Human AB Research Articles

Characterization of New Monoclonal PF4-Specific Antibodies as Useful Tools for Studies on Typical and Autoimmune Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is typically caused by platelet-activating immunoglobulin G (IgG) antibodies (Abs) against platelet factor 4 (PF4) complexed with heparin (H). Much less frequent "autoimmune" HIT is distinguished from typical HIT by platelet activation without heparin and the presence of both anti-PF4/H and anti-PF4 IgG. We developed three murine monoclonal anti-PF4 Abs with a human Fc-part, 1E12, 1C12, and 2E1, resembling autoimmune HIT Abs. To characterize 1E12, 1C12, and 2E1 in comparison to the heparin-dependent monoclonal anti-PF4/H Abs 5B9 and KKO, and polyclonal Abs from patients with typical HIT (group-2) and autoimmune HIT (group-3). Interactions of Abs with PF4 and PF4/H were studied by enzyme-linked-immunosorbent assay, single-molecule force spectroscopy, isothermal titration calorimetry, and dynamic light scattering. Serotonin release assay and heparin-induced platelet activation assay were used to assess platelet activation. The binding sites of monoclonal Abs on PF4 were predicted in silico (MAbTope method). 1C12, 1E12, and 2E1 displayed higher affinity for PF4/H complexes than 5B9 and KKO, comparable to human group-3 Abs. Only 1C12, 1E12, 2E1, and group-3 Abs formed large complexes with native PF4, and activated platelets without heparin. The predicted binding sites of 1C12, 1E12, and 2E1 on PF4 differed from those of KKO and 5B9, but were close to each other. 2E1 exhibited unique bivalent binding, involving its antigen recognition site to PF4 and charge-dependent interactions with heparin. 1C12, 1E12, and 2E1 are tools for studying the pathophysiology of autoimmune HIT. 2E1 provides evidence for a new binding mechanism of HIT Abs.

ANTI-IL-6 ANTIBODY REDUCES ENDOTHELIAL MEDIATED PRO-INFLAMMATORY RESPONSES AND AFTER BINDING OF HLA-SPECIFIC DSA TO ENDOTHELIAL CELLS

Endothelial cell activation and microvascular inflammation are hallmarks of antibody-mediated rejection and expression of activation associated transcripts by intragraft endothelial cells is observed whether or not complement activation is indicated. HLA expression is strongly upregulated by the allograft microvasculature during rejection and is a target for Donor Specific Antibody (DSA) binding. DSA specific for HLA antigens are prevalent, are deleterious to the allograft and both complement-dependent and -independent pathways of DSA-mediated damage have been reported. We have developed an in vitro model of human microvascular endothelial cells to reveal that inflammation-induced HLA-DR expression results in the activation and differentiation of allogeneic CD4+-T into both IL-6-dependent pro-inflammatory Th17 and anti-inflammatory Treg (Taflin C. 2011). Endothelial binding of HLA-II-specific antibodies further increased IL-6 secretion and amplification of pro-inflammatory Th17 and Th1 (Lion J. 2016, Cross AR. 2019). In the current study we have evaluated whether a humanized monoclonal anti-IL-6 antibody (clazakizumab) can alter the outcome of HLA-specific antibody binding to endothelial cells. Human microvascular endothelial cells cells (HMEC-1 and primary renal glomerular cells) were cultured in the presence or absence of non-HLA matched PBMC as described (Taflin C 2011). Soluble factors were assesed by ELISA assay and CD4+-T sub-populations identified by intracellular cytokine labelling. C4d and C5b-9 deposition on endothelial cells were measured in the presence or absence of complement-containing human AB serum. Antibodies specific for HLA antigens were either monoclonal or purified DSA from transplant patient serum or plasma-exchange products. When endothelial cells were co-cultured with PBMC from non-HLA matched donors, addition of clazakizumab strongly reduced IL-6 at 24hrs and the decrease was maintained even after 72hrs. MCP-1 levels were also reduced. Activation of endothelial cells by HLA class I and II antibodies prior to co-culture further increased IL-6 and this was also decreased by clazakizumab. The cell-surface phenotype of endothelial cells in co-cultures was unaltered by clazakizumab whereas differentiation of Th17 and Th1 were both decreased (particularly IFNY+Th1). HLA-specific antibody binding activated complement, and clazakizumab did not have a direct effect on C4d or C5b-9 deposition on endothelial cells Neither steady-state expression of the complement regulatory proteins CD46, CD55 and CD59 nor their expression under conditions of complement activation were altered by clazakizumab. These data indicate that anti-IL-6 antibody may limit endothelial damage mediated by DSA binding to HLA class I or II. The results reveal the ability of clazakizumab to alter endothelial immunogenicity leading to reduced differentiation of pro-inflammatory CD4+-T. Moreover the decreased level of MCP-1 may limit recuitment of myeloid cells within the allograft. Société Française de Transplantation. EU-COST 17138. Vitaeris Inc. Vaincre la Mucoviscidose.

Abstract 6530: Old target, but new approach: Targeting cancer-associated MUC1 with ADC and Car-T

Abstract Purpose: The characteristics of aberrant overexpression and distinct features of cancer-associated (CA)-MUC1 has been often considered as a promising target in most of the solid cancers. MUC1 is composed of α and β subunits to form a full length of MUC1, and α subunit is released and circulated into the blood in cancer, which is used as biomarkers such as CA 15-3 and CA 27-29. As those α subunit-targeting strategies were failed in the numerous clinical trials due to, at least in part, the diminished α subunit of MUC1 in cancer, we have developed a humanized Ab, PAb001 targeting the residual β subunit of MUC1, called as an onco-tethered (OT)-MUC1. To prove the concept of this strategy, an antibody-drug conjugate (ADC) and chimeric antigen receptor (CAR) T cells were applied to assess its efficacies. Methods: We produced a monoclonal antibody (mAb) against OT-MUC1 from mouse and sequentially generated its derivative humanized antibody, PAb001. Then, the target product profiles (TPP) were determined by SPR, ELISA, FACS, immunohistochemical analysis, pilot tissue cross-reactivity, and pharmacokinetics (PK) analysis. Then, to evaluate anti-cancer efficacies, we either conjugated PAb001 with Monomethyl auristatin E (MMAE) or generated the second-generation CAR T cells and then sequentially tested in vitro and in vivo cancer models. Results: Based on the TPP criteria, PAb001 antibody was chosen over a hundred of candidate antibodies tested. Next, it was conjugated with MMAE, treated and measured its cytotoxicity in ovarian and breast cancer cell lines, including triple-negative breast cancers (TNBC). As a result, PAb001-ADC selectively inhibited the growth of cancer cell lines, depending on the expression level of MUC1. Also, it showed 80-100 % tumor growth inhibition rates (TGI) in the cell lines- and patient-derived xenograft models. Lastly, OT-MUC1 Car-T cells showed significant and selective growth inhibition of cancer cells. Conclusion: Taken together, the preclinical studies of PAb001-ADC and Car-T indicate the high therapeutic potentials targeting OT-MUC1 in cancer, which further provided the rationale for clinical development. Citation Format: Jin G Jung, Min-Seok Kim, Hwa-Chul Jung, Hoil Choi, Kyung-Duk Moon. Old target, but new approach: Targeting cancer-associated MUC1 with ADC and Car-T [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 6530.

Identification of Structurally Related Antibodies in Antibody Sequence Databases Using Rosetta-Derived Position-Specific Scoring

The amount of antibody (Ab) variable gene sequence information is expanding rapidly, but our ability to predict the function of Abs from sequence alone is limited. Here, we describe a sequence-to-function prediction method that couples structural data for a single Ab/antigen (Ag) complex with repertoire data. We used a position-specific structure-scoring matrix (P3SM) incorporating structure-prediction scores from Rosetta to identify Ab variable loops that have predicted structural similarity to the influenza virus-specific human Ab CH65. The P3SM approach identified new members of this Ab class. Recombinant Ab expression, crystallography, and virus inhibition assays showed that the HCDR3 loops of the newly identified Abs possessed similar structure and antiviral activity as the comparator CH65. This approach enables discovery of new human Abs with desired structure and function using cDNA repertoires that are obtained readily with current amplicon sequencing techniques.

Anti-influenza H7 human antibody targets antigenic site in hemagglutinin head domain interface.

Although broadly protective, stem-targeted Abs against the influenza A virus hemagglutinin (HA) have been well studied, very limited information is available on Abs that broadly recognize the head domain. We determined the crystal structure of the HA protein of the avian H7N9 influenza virus in complex with a pan-H7, non-neutralizing, protective human Ab. The structure revealed a B cell epitope in the HA head domain trimer interface (TI). This discovery of a second major protective TI epitope supports a model in which uncleaved HA trimers exist on the surface of infected cells in a highly dynamic state that exposes hidden HA head domain features.

Preliminary results of a phase II study of neoadjuvant immune checkpoint inhibitor IMC-001 (anti-PD-L1 monoclonal antibody) in patients with resectable gastrointestinal cancers (NeoChance Study).

e16542 Background: Immune checkpoint inhibitors have shown survival benefits in unresectable/metastatic gastric cancer (GC), esophageal cancer (EC), and hepatocellular carcinoma (HCC). Based on scientific rationale for neoadjuvant immunotherapy, including enhanced immune recognition, we initiated a phase II study of IMC-001, a novel PD-L1 targeting fully human monoclonal Ab in the neoadjuvant setting for resectable GC, EC, and HCC. Methods: This is a prospective, open-label, phase II study of neoadjuvant IMC-001 (20 mg/kg iv every 2 weeks for 2 cycles) across three cohorts of resectable gastrointestinal cancers (GC, EC, HCC). The primary endpoint is major pathologic response rate ( < 10% of viable tumor cells) and secondary endpoints include safety, feasibility, R0 resection rate, clinical tumor response rate/disease control rate (DCR), progression-free survival, relapse-free survival, and overall survival. Exploratory endpoints include immune monitoring and biomarker analysis in tumor tissues, blood, and stool. Results: From Sep. 2019 to Feb. 2020, 14 eligible patients (pts) (6 HCC; 5 GC; 3 EC) were enrolled; male (79%), median age = 63 yrs (range, 40-72), clinical stage (AJCC 8th) I (57%)/II (21%)/III (21%). 12 pts completed 2 cycles of neoadjuvant IMC-001 and one pt stopped after one cycle due to G3 autoimmune hepatitis, which was only G3 adverse event (AE) and resolved with steroid. Other AE included G2 hyperthyroidism (n = 1), G1 pruritus (n = 2), G1 rash (n = 1), G1 myalgia (n = 1), G1 arthralgia (n = 1), G1 diarrhea (n = 1), G1 cough (n = 1), G1 palate discomfort (n = 1), and G1 chest discomfort (n = 1). So far, 10 clinical response-evaluable pts showed a 100% DCR and underwent surgery, which was all R0 resection. Post-treatment surgery specimens showed various degrees of lymphocyte infiltration in intratumoral or peritumoral areas ranging from 10% to 90%, which was increased compared to pre-treatment biopsies. In HCC pts, adjacent hepatic parenchyma showed mild to moderate, diffuse portal inflammation regardless of Hepatitis B virus status and minimal and moderate fatty change. IHC for PD-L1 using 22C3, SP263 and SP142 clones showed similar results among three Abs with relatively low expression regardless of tumor types (range, 0-20%). Conclusions: Neoadjuvant IMC-001 seems to be well tolerated and have preliminary immune modulating activity in resectable GC, EC, and HCC pts. The study is ongoing and the updated clinical and immunologic results will be presented. Clinical trial information: NCT04196465 .

Heterosubtypic human antibody response to influenza H7N9 in survivors of natural infection

Abstract H7N9 avian influenza virus causes severe infections in humans after cross-species transmission from bird reservoirs. Previous studies revealed that infection with H7N9 elicits a heterosubtypic polyclonal antibody (Ab) response between H7 and H3 or H1 hemagglutinins (HAs) in humans. However, relatively little is known about the functional activity, targeted epitopes, and breadth of reactivity of human monoclonal Abs (mAbs) against H7N9. In this study, we isolated 18 H7-reactive mAbs from survivors of natural H7N9 infection. The majority (14 of 18) of H7-reactive mAbs within the panel possessed neutralizing activity, mediated by inhibition of attachment or egress of the virus. Binding analysis of the isolated mAbs against fourteen influenza virus HAs representing group 1 and 2 revealed cross-reactivity for diverse strains within the H7 subtype and an extended response to H15 HA. Seven identified cross-reactive mAbs targeted the HA head domain, including neutralizing and non-neutralizing mAbs. Two neutralizing mAbs that are specific to the HA stem region revealed heterosubtypic reactivity against H7, H15 and H3 HAs. Further analysis identified the epitopes for heterosubtypic mAbs. This study provides insights into the basis for heterosubtypic antibody responses against H7N9 in human infection survivors and may inform efforts to incorporate novel epitope targets in broadly protective influenza vaccines.

Human Platelet Releasate (hPR) - A xeno-free supplement for the expansion of stem cells

Background & Aim Background Recent regulatory guidelines for cell therapy applications recommend the use of xeno-free materials to mitigate the risk of immune reaction to animal-derived additives i.e. fetal bovine serum (FBS). On this note, human platelet lysate (hPL) has been accepted as a xeno-free alternative for stem cell expansion. With several methods available to manufacture hPL, the lack of standardization amongst manufacturers has led to inconsistencies in expansion rate, phenotype, and immunomodulatory properties of stem cells; altering the intrinsic therapeutic potential of cells. Further, some platelet lysates induce “conditioning of cells”, where the cell expansion occurs with a bias towards a lineage of differentiation, as directed by the composition and concentration of growth factors, cytokines and chemokines present in the hPL. We have developed a cGMP process to manufacture human platelet releasate (hPR), a potential alternative to hPL and FBS. hPR is a xeno-free, non-conditioning media supplement derived from expired-human platelets. Methods, Results & Conclusion Methods We have developed a proprietary closed-system manufacturing process designed for optimal extraction of growth factors, cytokines and chemokines (Fig.1). Every lot is tested for sterility, endotoxin, mycoplasma, total protein content, and growth factors. The performance characteristics was evaluated by expanding bone marrow derived MSCs (BM-MSCs) in basal growth medium (DMEM) supplemented with hPR and benchmarked with equivalent strengths of three commercially available non-heparin-requiring human platelet lysates, AB serum, and FBS. Cell proliferation and population doubling time were measured using Luna II automated cell counter. Tri-lineage differentiation potential of these cells was assessed with adipogenesis, chondrogenesis and osteogenesis assay. The phenotypic, and biochemical markers of BM-MSCs were quantified using flow cytometry and multiplex ELISA quantibody assay (Fig.2). The potency of expanded cells was assessed by i) measuring human indoleamine 2, 3-dioxygenase (IDO) using kynurenine assay and luminex assay (IDO human ProcartaPlex), ii) mixed lymphocyte reaction assay (a lab-developed co-culture assay), and regulatory T-cell conversion assay using flow cytometry. Results We have developed a cGMP-compliant process to manufacture consistent lots of hPR. Our results indicate that the potency, and proliferation rate of MSCs is higher in basal medium supplemented with hPR compared to FBS or hPL (Fig.3).

Translation of small-scale CAR-T cell manufacturing methods to a clinical-scale production platform

Background & Aim CAR-T cells have displayed potent antitumor effects in liquid but not in solid tumors. Variables like technical and immuno-oncological factors contribute to the modest clinical effect of CAR-Ts in solid tumors. To improve their function, manufacturing conditions have been optimized to yield a favorable CAR-T cell phenotype. A product enriched in stem cell- and central memory T cells (TCM) is believed to improve durability of antitumor responses and engraftment of the adoptive cells, where highly differentiated T cell products may be prone to exhaustion and senescence. Finding optimal manufacturing conditions requires testing of media components and selection of appropriate bioreactor system for scale-up. To avoid the substantial costs of multiple bioreactor runs, screening typically relies on flask scale. We set out to determine the predictive ability of the flask-scale screening on the process performance in clinical scale. Glioblastoma-specific CAR-Ts were produced from six healthy donors in various medium compositions and compared to those produced in closed-system bioreactors. Methods, Results & Conclusion Lentivirally-transduced CAR-T cells were cultured in flasks in Miltenyi TexMACS medium with or without human AB serum (HABS) and either IL2 or IL7 and IL15. In the bioreactor process, T cells were purified and transduced in the Miltenyi CliniMACS Prodigy system, then further expanded in IL7 and IL15 containing HABS-free media in a suspension bioreactor. We characterized the cells by CAR expression level, viability, expansion rate, memory phenotype and in vitro functionality. All CAR-T products were highly viable and specifically cytotoxic, though biological differences between donors can cause variation. The main difference between culture conditions was in expansion rates. The phenotype was substantially effector memory T cell-biased in all flask cultures. The presence or absence of HABS rather than cytokine selection appeared to be the main determinant of the product characteristics. In our bioreactor scale pilot the lead media composition (IL7, IL15, no HABS) yielded a markedly different product, seen as superior expansion and significant enrichment of the TCM fraction. Our results suggest that small-scale screening may translate poorly to bioreactor systems. Such factors as medium replenishment, oxygenation, cell density and culture vessel rocking may have an impact on the product. Early-stage scale-up and testing using the final platform is essential for product success.

De-risking the immunotherapy supply chain: development of virus inactivated human AB serum for immune cell expansion

Background & Aim FBS and Calf bovine serum from USDA-approved animals are used in a broad range of cell culture applications; they have been demonstrated to be safe and effective growth stimuli. However, the use of xenogenic serum always implies a certain degree of risk associated with a pathogen-driven immune response or the unwanted incorporation of foreign proteins into human primary cells, such as stem cells, T cells and NK cells. These risks have driven demand for xenogeneic-free culture media for clinical handling of these cells. Akron Biotech has developed several off-the-shelf AB serum options that allow cell therapy developers to avoid the risk inherent in xenogenic sera. Akron's latest development is the development of a virus inactivated human AB serum that incorporates solvents/detergents to improve the safety of this critical supplement for immunotherapy development without compromising its efficacy in promoting cell growth. Methods, Results & Conclusion Methods Process development schematic figure is attached. The biochemical profile and quality of AB serum (native components #1, #2, and #3 as well as additional components) batches were analyzed. Their ability to support cell growth after two days cell culture was evaluated by crystal violet assay and cell proliferation assay. Cell lines: 293T, Jurkat T cell and PBMC-derived T cells. Results The three processed serum sources (#1, #2, #3) were demonstrated to effectively promote cell growth. The three cell lines performed similarly or better than converted AB serum. Overall, our results indicate that virus inactivated AB serum is a safe and efficient material for inducing cell proliferation that does not negatively impact cell quality. Given the wellunderstood problems associated with using FBS and other animal-derived materials, we believe that our virus inactivated AB serum, developed through extensive process development, is the safest alternative for the proliferation of cells critical to immunotherapy development and clinical application.

High yield of human umbilical cord-derived mesenchymal stromal cells in an animal component-free culture medium

Background & Aim Mesenchymal stromal cells (MSCs) can be isolated from adult and new born tissue sources. Unlike adult bone marrow, the collection of human umbilical cord (UC) tissue offers the advantage of being noninvasive. MSCs isolated from UC tissues are also not negatively impacted by advanced age and certain diseases, which serve their banking for future clinical applications. We have optimized MesenCult™-ACF Plus (MACFP), an improved animal component-free (ACF) culture medium for the efficient derivation and expansion of MSCs from UC tissue. Methods, Results & Conclusion Wharton's Jelly was dissected from the UC, cut into 1-2 mm3 fragments and placed into individual wells of a 24-well pre-coated tissue culture plate in UC-MesenCult™-ACF Plus (UC-MACFP) medium supplemented with an optimized cytokine cocktail, or in control medium containing fetal bovine serum (FBS). MSCs migrated from the explanted fragments in 10-14 days and were replated at 1.5–3.0 × 103 cells/cm2 for long-term expansion in each medium. The proliferative potential of UC-derived MSCs was measured by counting cells at each passage (P) up to P6 in both media and their phenotype was assessed. MSC differentiation potential was examined by culturing these cells in adipogenic and osteogenic differentiation media. The average yield of MSCs from UC was higher in UC-MACFP than in FBS medium (9.9±3.4 × 105 vs. 4.4±1.8 × 105 cells/g [mean ± SEM]; n=11 and 5, respectively). Cell outgrowth was further increased to 2.6±1.0 × 106 cells/g (n=9) with addition of 2.5% human AB serum to UC-MACFP medium. The average fold-expansion of UC-MSCs at each subculture over 6 passages was comparable in UC-MACFP (13.5 ± 0.9; n=13) and in FBS media (12.8±0.6; n=6). The phenotype of MSCs was similar in both UC-MACFP and FBS media, with >95% of cells positively expressing CD105, CD73 and CD90, and

Successful Use of Human AB Serum to Support the Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cell in a Microcarrier-Based Platform.

Mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and for the promotion of tissue repair, hence the increase of clinical trials in a worldwide scale. In particular, adipose tissue-derived stem/stromal cells (AT MSC) present easy accessibility and a rather straightforward process of isolation, providing a clear advantage over other sources. The high demand of cell doses (millions of cells/kg), needed for infusion in clinical settings, requires a scalable and efficient manufacturing of AT MSC under xenogeneic(xeno)-free culture conditions. Here we describe the successful use of human AB serum [10%(v/v)] as a culture supplement, as well as coating substrate for the expansion of these cells in microcarriers using (i) a spinner flask and (ii) a 500-mL mini-bioreactor (ApplikonTM Biotechnology). Cells were characterized by immunophenotype and multilineage differentiation potential. Upon an initial cell adhesion in the spinner flask of 35 ± 2.5%, culture reached a maximal cell density of 2.6 ± 0.1 × 105 at day 7, obtaining a 15 ± 1-fold increase. The implementation of the culture in the 500-mL mini-bioreactor presented an initial cell adhesion of 22 ± 5%, but it reached maximal cell density of 2.7 ± 0.4 × 105 at day 7, obtaining a 27 ± 8-fold increase. Importantly, in both stirred systems, cells retained their immunophenotype and multilineage differentiation potential (osteo-, chondro- and adipogenic lineages). Overall, the scalability of this microcarrier-based system presented herein is of major importance for the purpose of achieving clinically meaningful cell numbers.

A New Humanized Mouse Model Mimics Humans in Lacking α-Gal Epitopes and Secreting Anti-Gal Antibodies

Mice have been used as accepted tools for investigating complex human diseases and new drug therapies because of their shared genetics and anatomical characteristics with humans. However, the tissues in mice are different from humans in that human cells have a natural mutation in the α1,3 galactosyltransferase (α1,3GT) gene and lack α-Gal epitopes on glycosylated proteins, whereas mice and other nonprimate mammals express this epitope. The lack of α-Gal epitopes in humans results in the loss of immune tolerance to this epitope and production of abundant natural anti-Gal Abs. These natural anti-Gal Abs can be used as an adjuvant to enhance processing of vaccine epitopes to APCs. However, wild-type mice and all existing humanized mouse models cannot be used to test the efficacy of vaccines expressing α-Gal epitopes because they express α-Gal epitopes and lack anti-Gal Abs. Therefore, in an effort to bridge the gap between the mouse models and humans, we developed a new humanized mouse model that mimics humans in that it lacks α-Gal epitopes and secretes human anti-Gal Abs. The new humanized mouse model (Hu-NSG/α-Galnull) is designed to be used for preclinical evaluations of viral and tumor vaccines based on α-Gal epitopes, human-specific immune responses, xenotransplantation studies, and in vivo biomaterials evaluation. To our knowledge, our new Hu-NSG/α-Galnull is the first available humanized mouse model with such features.

Novel Abs targeting the N-terminus of fibroblast growth factor19 inhibit hepatocellular carcinoma growth without bile-acid-related side-effects.

Hepatocellular carcinoma (HCC) is a common and particularly fatal form of cancer for which very few drugs are effective. The fibroblast growth factor 19 (FGF19) has been viewed as a driver of HCC development and a potential Ab target for developing novel HCC therapy. However, a previously developed anti‐FGF19 Ab disrupted FGF19’s normal regulatory function and caused severe bile‐acid‐related side‐effects despite of having potent antitumor effects in preclinical models. Here, we developed novel human Abs (G1A8 and HS29) that specifically target the N‐terminus of FGF19. Both Abs inhibited FGF19‐induced HCC cell proliferation in vitro and significantly suppressed HCC tumor growth in mouse models. Importantly, no bile‐acid‐related side effects were observed in preclinical cynomolgus monkeys. Fundamentally, our study demonstrates that it is possible to target FGF19 for anti‐HCC therapies without adversely affecting its normal bile acid regulatory function, and highlights the exciting promise of G1A8 or HS29 as potential therapy for HCC.

The effects of Japanese encephalitis virus antibodies on Zika virus infection

Recently, Zika virus (ZIKV) has become more widespread, thus attracting global attention. The vaccine against Japanese encephalitis virus (JEV) is currently used in China, being included in planned immunisation regimes. Although ZIKV and JEV are closely related mosquito-borne Flaviviruses, and a complex cross-immune response within flaviviruses has been demonstrated, the effect of JEV vaccination on ZIKV infection has not been well described. Thus, this study aimed to explore the impact of different titres of anti-JEV antibodies (Abs) against ZIKV infection using sera from healthy human donors in Guangzhou and anti-JEV rabbit polyclonal antibodies (pAbs) in vitro and vivo. Human anti-JEV Ab titres were tested at decreasing concentrations as the age increased. A neutralising effect on ZIKV infection was observed when anti-JEV Ab titres in human sera or rabbit pAbs were high (the corresponding age was under 30years). Even though a lower titre in human sera showed no apparent effect, whereas rabbit pAbs had an antibody-dependent enhancement(ADE)effect, we proved an ADE effect in vivo for the first time. This study suggests that individuals over 60years of age are at high risk for JEV and ZIKV infection, and screening this age group for infection should strengthen. Furthermore, a deep exploration of the relationship between anti-JEV Abs and ZIKV infection is needed.

Lesional Inflammatory Profile in Hidradenitis Suppurativa Is Not Solely Driven by IL-1

Lesional Inflammatory Profile in Hidradenitis Suppurativa Is Not Solely Driven by IL-1

A LAG-3-Specific Agonist Antibody for the Treatment of T Cell-Induced Autoimmune Diseases.

T cells chronically stimulated with the same peptide tend to express exhaustion markers such as PD-1 or LAG-3. Deficiencies in the PD-1 and LAG-3 pathways have been linked to the development of autoimmune diseases. IMP761 is a LAG-3-specific humanized agonist Ab with immunosuppressive properties both in vitro and in vivo in an Ag-specific delayed-type hypersensitivity (DTH) model in the cynomolgus macaque (Macaca fascicularis). IMP761 inhibits TCR-mediated NFAT activation and Ag-induced human T cell proliferation and activation. In the DTH model, assessment of T cell infiltration and gene expression profile at the DTH biopsy site corresponds to immunosuppression of an Ag-induced T cell response. IMP761 is the first LAG-3-specific agonist product candidate, acting upstream on activated T cells, the root cause of self-Ag-specific T cell-induced autoimmune diseases.

Profiles of Long Non-Coding RNAs and mRNA Expression in Human Macrophages Regulated by Interleukin-27.

Macrophages play an essential role in the immune system. Recent studies have shown that long non-coding RNAs (lncRNAs) can regulate genes encoding products involved in the immune response. Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines with broad anti-viral effects that inhibits human immunodeficiency virus (HIV) type-1 and herpes simplex virus (HSV). However, little is known about the role of lncRNAs in macrophages affected by IL-27. Therefore, we investigated the expression profiles of mRNA and lncRNA in human monocyte-derived macrophages (MDMs) regulated by IL-27. Monocytes were differentiated in the presence of macrophage-colony stimulatory factor (M-CSF)- or human AB serum with or without IL-27, and these cells were the subject for the profile analysis using RNA-Seq. We identified 146 lncRNAs (including 88 novel ones) and 434 coding genes were differentially regulated by IL-27 in both M-CSF- and AB serum-induced macrophages. Using weighted gene co-expression network analysis, we obtained four modules. The immune system, cell cycle, and regulation of complement cascade pathways were enriched in different modules. The network of mRNAs and lncRNAs in the pathways suggest that lncRNAs might regulate immune activity in macrophages. This study provides potential insight into the roles of lncRNA in macrophages regulated by IL-27.

Abstract B057: BMS-986148, an anti-mesothelin antibody-drug conjugate (ADC), alone or in combination with nivolumab demonstrates clinical activity in patients with select advanced solid tumors

Abstract Background: Mesothelin, a GPI-anchored cell surface protein, is highly expressed in several tumor types and can be targeted for antibody (ab)-based cancer therapy. BMS-986148 is a fully human IgG1 anti-mesothelin monoclonal ab conjugated to tubulysin to promote selective cytotoxic delivery to tumor cells. In preclinical models, combination of anti–mesothelin-tubulysin with anti–PD-1 promoted a synergistic antitumor response and influx of tumor-infiltrating lymphocytes. Here, we present initial data for BMS-986148 ± nivolumab (NIVO; anti–PD-1) from a phase 1/2a trial in a biomarker-defined population of patients (pts) with select advanced solid tumors (NCT02341625). Methods: During dose escalation (ESC), pts received BMS-986148 0.1 to 1.6 mg/kg IV Q3W, BMS-986148 0.4 or 0.8 mg/kg IV Q1W, or BMS-986148 0.8 mg/kg + NIVO 360 mg IV Q3W. During monotherapy (mono) dose expansion (EXP), pts with mesothelin-selected mesothelioma (ie, H score ≥ 100 for tumor mesothelin expression), non-small cell lung cancer (NSCLC), or ovarian cancer received BMS-986148 1.2 mg/kg mono IV Q3W, and during combination (combo) dose EXP, pts with mesothelin-selected mesothelioma or pancreatic cancer received BMS-986148 0.8 mg/kg + NIVO 360 mg combo IV Q3W. The primary endpoint was safety. Secondary endpoints included pharmacokinetics (PK) and antitumor activity. Results: As of April 1, 2019, 126 pts have been treated in this trial; 96 pts received BMS-986148 mono (Q3W, n = 84; Q1W, n = 12), and 30 pts received BMS-986148 + NIVO combo. PK analysis in pts treated during ESC demonstrated that total antibody and active ADC exposures increased in a dose-proportional manner. Unconjugated tubulysin concentrations were low but sustained over the dosing interval. The maximum tolerated dose (MTD) for mono was 1.2 mg/kg Q3W, and the 0.8 mg/kg dose was determined to be tolerable for the combo. Across all cohorts, any-grade and grade 3/4 treatment-related AEs (TRAEs) were reported in 87% and 44% of all pts, respectively. Overall, the most common TRAEs (any grade; grade 3-4) reported in ≥ 15% of pts were AST increased (43%; 17%), ALT increased (41%; 16%), fatigue (37%; 5%), nausea (29%; 0%), decreased appetite (22%; 1%), and blood alkaline phosphatase increased (18%; 4%). The frequency of TRAEs and dose-limiting toxicities related to liver function tests increased with increasing dose of BMS-986148. Most TRAEs resolved with dose interruption, dose reduction, or treatment discontinuation. Serious TRAEs were reported in 18% of pts with mono and 23% of pts with combo. Fifteen percent of pts discontinued due to TRAEs, and 1 pt died due to a TRAE (pneumonitis; 1.2 mg/kg Q3W mono). The ORR was 6% (0 CR, 3 PR) with mono in EXP and 20% (0 CR, 6 PR) with the combo in ESC and EXP. Among pts with mesothelioma, the ORR was 4% with mono in EXP and 31% with combo in ESC and EXP. Among pts with ovarian carcinoma, the ORR was 9% with mono in EXP. Of note, durable responses lasting up to ≈ 20 mo in pts with ovarian cancer with mono and ≈ 9 mo in pts with mesothelioma with mono and combo were observed. Conclusions: BMS-986148 ± NIVO was tolerable and demonstrated a clinically manageable safety profile. Preliminary clinical activity was observed with BMS-986148 ± NIVO in select patient populations, including pts with mesothelioma. Citation Format: Jeffrey Clarke, Siu-Chung Chu, Lillian L Siu, Jean-Pascal Machiels, Benjamin Markman, Kimberley Heinhuis, Michael Millward, Martijn Lolkema, Sandip Pravin Patel, Paul de Souza, Giuseppe Curigliano, Armando Santoro, Michelle Brown, Ronald Fleming, Heather Vezina, Chunsheng He, Sylvie Rottey. BMS-986148, an anti-mesothelin antibody-drug conjugate (ADC), alone or in combination with nivolumab demonstrates clinical activity in patients with select advanced solid tumors [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 B057. doi:10.1158/1535-7163.TARG-19-B057

Antigen-binding affinity and thermostability of chimeric mouse-chicken IgY and mouse-human IgG antibodies with identical variable domains

Constant (C)-region switching of heavy (H) and/or light (L) chains in antibodies (Abs) can affect their affinity and specificity, as demonstrated using mouse, human, and chimeric mouse-human (MH) Abs. However, the consequences of C-region switching between evolutionarily distinct mammalian and avian Abs remain unknown. To explore C-region switching in mouse-chicken (MC) Abs, we investigated antigen-binding parameters and thermal stability of chimeric MC-6C407 and MC-3D8 IgY Abs compared with parental mouse IgGs and chimeric MH Abs (MH-6C407 IgG and MH-3D8 IgG) bearing identical corresponding variable (V) regions. The two MC-IgYs exhibited differences in antigen-binding parameters and thermal stability from their parental mouse Abs. However, changes were similar to or less than those between chimeric MH Abs and their parental mouse Abs. The results demonstrate that mammalian and avian Abs share compatible V-C region interfaces, which may be conducive for the design and utilization of mammalian-avian chimeric Abs.