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Introduction: Inducible co-stimulator (ICOS), an important co-stimulatory receptor on effector T cells (Teffs), may also contribute to tumor growth due to its high expression on regulatory T cells (Tregs). This study explored the clinical significance of ICOS-expressing Tregs in hepatocellular carcinoma (HCC). Methods: Tumor tissues from HCC patients who received curative hepatectomy were obtained at a referral center. Dual immunohistochemistry was performed to evaluate the expression of ICOS and Foxp3. The cell densities and proximities between stained cells in regions of interest were measured by digital pathology and the associations with clinical outcome were analyzed. Results: A total of 142 patients (male:female = 112: 30, median age of 61.0 years) were enrolled. Among them, 87 (61.3%) had chronic hepatitis B virus infection and 33 (23.2%) had chronic hepatitis C infection. Low α-fetoprotein level (+Foxp3+ cells and the ratio of ICOS+Foxp3+/total Foxp3+ cells were significantly higher (p < 0.001) in the tumor center than in the peritumor area. Patients with a high density of ICOS+Foxp3+ cells or a high ratio of ICOS+Foxp3+/total Foxp3+ cells in the tumor center trended to have a shorter OS. A shorter distance between ICOS+Foxp3+ cells and ICOS+Foxp3− cells (likely Teffs) in the tumor center was significantly associated with a shorter OS (p = 0.030), suggesting active immunosuppression of ICOS+ Tregs on ICOS+ Teffs. Conclusion: An increased abundance of ICOS+ Tregs in the tumor center in comparison to the peritumor area indicates a strong immunosuppressive tumor microenvironment of HCC. A high proportion of ICOS+Foxp3+ cells and a shorter distance between ICOS+ Tregs and other ICOS+ cells were associated with a poor OS, suggesting that depleting ICOS+ Tregs might provide clinical benefit for patients with HCC.

2571 Background: Leukocyte Ig-like receptor B1 [LILRB1; ILT2] is an inhibitory receptor expressed on various immune cells. ILT2 binds to classical and nonclassical MHC class I molecules, with highest affinity to HLA-G. ILT2-mediated inhibition leads to impairment of immune cell proliferation, differentiation, phagocytosis, cytotoxicity and cytokine secretion. Antagonism of ILT2 signaling may serve as a novel target for anti-cancer immunotherapy. SAR444881 (BND-22) is a novel humanized IgG4 monoclonal antagonist antibody which selectively binds to ILT2 and blocks its interaction with MHC I molecules. SAR444881 induces robust macrophage and lymphocyte-driven anti-tumor activity in in vitro and in vivo models. To overcome the limitation that LILRB proteins are not expressed in rodents, we conducted a series of in vivo studies using humanized mouse models, cancer patient biopsies and ex vivo co-culture systems to interrogate the pharmacodynamic (PD) response of ILT2 antagonism as well as inform the combinatorial and patient enrichment strategies for SAR444881. Methods: SAR444881-induced modulation of PD biomarkers was evaluated in humanized xenograft models. Ex vivo co-culture system has been established using patient tumor tissues and isolated PBMC or other immune cells. Biomarker expression on immune cells and secreted soluble proteins were monitored by flow cytometry and ELISA, respectively. Procured tumor samples of patients with various advanced solid tumors, including Head and neck squamous cell carcinoma (HNSCC), Gastric Cancer (GC), Non-Small Cell Lung Cancer (NSCLC) and Colorectal Cancer (CRC) were utilized to evaluate protein or gene expression levels of potential predictive biomarkers (ILT2, HLA = G, PD-L1) using immunohistochemistry (IHC) and whole transcriptomic analysis (RNAseq). Results: 1) PD biomarkers: in murine models, SAR444881 modulates intra-tumoral sub-populations of CD8+ T cells, NK cells and Macrophage polarization; 2) Combination strategy: functional study in an ex vivo system showed that addition of an anti-PD-1 antibody induced an increased production of pro-inflammatory cytokines, including IFNγ and TNFα, compared to SAR444881 or anti-PD-1 alone. Concomitantly, combining SAR444881 with cetuximab resulted in increased phagocytosis compared to isotype control. 3) Predictive biomarkers: protein and gene profiling results suggest enriched expression of ILT2 in majority of biopsies from several tumor types including NSCLC, HNSCC and CRC. Conclusions: These data inform the PD response biomarkers, combination, and patient enrichment strategies for the clinical development of SAR444881 to maximize its benefits for cancer patients. An ongoing phase 1/2 trial of SAR444881 mono- and combination therapy in patients with advanced solid tumors is testing these concepts in the clinic (NCT04717375).

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, via the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.

Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.

Sanjuan Nandin et al. describe an innovative approach based on antigen-dependent activation of human memory B cells in culture. It results in the rapid generation of human antibodies against infectious agents and offers the potential for therapeutic antibody production and vaccine development., Vaccines remain the most effective tool to prevent infectious diseases. Here, we introduce an in vitro booster vaccination approach that relies on antigen-dependent activation of human memory B cells in culture. This stimulation induces antigen-specific B cell proliferation, differentiation of B cells into plasma cells, and robust antibody secretion after a few days of culture. We validated this strategy using cells from healthy donors to retrieve human antibodies against tetanus toxoid and influenza hemagglutinin (HA) from H1N1 and newly emergent subtypes such as H5N1 and H7N9. Anti-HA antibodies were cross-reactive against multiple subtypes, and some showed neutralizing activity. Although these antibodies may have arisen as a result of previous influenza infection, we also obtained gp120-reactive antibodies from non–HIV-infected donors, indicating that we can generate antibodies without prior antigenic exposure. Overall, our novel approach can be used to rapidly produce therapeutic antibodies and has the potential to assess the immunogenicity of candidate antigens, which could be exploited in future vaccine development.

2626 Background: Inducible T-cell co-stimulator (ICOS) is an important co-stimulatory receptor on effector T cells (Teffs) that also promotes tumor growth due to its high expression on regulatory T cells (Tregs). KY1044 is a fully human IgG1 that targets ICOS, acting via a dual mode of action (MoA) by depleting ICOShigh Tregs and stimulating ICOSLow Teffs. A Phase 1/2 clinical trial (NCT03829501) is currently assessing the safety and preliminary efficacy of KY1044, as a single agent and in combination with atezolizumab, in subjects with advanced relapsed/refractory malignancies. Using longitudinal blood samples and tumor biopsies, we aim to correlate KY1044 target engagement levels with pharmacodynamic (PD) properties (e.g. dual MoA) in the tumor microenvironment (TME) and the circulation. Methods: Phase 1 subjects were enrolled in dose escalation and enrichment cohorts to evaluate the effect of KY1044 as monotherapy (0.8 – 240 mg) Q3W and in combination (0.8 – 80 mg) with atezolizumab (1200 mg) Q3W. PBMCs, plasma and tumor biopsies were collected over the first 3 cycles to confirm target engagement and KY1044 MoA. The sample analysis included: immunohistochemistry (IHC) of tumor samples (ICOS, FOXP3 and CD8); circulating T cell immunoprofiling and receptor occupancy by chip-cytometry; PBMC and tumor sample pre- and post-treatment transcriptomic analysis; and the assessment of circulating cytokines (e.g. GM-CSF). Results: As assessed in PBMCs, full/prolonged ICOS target engagement on T cells was confirmed in subjects receiving a flat dose of 8 to 240 mg, while partial/transient saturation was observed at lower doses (0.8-2.4 mg). The target engagement was not affected by atezolizumab. The immune cell profiling showed changes in some populations, but there was no significant depletion of peripheral ICOS+ cells. In contrast, pre- and post-treatment IHC analysis of ICOS+/FOXP3+ cells in tumor biopsies confirmed a KY1044-dose dependent reduction of ICOS+ Tregs and maintenance of CD8+ T cells in the TME. Together, this resulted in an increased intratumoral CD8+/ICOS+ Treg ratio at all doses, plateauing from subjects receiving a flat KY1044 dose of 8 mg. KY1044-dependent agonism was indirectly assessed by measuring circulating cytokine levels. A post-dosing transient induction of GM-CSF was evident in subjects dosed with KY1044 at the 0.8 and 2.4 mg dose, whereas minimal induction was observed at dose of 8 mg and higher. Conclusions: LongitudinalPDdata confirmed the expected KY1044 MoA, namely ICOS Treg depletion and increased CD8/ICOS Treg ratio in the TME as well as T cell co-stimulation. The observed PD responses are currently being further explored in a more homogenous patient population.

Background: Immunotherapy targeting co-stimulatory receptors of T cells is under active clinical investigation. Inducible co-stimulator (ICOS) is an important co-stimulatory receptor on effector T cells (Teffs) that also promotes tumor growth due to its high expression on regulatory T cells (Tregs). Human IgG1 anti-ICOS therapeutic antibodies may therefore induce antitumor immunity by stimulating ICOSLow Teffs and depleting ICOSHigh Tregs. This study explored the clinical significance of ICOS expression and Treg content in hepatocellular carcinoma (HCC). Patients and Methods: We collected tumor tissues from HCC patients who received curative hepatectomy at the National Taiwan University Hospital, Taipei, Taiwan. Dual immunohistochemistry (IHC) was performed to evaluate the expression of ICOS and Foxp3. The cell density and distances between single- and dual-expressing cells in the tumor center, margin, and the peritumor area were quantitated by digital pathology. Associations between clinical outcome or clinical metadata and ICOS/Foxp3 expression were analyzed. Results: A total of 142 patients (male: female= 112: 30, median age of 61.0 years) were enrolled. Among them, 87 (61.3%) had chronic hepatitis B virus (HBV) infection, 33 (23.2%) had chronic hepatitis C (HCV) infection, and 22 (15.5%) had no HBV/HCV infection. In this cohort, low AFP level (< 20 ng/ml) and early stage, but not age, gender, or viral etiology, were significantly associated with improved overall survival (OS). However, the density of ICOS+Foxp3+ cells and the ratio of ICOS+Foxp3+/total Foxp3+ cells were significantly higher (p Conclusions: A high proportion of Tregs in HCC tumors expresses ICOS, implying a strong immunosuppressive environment. Importantly, high ICOS expression in HCC tumors, a high ratio of ICOS+Foxp3+/total Foxp3+ cells and a shorter distance between ICOS+ Tregs and other ICOS+ cells are all associated with a poor OS, suggesting that targeting ICOS in this indication may provide clinical benefit (This work is partly supported by MOST 108-2314-B-002-073). Citation Format: Li-Chun Lu, Cecilia Deantonio, Laura Mitchell, Yi-Hsuan Lee, Yu-Yun Shao, Ron Chen, Marianne Cowan, Matthew Corser, Lorcan Sherry, Ann-Lii Cheng, Chia-Chi Lin, Sonia Quaratino, Richard C. Sainson, Chih-Hung Hsu. High ICOS/FOXP3 Tregs content in the tumor microenvironment is associated with poorer survival in patients with hepatocellular carcinoma [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 1590.

The identification of effective biomarkers for early diagnosis, prognosis, and response to treatments remains a challenge in ovarian cancer (OC) research. Here, we present an unbiased high-throughput approach to profile ascitic fluid autoantibodies in order to obtain a tumor-specific antigen signature in OC. We first reported the reactivity of immunoglobulins (Igs) purified from OC patient ascites towards two different OC cell lines. Using a discovery set of Igs, we selected tumor-specific antigens from a phage display cDNA library. After biopanning, 700 proteins were expressed as fusion protein and used in protein array to enable large-scale immunoscreening with independent sets of cancer and noncancerous control. Finally, the selected antigens were validated by ELISA. The initial screening identified eight antigenic clones: CREB3, MRPL46, EXOSC10, BCOR, HMGN2, HIP1R, OLFM4, and KIAA1755. These antigens were all validated by ELISA in a study involving ascitic Igs from 153 patients (69 with OC, 34 with other cancers and 50 without cancer), with CREB3 showing the highest sensitivity (86.95%) and specificity (98%). Notably, we were able to identify an association between the tumor-associated (TA) antibody response and the response to a first-line tumor treatment (platinum-based chemotherapy). A stronger association was found by combining three antigens (BCOR, CREB3, and MRLP46) as a single antibody signature. Measurement of an ascitic fluid antibody response to multiple TA antigens may aid in the identification of new prognostic signatures in OC patients and shift attention to new potentially relevant targets.

During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection.

Anemia of chronic disease (ACD) is the most common cause of anemia in hospitalized patients. The underlying pathophysiological mechanisms are manifold, including reduced Erythropoietin (EPO) availability and sensitivity, direct negative effects of inflammatory cytokines on erythropoiesis and functional iron deficiency due to iron restriction mainly in the reticuloendothelial system (RES). The latter can be ascribed to increased hepcidin levels, a small liver derived peptide that has been found to be the key iron regulator. Up to date, most patients suffering from ACD are treated with a combination of both ESA (Erythropoietin Stimulating Agent) and intravenous (i.v.) iron to maintain hemoglobin (Hb) levels. Despite this combination therapy a significant number of patients require increasing doses of i.v. iron and ESA during their medical history - often resulting in continuous, potential toxic iron overload and ESA doses that exceed the acceptable range. As hepcidin is central to the iron-restrictive phenotype in ACD, several therapeutic approaches of hepcidin modulation have been investigated to overcome iron overload and EPO resistance. Some of these therapies are currently investigated in early clinical trials. We here report the effects of a fully human anti-BMP6 antibody on anemia, iron metabolism, erythropoiesis and ESA dosing in two different, well established rodent models of ACD. As BMPs, mainly BMP2 and BMP6, have been reported to be involved in hepcidin control, with knock out mice showing very low hepcidin levels even in an inflammatory setting, a fully human anti-BMP6 antibody has been developed to suppress hepcidin levels. We tested the antibody treatment in two distinct ACD animal models: First, a rat arthritis model (PG-APS in Lewis rats) and second, a Chronic Kidney Disease (CKD) mouse anemia model (Adenine model in C57BL/6 mice). Both models present with long-lasting anemia as seen in humans suffering from ACD. Mice and rats were treated with different doses of the anti-BMP6 antibody with and without ESA. Whole blood count, serum iron parameters (including hepcidin), bone marrow erythropoiesis determined by FACS analysis, cytokine expression and iron metabolism gene expression in spleen, liver and kidney were analyzed. Anti-BMP6 as well as ESA monotherapy resulted in a net increase in Hb level but only anti-BMP6 treatment significantly increased MCV and MCH, which can be ascribed to effective iron mobilization. In contrast, ESA therapy raised Hb levels by increasing red blood cell numbers. Of note, mere i.v. iron supplementation (sodium ferric gluconate), even at high doses, was not able to restore Hb levels to the same extent as the anti-BMP6 monotherapy. Strikingly, combination of both, ESA and anti-BMP6 treatment, had a synergistic effect on Hb levels, especially in the rat PG-APS model. Combination therapy of low ESA doses that only had a modest effect as a monotherapy, led to a dramatic increase in Hb levels, even exceeding those seen in healthy rats. Based on these results, additional experiments were performed to investigate the potential of this combination treatment to reduce ESA doses. Indeed, when anti-BMP6 was combined with a significantly reduced total ESA dose Hb levels were corrected to normal values in disease animals. Anti-BMP6 treatment also led to a significant decrease of iron deposition in the spleen with no iron deposition in parenchymal organs, indicating that the freed-up iron was effectively used for erythropoiesis and not just distributed elsewhere. In summary, anti-BMP6 therapy worked synergistically with ESA treatment in two different models of ACD leading to significantly increased Hb levels, a reduced ESA need as well as reduced iron overload in the RES. Furthermore, these experiments clearly show that treatment of ACD, being a complex multifactorial disease, benefits from using a combination of diversified approaches to overcome anemia and significantly reduce the dose of each therapeutic. Disclosures Wake: Kymab Ltd.: Employment. Bayliss:Kymab Ltd.: Employment. Papworth:Kymab Ltd.: Employment. Carvalho:Kymab Ltd.: Employment. Deantonio:Kymab Ltd.: Employment. Weiss:Kymab Ltd.: Consultancy. Germaschewski:Kymab Ltd.: Employment. Theurl:Kymab Ltd.: Consultancy, Research Funding.

The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.

BACKGROUND: Over the last few years High-Throughput Protein Production (HTPP) has played a crucial role for functional proteomics. High-quality, high yield and fast recombinant protein production are critical for new HTPP technologies. Escherichia coli is usually the expression system of choice in protein production thanks to its fast growth, ease of handling and high yields of protein produced. Even though shake-flask cultures are widely used, there is an increasing need for easy to handle, lab scale, high throughput systems. RESULTS: In this article we described a novel minifermenter system suitable for HTPP. The Air-Well minifermenter system is made by a homogeneous air sparging device that includes an air diffusion system, and a stainless steel 96 needle plate integrated with a 96 deep well plate where cultures take place. This system provides aeration to achieve higher optical density growth compared to classical shaking growth without the decrease in pH value and bacterial viability. Moreover the yield of recombinant protein is up to 3-fold higher with a considerable improvement in the amount of full length proteins. CONCLUSIONS: High throughput production of hundreds of proteins in parallel can be obtained sparging air in a continuous and controlled manner. The system used is modular and can be easily modified and scaled up to meet the demands for HTPP.

During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection.

During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection.

The aim of this study was to dissect the autoantibody response in celiac disease (CD) that remains largely unknown, with the goal of identifying the disease-specific autoantigenic protein pattern or the so called epitome. Sera from CD patients were used to select immunoreactive antigens from a cDNA phage-display library. Candidate genes were identified, the corresponding proteins produced and their immunoreactivity validated with sera from CD patients and controls. Thirteen CD-specific antigens were identified and further validated by protein microarray. The specificity for 6 of these antigens was confirmed by ELISA. Furthermore we showed that this antibody response was not abolished on a gluten free diet and was not shared with other autoimmune diseases. These antigens appear to be CD specific and independent of gluten induction. The utility of this panel extends beyond its diagnostic value and it may drive the attention to new targets for unbiased screens in autoimmunity research.