Humanized Research Articles

High-resolution African HLA resource uncovers HLA-DRB1 expression effects underlying vaccine response.

How human genetic variation contributes to vaccine effectiveness in infants is unclear, and data are limited on these relationships in populations with African ancestries. We undertook genetic analyses of vaccine antibody responses in infants from Uganda (n = 1391), Burkina Faso (n = 353) and South Africa (n = 755), identifying associations between human leukocyte antigen (HLA) and antibody response for five of eight tested antigens spanning pertussis, diphtheria and hepatitis B vaccines. In addition, through HLA typing 1,702 individuals from 11 populations of African ancestry derived predominantly from the 1000 Genomes Project, we constructed an imputation resource, fine-mapping class II HLA-DR and DQ associations explaining up to 10% of antibody response variance in our infant cohorts. We observed differences in the genetic architecture of pertussis antibody response between the cohorts with African ancestries and an independent cohort with European ancestry, but found no in silico evidence of differences in HLA peptide binding affinity or breadth. Using immune cell expression quantitative trait loci datasets derived from African-ancestry samples from the 1000 Genomes Project, we found evidence of differential HLA-DRB1 expression correlating with inferred protection from pertussis following vaccination. This work suggests that HLA-DRB1 expression may play a role in vaccine response and should be considered alongside peptide selection to improve vaccine design.

Immunoadsorption as a method of antibody donation during the COVID-19 pandemic.

Initial therapeutic efforts to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) included the use of plasma from convalescent donors containing anti-SARS-CoV-2 antibodies. High-neutralizing antibody titres are required for therapeutic efficacy. This study aims to show that immunoadsorption followed by tangential flow filtration can be used to obtain antibody concentrates with high-neutralizing capacities. Eligible donors (n = 10, five males and three females) underwent immunoadsorption using adsorber columns specific for human antibodies. Glycine-washed out eluates of 1.5 L volume were further concentrated by tangential flow filtration using 30 kDa ultrafiltration membranes. The same membranes were applied for diafiltrations to exchange residual glycine for 0.9% normal saline. Antibody concentrates were obtained within 8 h from the start of donation and had, 4.58 ± 1.95, 3.28 ± 1.28 and 2.02 ± 0.92 times higher total IgG, IgA and IgM concentrations, 3.29 ± 1.62 and 3.74 ± 0.6 times higher SARS-CoV-2 N and S antibody concentrations and 3.85 ± 1.71 times higher SARS-CoV-2 S-specific IgG concentrations compared to the donors' peripheral blood. The specific SARS-CoV-2 virus neutralization capacities increased in all but one concentrate. All antibody concentrates (50-70 mL final volume) passed microbiological tests, were free of hazardous glycine levels and could be stored at -80°C and 4°C for 1 year with 20 ± 3% antibody loss. Immunoadsorption followed by tangential flow filtration is a feasible procedure to collect IgG, IgA and IgM as well as SARS-CoV-2 N- and S-specific antibody concentrates of low volume, free of albumin and coagulation factors. Whether these concentrates can be used as passive immunisation in infected patients remains to be elucidated.

Human Antibodies against Herpes Simplex Virus 2 Glycoprotein G Do Not Neutralize but Mediate Antibody-Dependent Cellular Cytotoxicity

Human Antibodies against Herpes Simplex Virus 2 Glycoprotein G Do Not Neutralize but Mediate Antibody-Dependent Cellular Cytotoxicity

An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries

Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the in silico design of high affinity binders against nearly any proteinaceous surface. Yet, publicly available experiment antibody sequence-binding datasets may not contain the mutagenic, antigenic, or antibody sequence diversity necessary for deep learning approaches to capture molecular recognition. In part, this is because limited experimental platforms exist for assessing quantitative and simultaneous sequence-function relationships for multiple antibodies. Here we present MAGMA-seq, an integrated technology that combines multiple antigens and multiple antibodies and determines quantitative biophysical parameters using deep sequencing. We demonstrate MAGMA-seq on two pooled libraries comprising mutants of nine different human antibodies spanning light chain gene usage, CDR H3 length, and antigenic targets. We demonstrate the comprehensive mapping of potential antibody development pathways, sequence-binding relationships for multiple antibodies simultaneously, and identification of paratope sequence determinants for binding recognition for broadly neutralizing antibodies (bnAbs). MAGMA-seq enables rapid and scalable antibody engineering of multiple lead candidates because it can measure binding for mutants of many given parental antibodies in a single experiment.

Transforming waste into valuables: Preparation and evaluation of dual-ligand hydrophobic charge-induction chromatography using two poor performing ligands

In conventional chromatographic ligand screening, underperforming ligands are often dismissed. However, this practice may inadvertently overlook potential opportunities. This study aims to investigate whether these underperforming ligands can be repurposed as valuable assets. Hydrophobic charge-induction chromatography (HCIC) is chosen as the validation target for its potential as an innovative chromatographic mode. A novel dual-ligand approach is employed, combining two suboptimal ligands (5-Aminobenzimidazole and Tryptamine) to explore enhanced performance and optimization prospects. Various dual-ligand HCIC resins with different ligand densities were synthesized by adjusting the ligand ratio and concentration. The resins were characterized to assess appearance, functional groups, and pore features using SEM, FTIR, and ISEC techniques. Performance assessments were conducted using single-ligand mode resins as controls, evaluating the selectivity against human immunoglobulin G and human serum albumin. Static adsorption experiments were performed to understand pH and salt influence on adsorption. Breakthrough experiments were conducted to assess dynamic adsorption capacity of the novel resin. Finally, chromatographic separation using human serum was performed to evaluate the purity and yield of the resin. Results indicated that the dual-ligand HCIC resin designed for human antibodies demonstrates exceptional selectivity, surpassing not only single ligand states but also outperforming certain high-performing ligand types, particularly under specific salt and pH conditions. Ultimately, a high yield of 83.9 % and purity of 96.7 % were achieved in the separation of hIgG from human serum with the dual-ligand HCIC, significantly superior to the single-ligand resins. In conclusion, through rational design and proper operational conditions, the dual-ligand mode can revitalize underutilized ligands, potentially introducing novel and promising chromatographic modes.

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

Abstract PO4-06-11: Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation

Abstract Triple negative breast cancer (TNBC) is characterized by invasiveness and poor survival. Identifying novel TNBC targeted therapies to potentiate standard of care (SOC) therapy, is an unmet need. In the past 4 years, several targeted therapies for TNBC have been approved by the Food and Drug Administration (FDA). However, the identification of targeted therapies against other biological drivers of TNBC remains important. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in breast cancer. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes such as progression of disease and shortened survival. PGRN/GP88 expression is elevated in 30% TNBC. The importance of inhibiting PGRN/GP88 effect on the proliferation and tumor growth of triple negative breast cancer cells was previously established. In the current study, in association with Precision Antibody, we developed anti-PGRN/GP88 fully human monoclonal antibodies by immunizing TC transgenic humanized mice with recombinant human progranulin. Within 60 days since the start of immunization, several fully human monoclonal antibodies were successfully developed, characterized by several functional assays including neutralization, inhibition of PGRN/GP88 cell surface binding, proliferation, migration in MDA-MB-231 cells in vitro and in vivo. Results: The inhibition of PGRN/GP88 action by fully human monoclonal antibodies inhibited proliferation and migration in a dose-and time-dependent fashion in TNBC cells. Octet analysis determined Kd in the range of 10−11 M to 10−10 M. Transwell assay showed that anti-PGRN treatment inhibited migration In vivo xenograft studies with MDA-MB-231 cells injected in athymic nude mice showed that several fully human anti-PGRN antibodies inhibited tumor growth when compared to antibody control treated mice. In vivo dose response of AG01 in MDAMB-231 tumor bearing mice will be provided. Conclusion: PGRN/GP88 represents a therapeutic target for TNBC with two companion diagnostics (tissue test and ELISA to measure GP88 circulating levels). The use of the TC transgenic mice allows for the rapid development of fully human high affinity monoclonal antibodies bypassing the need for chimerization or humanization of mouse monoclonal antibodies. The development of fully human anti-progranulin antibodies provides novel targeted therapeutic option for TNBC. This work is supported by a an SBIR grant 5R44 CA 224718 from the National Cancer Institute to GS. Citation Format: Ginette Serrero, Jianping Dong, Jun Hayashi, Chun Dong. Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-11.

Abstract PS11-07: High prevalence of HER2-Low and AR expression in breast cancer brain metastases provide novel therapeutic options

Abstract Background: Although survival rates for patients with advanced breast cancer (BC) have improved overall, rates of BC brain metastases (BCBM) have increased over the past two decades, occurring in up to 15% to 30% of patients. BCBM greatly reduce quality of life and overall survival (OS) and are now the leading cause of BC patient mortality. Patients with primary HER2+ and triple negative BC (TNBC) are at highest risk for BM. Locoregional therapy with surgery and radiation are currently standard of care (SOC), but these treatments have significant morbidity and BCBM progressing despite these interventions are hard to treat. Systemic therapies to reduce cognitive impairment and increase survival are desperately needed, particularly in HER2-negative patients. Currently, immune checkpoint inhibitors (ICIs) and small molecules such as PARP inhibitors are among the limited systemic options that have BCBM activity in HER2-negative BC. However, the benefit of these therapies is limited to a select group of patients. For example, androgen receptor (AR) is expressed in 60-80% of all BC and, in our recently completed clinical trial (NCT03206203), patients with AR+ metastatic TNBC treated with carboplatin +/- atezolizumab received no clinical benefit from the addition of atezolizumab. Alternatives to ICIs are needed for patients with AR+ TNBC. HER2-low (H2L) BC is a newly defined subset of HER2-negative BC with a HER2 immunohistochemical (IHC) score of 1+ ( >10%) or 2+/in situ hybridization (ISH) negative phenotype. Recent clinical trials have demonstrated significant clinical benefit from novel HER2 antibody-drug conjugates (ADCs) in treating H2L BC. Trastuzumab-deruxtecan (T-Dxd), a HER2-directing ADC (topoisomerase I inhibitor conjugated to a humanized HER2-targeted antibody) was recently FDA approved as the first targeted therapy to treat H2L BC and can penetrate the blood–brain barrier. DESTINY-Breast04 demonstrated notable PFS and OS benefit of T-DXd compared to SOC in patients with H2L metastatic BC and included 61 patients with BCBM (results anticipated shortly). The overall rates of AR and H2L expression in patients with BCBM is unknown. The goal of this study is to investigate the prevalence of AR and HER2-low expression in a large cohort of BCBM treated at our institution. Methods: We retrospectively assessed rates of H2L and AR positivity (AR+) among patients with BCBM whose treatment included surgery at our institution between 2005-2023. Results were correlated with clinical receptor status (HR+/HER2-, HER2+/HR+, HER2+/HR- and TNBC) and clinical characteristics. Results of ER, PR, and HER2 testing performed for clinical purposes on the brain metastases and primary tumors were obtained from pathology reports. We retrieved formalin-fixed paraffin-embedded tissue blocks of the brain metastases and performed IHC for AR (SP107) and prognostic markers for cases with missing data. Results: We identified 101 cases of BCBM. Based on clinical prognostic markers, BCBM subtypes were HR+/HER2- (22%), HR+/HER2+ (14%), HER2+/HR- (21%) and TNBC (43%). Forty-five percent of the BCBM (n=45) were AR+ ( >10%) and 24% (n=24) demonstrated > 50% AR+. Among tumors with >50% AR+, 42% (n=10) are H2L (7 HR+[30%] and 3 TNBC [13%]). Forty-two percent of BCBM were H2L. Among the HER2-negative BCBM (n= 66), 64% (n=42) were H2L, including 23 of 44 TNBC (52%) and 19 of 22 HR+ (86)%. Fifteen percent of BMBC changed clinical subtype from the primary, including conversion to TNBC and H2L. Conclusion: AR+ positive (24% > 50% expression) and HER2-Low expressing (42%) tumors represent a significant proportion of our BCBM cohort providing exciting new options for treatment. Trials re-evaluating AR antagonists in combination with SOC utilizing high AR+ thresholds may benefit patients with BCBM. At least 10% of BCBM are both H2L and demonstrate >50% AR positivity providing the rationale for the combination of ADCs such as T-DXd and AR-targeted therapies for BCBM. Citation Format: Guadalupe Garcia, Paula Gonzalez-Ericsson, Brian Lehmann, Bret Mobley, Jennifer Pietenpol, Laura Kennedy, Ben Park, Melinda Sanders. High prevalence of HER2-Low and AR expression in breast cancer brain metastases provide novel therapeutic options [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-07.

Abstract PO2-06-13: Promising clinical performance of 89Zirconium-labelled Girentuximab PET-CT for imaging metastatic triple negative breast cancer patients (OPALESCENCE)

Abstract Aim: Triple Negative Breast Cancer (TNBC) is aggressive, often with a metastatic evolution and drug-resistant with limited therapeutic options. Evaluating new markers is an unmet need. Recently, the combination of anti-angiogenic and targeted hypoxia effectors agents proved to enhance therapy response. Indeed, anhydrase Carbonic-IX (CA-IX), a hypoxia-mediated breast tumor growth regulator, is important for the BC stem cells maintenance within hypoxic region and is highly overexpressed in TNBC. This pilot prospective study “OPALESCENCE” (NCT04758780) aimed at assessing PET/CT with 89Zr-labeled girentuximab (89Zr-TLX250) targeting CA-IX in 12 metastatic TNBC patients. Methods: Patients underwent FDG and 89Zr-TLX250 PET-CT and conventional imaging (CI) if needed (CT, US, mammography, brain MRI). Without any premedication or dietary preparation, patients received a single slow intravenous administration of 37±10% MBq 89Zr-TLX250 (10 mg). Day 3 or 5 post injection, a skull to mid-thigh PET/CT was acquired with 10 minutes acquisition time per bed position. The gold standard was determined by FDG PET/CT, CI and follow-up; lesion detected at least by 2 modalities was considered as true positive. Immunohistochemistry (IHC) was performed with Bond RX fully automated research stainer with CA-IX antibody (Leica, clone TH22). Staining was evaluated with semi-quantitative analysis (percentage and intensity of tumor cells expression). Human anti-chimeric antibody (HACA) were sampled but will be determined at study end. Results: Enrolment has been completed, a total of 273 lesions confirmed by gold standard as shown on table 1 were detected in 12 patients (231 by 89Zr-TLX250 and 264 by FDG PET/CT). Overall sensitivity of 89Zr-TLX250 PET/CT was 87.5%, with 100% sensitivity for breast, skin, adrenal gland and brain and 88.0%, 91.9% for nodes and bone respectively. Overall sensitivity for FDG-PET/CT was 96.7%. According to table 2, 6 patients had 100% of their lesions highlighted by 89Zr-TLX250 PET. In addition, for 2 other patients only one of the lesions was not detected by immunoPET. 89Zr-TLX250 PET therefore detected almost all of the metastatic lesions in 67% of the patients. For 8/12 patients, IHC was analysed and for 6 patients, CA-IX expression lesions was from 100% to 10% with an intensity from 3+ to 2+, whereas two patients presented no Ca-IX expression on tumour lesions. No 89Zr-TLX250 safety issue was reported. Conclusion: 89Zr-TLX250 PET/CT seems very promising because it allows the CA IX status lesions knowledge on whole body scale with a minimally invasive way, unlike the information provided by biopsies corresponding to only 1 or 2 lesions studied. Moreover, these results illustrated interest of 89Zr-TLX250 PET/CT as targeting agent for TNBC patients leading above all to a theranostic approach possibility. Study analysis (specially IHC, HACA and semi-quantitative PET/CT analysis) is ongoing and supplementary data will be presented during the congress. This is an investigator initiated trial. Investigational medicinal product was provided by Telix Pharmaceuticals. Table 1: Sensitivity of 89Zr-TLX250 PET, FDG PET, and conventional imaging (US, brain MRI and Mammography). Table 2: Number of confirmed lesions according to Gold Standard detected by 89Zr-TLX250 PET/CT (iPET), FDG PET/CT and CI for each patients. Citation Format: Caroline Rousseau, Marie Francoise Heymann, Jean Sebastien FRENEL, Elise Picot-Dilly, Maelle Le Thiec, Manon Taupin, Alexis Mouton, Emmanuelle Bourbouloux, Agnès Morel, Nadia Allam, Amélie Mallet, Mario Campone, Ludovic Ferrer, Françoise Kraeber-Bodéré. Promising clinical performance of 89Zirconium-labelled Girentuximab PET-CT for imaging metastatic triple negative breast cancer patients (OPALESCENCE) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-06-13.

Abstract PO2-04-03: BL-M07D1, an antibody-drug conjugate directed to HER2 in patients with locally advanced or metastatic Breast Cancer with HER2-positive/low-expression and other solid tumors: Results from a first-in-human phase 1 study

Abstract Background: BL-M07D1 is an anti-HER2 antibody-drug conjugate (ADC) comprised of a humanized anti-HER2 antibody, a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor (Ed-04). Methods: This study included subjects with locally advanced or metastatic HER2 expressing (positive/low) breast cancer (BC) and other solid tumors. BL-M07D1 would be administered at doses of 1.0mg/kg Day 1 & Day 8 every 3 weeks (D1D8 Q3W) or 2.6, 3.2, 3.8, 4.4, 5.0, 5.6, 6.2, 6.8 and 7.4 mg/kg Day 1 every 3 weeks (D1Q3W) during dose escalation (D-ESC). A subset of patients (pts) will be enrolled in the dose-expansion phase (D-EXP) at D1 Q3W regimens. Results: As of June 25, a total of 75 pts have been treated with at least one dose of BL-M07D1, with 22 pts in the D-ESC phase and 53 in the D-EXP phase. Among the 75 pts, 1 received 1.0 mg/kg D1D8Q3W and 74 were treated on the D1Q3W schedule. Dose-limiting toxicity (DLT) was observed at 6.2mg/kg (febrile neutropenia and G3 thrombocytopenia lasting >7 days in one patient). The maximum tolerated dose (MTD) has not been reached yet. D-EXP dose levels included 3.8, 4.4, 5.0 and 5.6mg/kg D1 Q3W. This study enrolled 62 patients with BC, 6 with gastric cancer, 4 with colorectal cancer and 3 with non-small cell lung cancer. The most common TEAEs ( >10%, all grade/≥G3) were leukopenia (79%/24%), neutropenia (69%/39%), anemia (63%/11%), nausea (47%/0%), thrombocytopenia (37%/9%), vomiting (37%/1%), decreased appetite (27%/0%), lymphopenia (24%/8%), alopecia (23%/0%), asthenia (19%/0), gamma-glutamyl transferase increased (17%/0%), aspartate aminotransferase increased (17%/0%), constipation (13%/0%), diarrhea (12%/0%), hypertriglyceridemia (12%/0%), urinary tract infection (11%/1%), COVID-19 (11%/0%), occult blood positive (11%/0%), pyrexia (11%/0%). No ILD was observed. Forty-five pts with BC were evaluated for efficacy (i.e., had at least one post-baseline tumor assessment). Updated efficacy and pharmacokinetic (PK) results will be presented at the meeting. Conclusions: BL-M07D1 demonstrated encouraging efficacy in heavily pretreated HER2 expressing cancers, especially in HER2+ BC. The safety profile showed adequate safety and tolerability. Clinical trial identification: NCT05461768. Efficacy in Patients with Breast Cancer a. 8/9 pts received prior HER2-ADC with microtubule inhibitor. 1/9 pts received prior HER2-ADC with TOPI inhibitor. b. 6 pts with tumor shrinkage are still on-going. c. All pts with tumor shrinkage are still on-going. Citation Format: Erwei Song, Herui Yao, Meili Sun, Hong Zong, Rongbo Lin, Wen Zou, Muran Ding, Jing Yu, Sa Xiao, Hongwei Wang, Hai Zhu, Martin Olivo, Yi Zhu. BL-M07D1, an antibody-drug conjugate directed to HER2 in patients with locally advanced or metastatic Breast Cancer with HER2-positive/low-expression and other solid tumors: Results from a first-in-human phase 1 study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-03.

Evaluation of the precision of the Plasmodium knowlesi growth inhibition assay for Plasmodium vivax Duffy-binding protein-based malaria vaccine development

Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted with four different anti-PvDBPII human monoclonal antibodies (mAbs) at concentrations of 0.016 to 2mg/mL, and three GIAs with eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95 %CI) for %GIA or GIA50 in repeat assays was calculated in this investigation. The error range determined in this study will help researchers to compare PkGIA results from different assays and studies appropriately, thus supporting the development of future blood-stage malaria vaccine candidates, specifically second-generation PvDBPII-based formulations.

Anti-CAMP1 IgG promotes macrophage phagocytosis of Cutibacterium acnes Type II

Among 5 types of the Christie-Atkins-Munch-Petersen factor (CAMP) of Cutibacterium acnes, CAMP1 is highly expressed in phylotype II as well as IB, and thought to be a virulence factor of opportunistic but fatal blood, soft tissue, and implant-related infections. The target of a human single-chain variable antibody fragment (scFv), recently isolated from a phage display library, has been identified as CAMP1 of phylotype II, using immunoprecipitation followed by mass spectrometry, phage display peptide biopanning, 3D-modelling, and ELISA. The IgG1 format of the antibody could enhance phagocytosis of C. acnes DMST 14916 by THP-1 human monocytes. Our results suggest that the antibody-dependent phagocytosis process is mediated by the caveolae membrane system and involves the induction of IL-1β. This is the first report on the study of a human antibody against CAMP1 of C. acnes phylotype II, of which a potential use as therapeutic antibody against virulence C. acnes infection is postulated.

IBI310 (anti-CTLA-4 antibody) monotherapy or in combination with sintilimab in advanced melanoma or urothelial carcinoma

IBI310 is a recombinant fully human IgG1 antibody against cytotoxic T-lymphocyte antigen 4 (CTLA-4). This study was conducted to evaluate IBI310 monotherapy or combination therapy with sintilimab in the patients with advanced melanoma or urothelial carcinoma (UC). Patients in phase Ia received IBI310 at 0.3/1/2/3 mg/kg intravenously (IV) every three weeks (Q3W) following the accelerated titration and 3 + 3 escalation design. Patients in phase Ib received IBI310 (1/2/3 mg/kg IV, Q3W) plus sintilimab (200 mg IV, Q3W) for 4 cycles, followed by sintilimab maintenance therapy. The phase Ib expansion of IBI310 plus sintilimab was performed in patients with advanced melanoma or UC. Overall, 53 patients were enrolled including 10 patients with melanoma in phase Ia, 34 with melanoma and 9 with UC in phase Ib. 94.3% (50/53) patients experienced at least one treatment-related adverse event (TRAE) with most being grade 1-2; 26.4% (14/53) patients experienced grade ≥3 TRAEs. In phase Ia, disease control rate (DCR) was 50.0% (95% CI: 18.7%-81.3%). In phase Ib, objective response rate (ORR) and DCR were 17.6% (95% CI: 6.8%-34.5%) and 44.1% (95% CI: 27.2%-62.1%) respectively for melanoma, and were 22.2% (95% CI: 2.8%-60.0%) and 66.7% (95% CI: 29.9%-92.5%) respectively for UC. IBI310 monotherapy or combination therapy with sintilimab was well-tolerated with favorable antitumor activity across patients with advanced melanoma and UC.

Spectroscopic analysis of the bacterially expressed head domain of rotavirus VP6.

The rotavirus capsid protein VP6 forms the middle of three protein layers and is responsible for many critical steps in the viral life cycle. VP6 as a structural protein can be used in various applications including as a subunit vaccine component. The head domain of VP6 (VP6H) contains key sequences that allow the protein to trimerize and that represent epitopes that are recognized by human antibodies in the viral particle. The domain is rich in β-sheet secondary structures. Here, VP6H was solubilised from bacterial inclusion bodies and purified using a single affinity chromatography step. Spectral (far-UV circular dichroism and intrinsic tryptophan fluorescence) analysis revealed that the purified domain had native-like secondary and tertiary structures. The domain could maintain structure up to 44°C during thermal denaturation following which structural changes result in an intermediate forming and finally irreversible aggregation and denaturation. The chemical denaturation with urea and guanidinium hydrochloride produces intermediates that represent a loss in the cooperativity. The VP6H domain is stable and can fold to produce its native structure in the absence of the VP6 base domain but cannot be defined as an independent folding unit.

The D Gene in CDR H3 Determines a Public Class of Human Antibodies to SARS-CoV-2

Public antibody responses have been found against many infectious agents. Structural convergence of public antibodies is usually determined by immunoglobulin V genes. Recently, a human antibody public class against SARS-CoV-2 was reported, where the D gene (IGHD3-22) encodes a common YYDxxG motif in heavy-chain complementarity-determining region 3 (CDR H3), which determines specificity for the receptor-binding domain (RBD). In this review, we discuss the isolation, structural characterization, and genetic analyses of this class of antibodies, which have been isolated from various cohorts of COVID-19 convalescents and vaccinees. All eleven YYDxxG antibodies with available structures target the SARS-CoV-2 RBD in a similar binding mode, where the CDR H3 dominates the interaction with antigen. The antibodies target a conserved site on the RBD that does not overlap with the receptor-binding site, but their particular angle of approach results in direct steric hindrance to receptor binding, which enables both neutralization potency and breadth. We also review the properties of CDR H3-dominant antibodies that target other human viruses. Overall, unlike most public antibodies, which are identified by their V gene usage, this newly discovered public class of YYDxxG antibodies is dominated by a D-gene-encoded motif and uncovers further opportunities for germline-targeting vaccine design.

Flow cytometric immunophenotypic differentiation patterns of bone marrow eosinophilopoiesis.

Flow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied. Fresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR-1, Siglec-8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL-5Rα), HLA-DR, using 10 parameter flow cytometry. Putative CD34-negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence-based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM. The eosinophilic lineage was first verified using the human eosinophil-specific antibody EMR-1 in combination with anti-IL-5Rα antibody. Then, a combination of Siglec-8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec-8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec-8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec-8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils. A novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.

Validation of an enzyme-linked immunosorbent assay test-system for bevacizumab concentration determing in biological fluids

Introduction. One of the reasons for the development of many diseases primarily malignant is the increased expression of Vascular Endothelial Growth Factor (VEGF). Bevacizumab is a drug that neutralizes the biological activity of VEGF. The molecular structure of bevacizumab is a recombinant humanized antibody. Its use reduces vascularization in the foci of increased VEGF expression which slows down tumor growth and also helps restore vision in a number of ophthalmic diseases. To determine the concentration of bevacizumab in human biological fluids a test system based on Enzyme-linked Immunosorbent Assay (ELISA) is presented.Aim. Aim of this study is the validation of this test system.Materials and methods. Blank sera of volunteers, bevacizumab solution, enzyme-linked immunosorbent assay, solid-phase sandwich ELISA kit, microplate photometer.Results and discussion. The following characteristics of the test system were determined: the lower limit of quantification is 2.0 mcg/ml, the upper calibration range is up to 200 mcg/ml, the accuracy and precision within one series and between series does not exceed 20 %, and the total error of the method – 30 %, short-term stability for samples at room temperature – 6 hours, long-term stability – 14 days at –20 °C, the ability to freeze/thaw of samples is up to three times, the ability to determine samples with a concentration above the upper calibrator after diluting in 2 times.Conclusion. The results obtained fully comply with international acceptance criteria and allow the use of the ELISA test system manufactured by LLC "Probiotek" for use in the field of clinical laboratory diagnostics.  

Structural basis of Zika virus NS1 multimerization and human antibody recognition

Zika virus (ZIKV) belongs to the Flavivirus genus of the Flaviviridae family along with the four serotypes of dengue virus (DENV1–4). The recent global outbreaks of contemporary ZIKV strains demonstrated that infection can lead to neurological sequelae in adults and severe abnormalities in newborns that were previously unreported with ancestral strains. As such, there remains an unmet need for efficacious vaccines and antiviral agents against ZIKV. The non-structural protein 1 (NS1) is secreted from the infected cell and is thought to be associated with disease severity besides its proven usefulness for differential diagnoses. However, its physiologically relevant structure and pathogenesis mechanisms remain unclear. Here, we present high-resolution cryoEM structures of ZIKV recombinant secreted NS1 (rsNS1) and its complexes with three human monoclonal antibodies (AA12, EB9, GB5), as well as evidence for ZIKV infection-derived secreted NS1 (isNS1) binding to High Density Lipoprotein (HDL). We show that ZIKV rsNS1 forms tetramers and filamentous repeats of tetramers. We also observed that antibody binding did not disrupt the ZIKV NS1 tetramers as they bound to the wing and connector subdomain of the β-ladder. Our study reveals new insights into NS1 multimerization, highlights the need to distinguish the polymorphic nature of rsNS1 and isNS1, and expands the mechanistic basis of the protection conferred by antibodies targeting NS1.

Clinical conundrums in pediatric kidney transplantation: What we know about the role of angiotensin II type I receptor antibodies in pediatric kidney transplantation and the path forward.

Antibodies to angiotensin II type 1 receptor (AT1R-Abs) are among the most well-studied non-HLA antibodies in renal transplantation. These antibodies have been shown to be common in pediatric kidney transplantation and associated with antibody-mediated rejection (AMR), vascular inflammation, development of human leukocyte donor-specific antibodies (HLA DSA), and allograft loss. As AT1R-Ab testing becomes more readily accessible, evidence to guide clinical practice for testing and treating AT1R-Ab positivity in pediatric kidney transplant recipients remains limited. This review discusses the clinical complexities of evaluating AT1R-Abs given the current available evidence.

A new TROP2-targeting antibody-drug conjugate shows potent antitumor efficacy in breast and lung cancers

Trophoblast cell surface antigen 2 (Trop2) is considered to be an attractive therapeutic target in cancer treatments. We previously generated a new humanized anti-Trop2 antibody named hIMB1636, and designated it as an ideal targeting carrier for cancer therapy. Lidamycin (LDM) is a new antitumor antibiotic, containing an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). AE and LDP can be separated and reassembled, and the reassembled LDM possesses cytotoxicity similar to that of native LDM; this has made LDM attractive in the preparation of gene-engineering drugs. We herein firstly prepared a new fusion protein hIMB1636-LDP composed of hIMB1636 and LDP by genetic engineering. This construct showed potent binding activities to recombinant antigen with a KD value of 4.57 nM, exhibited binding to Trop2-positive cancer cells and internalization and transport to lysosomes, and demonstrated powerful tumor-targeting ability in vivo. We then obtained the antibody-drug conjugate (ADC) hIMB1636-LDP-AE by molecular reconstitution. In vitro, hIMB1636-LDP-AE inhibited the proliferation, migration, and tumorsphere formation of tumor cells with half-maximal inhibitory concentration (IC50) values at the sub-nanomolar level. Mechanistically, hIMB1636-LDP-AE induced apoptosis and cell-cycle arrest. In vivo, hIMB1636-LDP-AE also inhibited the growth of breast and lung cancers in xenograft models. Moreover, compared to sacituzumab govitecan, hIMB1636-LDP-AE showed more potent antitumor activity and significantly lower myelotoxicity in tumors with moderate Trop2 expression. This study fully revealed the potent antitumor efficacy of hIMB1636-LDP-AE, and also provided a new preparation method for LDM-based ADC, as well as a promising candidate for breast cancer and lung cancer therapeutics.