72% TGI, compared to 47% and 27% TGI for tumors treated with 5 mg/kg and 2.5 mg/kg carfilzomib at day 29 post treatment.To elucidate the pathways and processes that may be underpinning the anti-tumor activity of AO-176, we performed bulk RNA sequencing on AO-176 or isotype control-treated tumors harvested at multiple time points from a MM xenograft model we previously reported as exhibiting profound sensitivity to AO-176 in vivo. Murine transcripts from harvested tumor RNA were evaluated to assess differences in immune infiltrate resulting from AO-176 treatment. Days 3 and 7 post treatment with AO-176 showed the greatest number of differentially expressed genes compared to control treated tumors. The top enriched pathway on day 3 was microglia phagocytosis. Principal component analysis of gene expression indicated partitioning of day 3 post AO-176 treatment from the rest of the groups. Furthermore, deconvolution of abundances of infiltrating immune cells using CIBERSORT via TIMER analytical tool showed an enrichment of macrophages relative to other cell types on day 3 post treatment.To extend our RNA sequencing findings, we then sought to evaluate intratumoral immune cell populations after AO-176 treatment in a subcutaneous MM xenograft model. MM cells were inoculated into NOD-SCID mice, then treated with 25 mg/kg AO-176 or human IgG2 isotype control. At 48 hours post treatment, tumors were harvested, and we observed an increase of macrophages in the AO-176 treated tumors, confirming our previous results.In summary, the robust preclinical data in DLBCL and MM warrants further development of AO-176 for treatment of hematological malignancies. AO-176 is being evaluated in phase 1/2 clinical trials for the treatment of patients with solid tumors (NCT03834948) and with MM (NCT04445701). DisclosuresWilson: Arch Oncology: Current Employment. Richards: Arch Oncology: Current Employment. Andrejeva: Arch Oncology: Current Employment. Capoccia: Arch Oncology: Current Employment. Puro: Arch Oncology: Current Employment. Donio: Arch Oncology: Current Employment. Hiebsch: Arch Oncology: Current Employment. Kashyap: Arch Oncology: Current Employment. Pereira: Arch Oncology: Current Employment.

The selection of variable regions affects effector mechanisms of IgA antibodies against CD20

AbstractBlockade of the CD47-SIRPα axis improves lymphoma cell killing by myeloid effector cells, which is an important effector mechanism for CD20 antibodies in vivo. The approved CD20 antibodies rituximab, ofatumumab, and obinutuzumab are of human immunoglobulin G1 (IgG1) isotype. We investigated the impact of the variable regions of these 3 CD20 antibodies when expressed as human IgA2 isotype variants. All 3 IgA2 antibodies mediated antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cellular cytotoxicity (ADCC) by polymorphonuclear cells. Both effector mechanisms were significantly enhanced in the presence of a CD47-blocking antibody or by glutaminyl cyclase inhibition to interfere with CD47-SIRPα interactions. Interestingly, an IgA2 variant of obinutuzumab (OBI-IgA2) was consistently more potent than an IgA2 variant of rituximab (RTX-IgA2) or an IgA2 variant of ofatumumab (OFA-IgA2) in triggering ADCC. Furthermore, we observed more effective direct tumor cell killing by OBI-IgA2 compared with RTX-IgA2 and OFA-IgA2, which was caspase independent and required a functional cytoskeleton. IgA2 variants of all 3 antibodies triggered complement-dependent cytotoxicity, with OBI-IgA2 being less effective than RTX-IgA2 and OFA-IgA2. When we investigated the therapeutic efficacy of the CD20 IgA2 antibodies in different in vivo models, OBI-IgA2 was therapeutically more effective than RTX-IgA2 or OFA-IgA2. In vivo efficacy required the presence of a functional IgA receptor on effector cells and was independent of complement activation or direct lymphoma cell killing. These data characterize the functional activities of human IgA2 antibodies against CD20, which were affected by the selection of the respective variable regions. OBI-IgA2 proved particularly effective in vitro and in vivo, which may be relevant in the context of CD47-SIRPα blockade.

IL‐10 induces IgG4 production in NOD‐scid Il2rγnull mice humanized by engraftment of peripheral blood mononuclear cells

IL‐10 induces IgG4 production in NOD‐<i>scid</i> Il2rγ^null mice humanized by engraftment of peripheral blood mononuclear cells

Discovery of new vascular disrupting agents based on evolutionarily conserved drug action, pesticide resistance mutations, and humanized yeast.

Thiabendazole (TBZ) is an FDA-approved benzimidazole widely used for its antifungal and antihelminthic properties. We showed previously that TBZ is also a potent vascular disrupting agent and inhibits angiogenesis at the tissue level by dissociating vascular endothelial cells in newly formed blood vessels. Here, we uncover TBZ's molecular target and mechanism of action. Using human cell culture, molecular modeling, and humanized yeast, we find that TBZ selectively targets only 1 of 9 human β-tubulin isotypes (TUBB8) to specifically disrupt endothelial cell microtubules. By leveraging epidemiological pesticide resistance data and mining chemical features of commercially used benzimidazoles, we discover that a broader class of benzimidazole compounds, in extensive use for 50 years, also potently disrupt immature blood vessels and inhibit angiogenesis. Thus, besides identifying the molecular mechanism of benzimidazole-mediated vascular disruption, this study presents evidence relevant to the widespread use of these compounds while offering potential new clinical applications.

Enhancement of epidermal growth factor receptor antibody tumor immunotherapy by glutaminyl cyclase inhibition to interfere with CD47/signal regulatory protein alpha interactions.

Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a “don't eat me” signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N‐terminal pyro‐glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell‐mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab‐mediated direct growth inhibition, complement‐dependent cytotoxicity, or Ab‐dependent cell‐mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα‐Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose‐dependent manner, suggesting that pyro‐glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab‐dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte‐mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell‐mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.

Development of a Macrophage-Based ADCC Assay.

Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies.

Accelerating ADCP screening using the iQue® Advanced Flow Cytometry Platform

Abstract Antibody-dependent cellular phagocytosis (ADCP) is an immune process stimulated by monoclonal antibodies (mAbs) that promotes specific recruitment of phagocytes to eliminate targeted cancer cells. In vitro functional assays are used extensively in drug development to characterize the effects of mAbs on a range of mechanisms of action, including ADCP. We describe a high throughput assay for ADCP quantification via co-localization analysis of labeled target cells with CD14 positive effector cells. Target cells were incubated in 96 or 384 well plates with clinically relevant mAbs prior to addition of PBMCs or isolated immune cells (e.g. monocytes or macrophages). Cells were analyzed using the iQue®3 Advanced Flow Cytometer and inbuilt software. Various effector:target (E:T) cell ratios were used to validate the assay. Maximal ADCP of non-adherent Ramos target cells by PBMCs in the presence of Truxima was 43 ± 2% with an E:T ratio of 20:1, reducing to 19 ± 6% with a 5:1 ratio. Further pharmacological data was obtained for adherent AU565 cells, comparing the response between anti-HER2 mAb isotypes. The potency of response to anti-HER2-IgG1 mAb (Trastuzumab) was five times greater than to another native human isotype, anti-HER2-IgA2, with EC50 values of 6.6 ng/mL and 35 ng/mL, respectively. These data are in line with previous characterizations of isotype effector functions which showed increased ADCP functionality with IgG1 compared to IgA2 mAbs. The iQue® platform provides a solution for rapid profiling of ADCP effector function in both adherent and suspension target cell models. This accelerates antibody discovery by highlighting potential efficacy and toxicity of antibody drug candidates, leading to greater productivity and insight.

Novel mutations in TUBB8 expand the mutational and phenotypic spectrum of patients with zygotes containing multiple pronuclei.

After fertilization, parental chromosomes decondense and form pronuclei. During these processes, germ cell genomes merge and give rise to the zygotic genome. Multiple pronuclei (MPN) formation is usually caused by polyspermic fertilization or oocyte-derived meiotic failure, and account for 15-18% of cytogenetically abnormal cases among spontaneous abortions. However, pathogenic gene mutations responsible for human MPN formation still need to be identified. Tubulin β eight class VIII (TUBB8) is the major β-tubulin isotype that assembles the human oocyte spindle. In this study, we identified 3 novel heterozygous missense mutations (c.524T>C [p.V175A], c.10_12delins CTT [p.I4L], and c.1045 G>A [p.V349I]) in TUBB8 that were associated with a new phenotype: MPN in zygotes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). These mutations were found in 3 independent female patients with infertility, and had experienced 2-3 failed IVF/ICSI attempts due to zygotic developmental arrest. These sites are evolutionarily conserved in primate TUBB8 genes as well as in other human β-tubulin isotypes, suggesting that they have important biochemical functions. This finding reveals previously unreported phenotypes caused by TUBB8 mutations and will be helpful for future genetic counseling of infertile patients with MPN.

Engineering the Human Fc Region Enables Direct Cell Killing by Cancer Glycan-Targeting Antibodies without the Need for Immune Effector Cells or Complement.

Murine IgG3 glycan-targeting mAb often induces direct cell killing in the absence of immune effector cells or complement via a proinflammatory mechanism resembling oncotic necrosis. This cancer cell killing is due to noncovalent association between Fc regions of neighboring antibodies, resulting in enhanced avidity. Human isotypes do not contain the residues underlying this cooperative binding mode; consequently, the direct cell killing of mouse IgG3 mAb is lost upon chimerization or humanization. Using the Lewisa/c/x -targeting 88mAb, we identified the murine IgG3 residues underlying the direct cell killing and increased avidity via a series of constant region shuffling and subdomain swapping approaches to create improved ("i") chimeric mAb with enhanced tumor killing in vitro and in vivo. Constant region shuffling identified a major CH3 and a minor CH2 contribution, which was further mapped to discontinuous regions among residues 286-306 and 339-378 that, when introduced in 88hIgG1, recapitulated the direct cell killing and avidity of 88mIgG3. Of greater interest was the creation of a sialyl-di-Lewisa-targeting i129G1 mAb via introduction of these selected residues into 129hIgG1, converting it into a direct cell killing mAb with enhanced avidity and significant in vivo tumor control. The human iG1 mAb, termed Avidimabs, retained effector functions, paving the way for the proinflammatory direct cell killing to promote antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity through relief of immunosuppression. Ultimately, Fc engineering of human glycan-targeting IgG1 mAb confers proinflammatory direct cell killing and enhanced avidity, an approach that could be used to improve the avidity of other mAb with therapeutic potential. SIGNIFICANCE: Fc engineering enhances avidity and direct cell killing of cancer-targeting anti-glycan antibodies to create superior clinical candidates for cancer immunotherapy.

Abstract 1782: N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target

Abstract N-myristoyltransferases (NMTs) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. This activity regulates numerous membrane-bound signal transduction pathways important in cancer biology, and the pan-NMT inhibitor PCLX-001 is in clinical development as a cancer therapy. The physiologic distribution and relative contributions of the two human NMTs, NMT1 and NMT2, remain poorly understood as previous studies used polyclonal antibodies with potential cross-reactivity. We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to the human isotypes of NMT1 and NMT2. These mAbs were used to perform an immunohistochemical (IHC) analysis of the abundance and distribution of NMT1 and NMT2 in normal human breast epithelial samples and a large (n=703) cohort of primary breast adenocarcinomas from the BCIRG001 study. While NMT1 protein was readily identifiable in most normal and transformed breast epithelial tissue, NMT1 abundance was associated with higher overall histologic grade, higher Ki67, and lower hormone expression. While NMT2 protein was readily detected in normal breast epithelial tissue, NMT2 protein was undetectable in the majority of malignant breast cancers, but detectable NMT2 protein correlated with significantly poorer overall survival outcomes (hazard ratio for death 1.36; p &amp;lt; 0.029) and significantly worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. NMT status was unrelated to HER2 status. NMT1 and NMT2 protein abundances were positively correlated with each other. Treatment of cultured breast cancer cells with the pan-NMT inhibitor PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts induced significant dose-dependent tumor growth inhibition in vivo. These results support the further evaluation of NMT immunohistochemistry for selection of patients for NMT inhibitor therapy, and clinical trials of NMT inhibition in breast cancer patients. Citation Format: John R. Mackey, Justine Lai, Utkarsh Chauhan, Wei-Feng Dong, Darryl Glubrect, Sunita Ghosh, Gilbert Bigras, Raymond Lai, Luc G. Berthiaume. N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1782.

Binding modes of cabazitaxel with the different human β-tubulin isotypes: DFT and MD studies.

Taxanes (paclitaxel, docetaxel, cabazitaxel) are anticancer drugs as microtubule inhibitors. Following our previous studies on paclitaxel and docetaxel, in this work, we examine cabazitaxel and compare these three taxenes. The binding interaction of three taxanes with various β-tubulin isotypes is studied by homology modeling, molecular docking, and molecular dynamics simulations. The results show that the effects of docetaxel on βI-tubulin (- 29.5kcal/mol) and of paclitaxel on βIIa-tubulin (- 25.5kcal/mol) are much stronger than their effects on βIII-tubulin (- 17.8kcal/mol and - 8.6kcal/mol, respectively). However, the effect of cabazitaxel on βIII-tubulin (- 23.0kcal/mol) is comparable with that on βI-tubulin (- 24.0kcal/mol) and βIIa-tubulin (- 25.9kcal/mol), consistent with the fact that overexpression of βIII-tubulin increases the drug resistance to paclitaxel and docetaxel, but has little influence for cabazitaxel. This theoretical research supports the use of cabazitaxel for patients who are resistant to the action of paclitaxel and docetaxel.

Structural Dynamics of the Lipid Antigen-Binding Site of CD1d Protein

CD1 molecules present lipid antigens to T-cells in early stages of immune responses. Whereas CD1‒lipid‒T-cell receptors interactions are reasonably understood, molecular details on initial trafficking and loading of lipids onto CD1 proteins are less complete. We present a molecular dynamics (MD) study of human CD1d, the isotype that activates iNKT cells. MD simulations and calculations of properties and Poisson-Boltzmann electrostatic potentials were used to explore the dynamics of the antigen-binding domain of the apo-form, CD1d complexes with three lipid–antigens that activate iNKT cells and CD1d complex with GM2AP, a protein that assists lipid loading onto CD1 molecules in endosomes/lysosomes. The study was done at pH 7 and 4.5, values representative of strongly acidic environments in endosomal compartments. Our findings revealed dynamic features of the entrance to the hydrophobic channels of CD1d modulated by two α helices with sensitivity to the type of lipid. We also found lipid- and pH-dependent dynamic changes in three exposed tryptophans unique to CD1d among the five human CD1 isotypes. On the basis of modelled structures, our data also revealed external effects produced by the helper protein GM2AP only when it interacts in its open form, thus suggesting that the own assistant protein also adapts conformation to association with CD1d.

Dynamic plasticity of the lipid antigen-binding site of CD1d is crucially favoured by acidic pH and helper proteins

CD1 molecules present lipid antigens for recognition by T-cell receptors (TCRs). Although a reasonably detailed picture of the CD1-lipid-TCR interaction exists, the initial steps regarding lipid loading onto and exchange between CD1 proteins remain elusive. The hydrophobic nature of lipids and the fact that CD1 molecules are unable to extract lipids from membranes raise the need for the assistance of helper proteins in lipid trafficking. However, the experimental study of this traffic in the endosomal compartments at which it occurs is so challenging that computational studies can help provide mechanistic insight into the associated processes. Here we present a multifaceted computational approach to obtain dynamic structural data on the human CD1d isotype. Conformational dynamics analysis shows an intrinsic flexibility associated with the protein architecture. Electrostatic properties together with molecular dynamics results for CD1d complexes with several lipids and helper proteins unravel the high dynamic plasticity of the antigen-binding site that is crucially favoured by acidic pH and the presence of helper proteins.

Optimizing TNFR2 antagonism for immunotherapy with tumor microenvironment specificity.

Most approved cancer immunotherapies lack T-regulatory (Treg) or tumor specificity. TNF receptor 2 (TNFR2) antibody antagonism is emerging as an attractive immunotherapy due to its tumor microenvironment (TME) specificity. Here we show that the human TNFR2 receptor is overexpressed on both human tumor cells and on human tumor-residing Tregs, but negligibly expressed on beneficial T effectors (Teffs). Further, we found widespread, if variable, TNFR2 expression on 788 human tumor cell lines from diverse cancer tissues. These findings provided strong rationale for developing a targeted immunotherapy using a TNFR2 antibody antagonist. We designed a novel, human-directed TNFR2 antibody antagonist and tested it for function using three cell-based TME assays. The antagonist showed TME specificity by killing of TNFR2-expressing tumor cells and Tregs, but sparing Teffs, which proliferated. However, the antagonist shuffled between five isoforms, only one of which showed the desirable function. We designed and tested several new chimeric human versions of the antagonist, finding that the IgG2 isotype functioned better than the IgG1 isotype. To further improve function, we introduced targeted mutations to its amino acid sequence to stabilize the natural variability of the IgG2 isotype's hinge. Altogether, our findings suggest that optimal TNFR2 antagonists are of the human IgG2 isotype, have hinge stabilization, and have wide separation of antibody arms to bind to newly synthesized TNFR2 on rapidly growing tumor cells. Antagonistic antibodies with these characteristics, when bound to TNFR2, can form a nonsignaling cell surface dimer that functions with high TME specificity.

Abstract LB-A01: Establishment of a novel prostate cancer bone metastasis model in humanized mice and early efficacy results of pembrolizumab

Abstract Background: Programmed cell death 1 (PD-1) and programmed death ligand 1 (PD-L1) expression is typically low in primary prostate cancer but increases at advanced stages. However, targeting PD-1 or PD-L1 has not proven efficacy in prostate cancer patients, and recent clinical trials are directed to combination therapies. About 85% of advanced prostate cancer patients develop skeletal metastases, and the focus in this context should be addressed to the treatment of these metastases. These efforts have been hindered by the lack of relevant preclinical bone metastasis models in immunocompetent mice. We aimed to establish a prostate cancer bone metastasis model in humanized mice and to assess pembrolizumab efficacy in the established model. Materials and methods: Two million LNCaP human prostate cancer cells were inoculated into tibia bone marrow of male CIEA NOG® mice engrafted with human CD34+ hematopoietic stem cells to generate humanized mice. Serum prostate-specific antigen (PSA) levels were measured at 4 weeks, and the mice were allocated to receive either pembrolizumab (anti-PD-1, Keytruda®, 5 mg/kg, Q5D) or human IgG4 isotype control for 6 weeks. Tumor growth was monitored by measuring serum PSA levels. Tumor-induced bone changes were monitored by measuring serum levels of the bone formation marker N-terminal propeptide of type I procollagen (PINP), and by X-ray imaging of tibia. Changes in quantity of circulating T cells was monitored by flow cytometry. At study termination, tissue samples were collected for histological and immunohistochemical evaluation of tumor-infiltrating lymphocytes (TILs). Results: A tumor take of 90% was observed in the humanized mice evaluated by serum PSA levels at endpoint. No significant differences were observed in serum PSA levels, but in a few mice treated with pembrolizumab, only modest tumor growth was observed. Intratibial LNCaP tumors induced osteoblastic-mixed lesions as evaluated by X-ray imaging. No differences were observed in the bone lesion area or in serum PINP levels between the isotype control and pembrolizumab groups. No changes in circulating levels of CD4+, CD8+, or CD4+CD8+ cells were observed. Histology will be performed to confirm tumor morphology and overall tumor viability, and immunohistochemical analysis will reveal the quantity and location of TILs in the tumor. Conclusions: A novel preclinical model of bone metastasis in humanized mice was established. Intratibial prostate cancer tumors induced osteoblastic mixed bone lesions and increased serum PSA levels, mimicking the clinical situation in patients. Resembling recent clinical findings, no or modest responses with pembrolizumab as monotherapy were observed. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Azusa Tanaka, Philip Dube, Jussi M Halleen, Jenni Bernoulli. Establishment of a novel prostate cancer bone metastasis model in humanized mice and early efficacy results of pembrolizumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A01. doi:10.1158/1535-7163.TARG-19-LB-A01

Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

Recurrent and persistent airway infections remain prevalent in patients with primary immunodeficiency (PID), despite restoration of serum immunoglobulin levels by intravenous or subcutaneous plasma-derived IgG. We investigated the effectiveness of different human Ig isotype preparations to protect mice against influenza when delivered directly to the respiratory mucosa. Four polyvalent Ig preparations from pooled plasma were compared: IgG, monomeric IgA (mIgA), polymeric IgA-containing IgM (IgAM) and IgAM associated with the secretory component (SIgAM). To evaluate these preparations, a transgenic mouse expressing human FcαRI/CD89 within the myeloid lineage was created. CD89 was expressed on all myeloid cells in the lung and blood except eosinophils, reflecting human CD89 expression. Intranasal administration of IgA-containing preparations was less effective than IgG in reducing pulmonary viral titres after infection of mice with A/California/7/09 (Cal7) or the antigenically distant A/Puerto Rico/8/34 (PR8) viruses. However, IgA reduced weight loss and inflammatory mediator expression. Both IgG and IgA protected mice from a lethal dose of PR8 virus and for mIgA, this effect was partially CD89 dependent. Our data support the beneficial effect of topically applied Ig purified from pooled human plasma for controlling circulating and non-circulating influenza virus infections. This may be important for reducing morbidity in PID patients.

COMPARISON OF HUMAN PLATELET LYSATE AND FETAL BOVINE SERUM IN CULTURE MEDIA FOR HUMAN DENTAL PULP STEM CELL PROLIFERATION

Objective: Ex vivo and in vitro cell cultures require a basal medium with added supplements containing growth factors, proteins, and enzymes tosupport attachment, growth, and proliferation. Fetal bovine serum (FBS) is used to supplement cell culture media. However, human platelet lysate(hPL) represents an attractive alternative as it is nonxenogeneic.Methods: Human third molars were collected from six healthy donors (19–35 years old) with no history of regular alcohol consumption or smoking.Human dental pulp stem cells (hDPSCs) at the second passage were divided into two culture media groups, 10% FBS and 5% hPL, as well as a controlgroup after 24 h of serum starvation. A flow cytometry analysis was conducted to measure CD90, CD105, CD73, CD34, CD45, and Human LeukocyteAntigen-DR isotype (HLA-DR). Cellular proliferation was evaluated on days 1, 3, and 5.Results: The flow cytometry analysis revealed that the majority of the cells expressed positive mesenchymal stem cell surface markers, includingCD73 (98.5%), CD90 (98.3%), and CD105 (71.0%), and lacked CD34, CD45, and HLA-DR. There were significant differences among the 5% hPL, 10%FBS, and control groups on days 1, 3, and 5.Conclusion: For a nonxenogeneic culture, 5% hPL can be used as an alternative in culture media for hDPSC proliferation.

More severe OA pathology in a humanized mouse model for APOE-ε4 as compared to APOE-ε3: APOE-isoforms as possible risk factor for inflammatory osteoarthritis development?

Purpose: Apolipoprotein E (APOE) is a protein that is closely involved in lipid metabolism. Absence of APOE results in increased serum levels of low-density lipoprotein (LDL) cholesterol, which we previously described to be associated with the development of osteoarthritis (OA) pathology. However, next to its function in lipid metabolism, APOE has been shown to dampen the innate immune system that is strongly involved in the development of OA pathology. In humans, three isoforms of the APOE gene exist (APOE-ε2, APOE-ε3 and APOE-ε4), with different efficacies in suppressing innate immunity (ε2>ε3>ε4). Therefore not surprisingly, the APOE-ε4 isotype has been associated with various inflammation-mediated diseases, including atherosclerosis, Alzheimer’s disease, and multiple sclerosis. However, whether the various APOE isoforms also affect the development of OA remains unknown. Therefore, in the present study we investigated the development of joint pathology after induction of collagenase-induced OA (CIOA) in APOE-ε3 and APOE-ε4 targeted replacement mice. Methods: Wild type, Apoe-/-, and humanized APOE-ε3 and APOE-ε4 targeted replacement mice in which the murine Apoe gene was replaced by the human APOE-ε3 and APOE-ε4 gene, all received a normal diet. CIOA was induced by intra-articular injection of collagenase and mice were sacrificed at day 42 after induction of the model. Serum cholesterol levels were determined using a colorimetric assay. Cartilage degeneration and osteophyte/ectopic bone formation were scored using Safranin O & Fast Green stained sections, representing the entire depth of the joint. Hematoxylin & eosin-stained sections were used to quantify synovial inflammation and chondrocyte death as the percentage of cartilage containing empty lacunae compared to the total cartilage area. Results: Apoe-/-mice showed markedly increased serum LDL levels as compared to their wild type controls (8.90 mmol/L and 0.40 mmol/L, respectively; p<0.001). Moreover, Apoe-/- mice showed significantly increased thickening of the synovium at end-stage OA. Whereas we did not observe differences in cartilage degeneration, we found a strongly increased ectopic bone formation, mainly in the medial collateral ligament. Next, we determined the effects of various human APOE isotypes on the development of joint pathology after induction of CIOA using humanized APOE-ε3 and APOE-ε4 targeted replacement mice. Both mouse strains showed comparable serum LDL levels, which were not raised as compared to wild type mice. Contralateral control joints showed comparable but low synovial inflammation, cartilage degeneration and osteophyte/ectopic bone formation. Interestingly, after induction of CIOA, APOE-ε4 showed increased synovial inflammation as compared to APOE-ε3 mice. In agreement with this finding, we observed significantly increased degeneration of the articular cartilage, which was associated with an increased chondrocyte death. Finally, we found a significantly increased surface area of ectopic bone and osteophyte formation. Conclusions: High LDL cholesterol levels by Apoe deficiency in mice results in increased synovial thickening and ectopic bone formation in end-stage OA. Mice with the human APOE-ε4 gene develop more severe OA pathology as compared to the APOE-ε3 isotype, which is in line with the decreased potency of APOE-ε4 to dampen innate immune responses. This indicates a possible role for the human APOE isotypes as risk factor for the development of OA that needs further investigation.