Rapid Mass Spectrometry-Based Multiattribute Method for Glycation Analysis with Integrated Afucosylation Detection Capability.

The multiattribute method (MAM) has emerged as a powerful tool for simultaneously screening multiple product quality attributes of therapeutic antibodies. One such potential critical quality attribute (CQA) is glycation, a common modification that can impact the heterogeneity, functional activity, and immunogenicity of therapeutic antibodies. However, current methods for monitoring glycation levels in MAM are rare and not sufficiently rapid and accurate. In this study, an improved mass spectrometry (MS)-based MAM was developed to simultaneously monitor glycation and other quality attributes including afucosylation. The method was evaluated using two therapeutic antibodies with different glycosylation site numbers. Treatment with IdeS, Endo F2, and dithiothreitol generated three distinct subunits, and the glycation results obtained were similar to those treated with PNGase F, which is routinely used to release glycans; the sample processing time was greatly reduced while providing additional quality attribute information. The MS-based MAM was also employed to assess the glycation progression following forced glycation in various buffer solutions. A significant increase in oxidation was observed when forced glycation was conducted in an ammonium bicarbonate buffer solution, and a total of 23 potential glycation sites and 4 significantly oxidized sites were identified. Notably, we found that ammonium bicarbonate was found to specifically stimulate oxidation, while glycation had a synergistic effect on oxidation. These findings establish this study as a novel methodology for achieving a technologically advanced platform and concept that enhances the efficacy of product development and quality control, characterized by its broad-spectrum, rapid, and accurate nature.

Development of a reliable cell-based reporter gene assay to measure the bioactivity of anti-HER2 therapeutic antibodies.

Human epidermal growth factor receptor 2 (HER2) is a key player in the pathogenesis and progression of breast cancer and is currently a primary target for breast cancer immunotherapy. Bioactivity determination is necessary to guarantee the safety and efficacy of therapeutic antibodies targeting HER2. Nevertheless, currently available bioassays for measuring the bioactivity of anti-HER2 mAbs are either not representative or have high variability. Here, we established a reliable reporter gene assay (RGA) based on T47D-SRE-Luc cell line that expresses endogenous HER2 and luciferase controlled by serum response element (SRE) to measure the bioactivity of anti-HER2 antibodies. Neuregulin-1 (NRG-1) can lead to the heterodimerization of HER2 on the cell membrane and induce the expression of downstream SRE-controlled luciferase, while pertuzumab can dose-dependently reverse the reaction, resulting in a good dose-response curve reflecting the activity of the antibody. After optimizing the relevant assay parameters, the established RGA was fully validated based on ICH-Q2 (R1), which demonstrated that the method had excellent specificity, accuracy, precision, linearity, and stability. In summary, this robust and innovative bioactivity determination assay can be applied in the development and screening, release control, biosimilar assessment and stability studies of anti-HER2 mAbs.

Improving product quality and productivity of an antibody-based biotherapeutic using inverted frustoconical shaking bioreactors.

The Chinese hamster ovarian (CHO) cells serve as a common choice in biopharmaceutical production, traditionally cultivated in stirred tank bioreactors (STRs). Nevertheless, the pursuit of improved protein quality and production output for commercial purposes demand exploration into new bioreactor types. In this context, inverted frustoconical shaking bioreactors (IFSB) present unique physical properties distinct from STRs. This study aims to compare the production processes of an antibody-based biotherapeutic in both bioreactor types, to enhance production flexibility. The findings indicate that, when compared to STRs, IFSB demonstrates the capability to produce an antibody-based biotherapeutic with either comparable or enhanced bioprocess performance and product quality. IFSB reduces shear damage to cells, enhances viable cell density (VCD), and improves cell state at a 5-L scale. Consequently, this leads to increased protein expression (3.70 g/L vs 2.56 g/L) and improved protein quality, as evidenced by a reduction in acidic variants from 27.0% to 21.5%. Scaling up the culture utilizing the Froude constant and superficial gas velocity ensures stable operation, effective mixing, and gas transfer. The IFSB maintains a high VCD and cell viability at both 50-L and 500-L scales. Product expression levels range from 3.0 to 3.6 g/L, accompanied by an improved acidic variants attribute of 20.6%-22.7%. The IFSB exhibits superior productivity and product quality, underscoring its potential for incorporation into the manufacturing process for antibody-based biotherapeutics. These results establish the foundation for IFSB to become a viable option in producing antibody-based biotherapeutics for clinical and manufacturing applications.

Highly drug/target-tolerant neutralizing antibody (NAb) assay development through target-based drug depletion and drug-based NAb extraction for an anti-EGFR therapeutic monoclonal antibody.

The reduction of immunogenicity is fundamental for the development of biobetter Erbitux, given that the development of an immune response reduces treatment efficacy and may lead to potential side effects. One of the requirements for the clinical research of a Erbitux biobetter candidate (CMAB009) is to develop a neutralizing antibody (NAb) assay, and sufficient drug and target tolerance for the assay is necessary. Here, we describe the development of a competitive ligand binding (CLB) assay for CMAB009 with high drug and target tolerance through target-based drug depletion and drug-based NAb extraction, the integrated experimental strategy was implemented to simultaneously mitigate drug interference and enhance target tolerance. Following troubleshooting and optimization, the NAb assay was validated for clinical sample analysis with the sensitivity of 92 ng/mL, drug tolerance of 70 µg/mL and target tolerance of 798 ng/mL. The innovative drug depletion and NAb extraction achieved though the combination of drug and target beads would enable the development of reliable NAb assays for many other therapeutics that overcome drug and its target interference for more precise and sensitive NAb assessment.

Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy.

CD47 and its receptor signal regulatory protein α (SIRPα) act as a dominant antiphagocytic, "don't eat me" signal. Recent studies reveal CD24 as a novel target for cancer immunotherapy by macrophages in ovarian cancer and breast cancer. However, whether simultaneous blockade of CD47 and CD24 by a bispecific antibody may result in a potential synergy is still unclear. In the present study, we for the first time designed and developed a bispecific antibody fusion protein, PPAB001 for cotargeting CD47 and CD24. Data demonstrate that simultaneous blockade of CD47/SIRPα and CD24/Siglec-10 signaling by PPAB001 potently promoted macrophage phagocytosis of tumor cells. Compared to single CD47 or CD24 targeting agents, PPAB001 was more effective in inhibiting tumor growth in both mouse 4T-1 syngeneic and human SK-OV-3 xenogeneic tumor models. Mechanistically, we found that PPAB001 therapy markedly increased the proportion of tumor-infiltrating macrophages and upregulated interleukin-6 and tumor necrosis factor-α levels that were representative macrophage inflammatory cytokines. Notably, an increased ratio of M1/M2 in tumor-infiltrating macrophages in the mice treated with PPAB001 suggested that the dual blockade may promote the transition of macrophages from M2 to M1. Taken together, our data supported the development of PPAB001 as a novel immunotherapeutic in the treatment of CD47 and CD24 double-positive cancers.

Population Pharmacokinetics of CMAB008 (an Infliximab Biosimilar) and Remicade® in Healthy Subjects and Patients with Moderately to Severely Active Rheumatoid Arthritis.

INTRODUCTION: CMAB008 is a monoclonal antibody developed as a biosimilar to infliximab (Remicade®, Janssen). The pharmacokinetic characteristics of CMAB008 and Remicade® in healthy subjects and patients with moderately to severely active rheumatoid arthritis (RA) were investigated using a population modeling approach, and the pharmacokinetic similarity of CMAB008 to Remicade® was assessed. METHODS: The population pharmacokinetic model was developed on the basis of intensive pharmacokinetic data from a phase 1 study in healthy male subjects and combined intensive and sparse pharmacokinetic data from a phase 3 study in patients with RA. RESULTS: A two-compartment model with first-order elimination adequately described CMAB008 and Remicade® concentration data in healthy subjects and patients with RA. The analysis of covariates identified anti-drug antibody (ADA), neutralizing antibody (NAB), real-time body weight (BWT), and real-time albumin (ALB) as significant covariates on clearance, and BWT was also a significant covariate for the central volume of distribution. The treatment type (CMAB008 versus Remicade®) and the study population (healthy subjects versus patients with RA) were not identified as significant covariates on the pharmacokinetics of infliximab, demonstrating pharmacokinetic similarity between CMAB008 and Remicade® in both study populations. The effect of BWT and ALB changes on exposures to infliximab was within the acceptable range, suggesting that the 3 mg/kg regimen is appropriate in clinical practice for patients with RA and BTW and ALB distribution within the range evaluated in the current analysis. CONCLUSIONS: The pharmacokinetic characteristics were similar between CMAB008 and Remicade® in healthy subjects and patients with RA. CMAB008 can be considered bioequivalent to Remicade®. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifiers NCT04779892, NCT03478111.

A Randomized, Single-dose, Phase I Clinical Comparison of a Trastuzumab Biosimilar With a Reference Trastuzumab Formulation in Healthy Chinese Male Volunteers.

The test drug, a recombinant humanized monoclonal antibody, is a biosimilar candidate for the reference drug. The purpose of this study was to evaluate the bioequivalence of these two drugs. The study was divided into two parts, a pre-study and a formal trial. The pre-study included two subjects who were each given a single intravenous infusion of 6 mg/kg test drug. The formal trial was designed to be a randomized, double-blind, parallel controlled trial in which 70 subjects were randomly assigned 1:1 to receive either test or reference drug as a single 6 mg/kg intravenous infusion. In the pre-study, the immunogenicity was negative in both subjects and the safety of the test drug was considered to be good. The two groups in the formal trial had similar demographic characteristics. The 90% confidence interval of geometric mean ratios of area under the serum concentration-time curve from the time 0 to the time of last quantifiable concentration, area under the serum concentration-curve from time 0 to infinity, and maximum observed serum concentration between the test group and the reference group fell between 80% and 125% and the bioequivalence was recognized. There was no significant difference in the positive rate of antidrug antibodies. The treatment-emergent adverse events in the test group were similar to those in the reference group. This study showed that the test drug has similar pharmacokinetics, immunogenicity, and safety to the reference drug in healthy male subjects.

Mass spectrometry-based multi-attribute method for mutation analysis in the early development of therapeutic proteins.

The early intervention is essential, and later development cannot compensate for this initial generation of an antibody drug. Especially for sequence variants (SVs), should cause concern during the early bioprocess development. The advancement of bioprocess development is paralleled by development of state-of-the-art analytical methods that will provide further information. In the present study, a mass spectrometry (MS)-based multi-attribute method (MAM) was used to simultaneously monitor the SVs and other quality attributes in the early bioprocess development of ofatumumab, and a sequence variant (SV) was detected by a subunit-based MAM. Subsequently, the variant was further identified by MS/MS and confirmed by adding a synthetic peptide. Furthermore, the content of the SV was detected via DNA sequencing. The levels of the variant (T175A mutant) in the light chain were demonstrate to be nearly consistent at the DNA and protein levels, suggesting that the mutation may have negligible effect on both the transcriptional and translational levels. Collectively, these results indicate that broad-spectrum, rapid, and accurate platform such as MS-based MAM should be implemented to quality control for the early development of therapeutic proteins, it will also be important to establish an effective and integrated MAM to control SVs during therapeutic proteins development.

A Randomized, Double-Blind, Parallel Controlled, Single-Dose Phase I Study Comparing the Pharmacokinetics, Safety, and Immunogenicity of the Infliximab Biosimilar CMAB008 and the Reference Product in Healthy Chinese Male Subjects.

This study aimed to evaluate the pharmacokinetics (PK), safety, and immunogenicity of the infliximab biosimilar CMAB008 compared to the reference product (Remicade) in healthy Chinese male subjects to provide the basis for the similarity evaluation of the 2 drugs. In this phase I randomized, double-blind, parallel-controlled, single-dose study, a total of 90 subjects were randomized 1:1 to receive CMAB008 or infliximab reference product with single intravenous injections (5 mg/kg). Blood samples were collected at designed time points for PK and immunogenicity assessment. If the 90%CI of the geometric mean ratio of area under the plasma concentration-time curve from 0 to the time of the last observation, maximum observed plasma concentration, area under the plasma concentration-time curve from 0 to infinity was completely within the range of 80% to 125%, the PK bioequivalence was established. Other PK parameters including time to maximum plasma concentration, half-life time, clearance, apparent volume of distribution, and last measurable concentration time point were also assessed. Adverse events (AEs) were recorded. Serum concentration-time profiles were similar across the 2 groups, and PK parameters were comparable in the 2 groups. The 90%CI of the geometric mean ratio of test to reference was within the predefined bioequivalence range of 80% to 125%. The AEs occurred similarly in 2 groups. One serious AE (rhabdomyolysis, grade 3) occurred in the test group. The total positive rates of antidrug antibody and neutralizing antibodies in the test group (85.7% and 5.6%, respectively) were numerically lower than infliximab reference product group (90.9% and 15%, respectively). The PK profile of the 2 groups is statistically equivalent. The preliminary safety and immunogenicity evaluation of the 2 drugs are comparable.

Identification, Efficacy, and Stability Evaluation of Succinimide Modification With a High Abundance in the Framework Region of Golimumab.

Succinimide (Asu) is the intermediate for asparagine deamidation in therapeutic proteins, and it can be readily hydrolyzed to form aspartate and iso-aspartate residues. Moreover, Asu plays an important role in the protein degradation pathways, asparagine deamidation, and aspartic acid isomerization. Here, Asu modification with a high abundance in the framework region (FR) of golimumab was first reported, the effect of denaturing buffer pH on the Asu modification homeostasis was studied, and the results revealed that it was relatively stable over a pH range of 6.0-7.0 whereas a rapid decrease at pH 8.0. Then, the peptide-based multi-attribute method (MAM) analyses showed that the Asu formation was at Asn 43 in the FR of the heavy chain. Meanwhile, the efficacy [affinity, binding and bioactivity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated cytotoxicity (ADCC) activity] and stability of the Asu modification of golimumab were evaluated, and the current results demonstrated comparable efficacy and stability between the Asu low- and high-abundance groups. Our findings provide valuable insights into Asu modification and its effect on efficacy and stability, and this study also demonstrates that there is a need to develop a broad-spectrum, rapid, and accurate platform to identify and characterize new peaks in the development of therapeutic proteins, particularly for antibody drugs.

N-Glycosylation and enzymatic activity of the rHuPH20 expressed in Chinese hamster ovary cells.

rHuPH20, a neutral pH-active hyaluronidase that degrades glycosaminoglycans under physiologic conditions, has six potential N-glycosylation sites. In this report, the rHuPH20 expressed in Chinese hamster ovary (CHO) cells was analyzed and characterized using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Removal of the N-linked glycans from rHuPH20 with PNGase F shifted the molecular weight from 66 kDa to approximately 52 kDa, its deduced molecular weight based on sequence analysis, suggesting that most, if not all, of the potential N-glycosylation sites are linked to oligosaccharides. Then the N-linked glycans released from the rHuPH20 by PNGase F were characterized by UPLC-FLR-MS, and the six N-glycosylation sites of the rHuPH20 were identified and characterized by UPLC-MS/MS at peptide levels. Subsequently, we found that the rHuPH20 increased the dispersion of locally subcutaneous injected drugs and the in vitro and in vivo bioactivity were decreased significantly after PNGase F treatment. In particular, rHuPH20 significantly augmented the absolute bioavailability of locally subcutaneous injected large protein therapeutics, while the bioavailability decreased after being digested by PNGase F. These results demonstrated that N-glycosylation is important for the bioactivity of the rHuPH20.

Biosimilar or Not: Physicochemical and Biological Characterization of MabThera and Its Two Biosimilar Candidates.

The development of therapeutic biosimilar antibodies has become an important driving force of the modern biopharmaceutical industry. In this study, physiochemical characteristics (amino acid sequence, intact/subunit molecular weight, isoelectric point, post-translation modification, and disulfide linkage pattern), purity (charge variants, high and low molecular weight variants), antigen binding activity, Fc receptor binding affinity and Fc-effector function (CDC and ADCC) were analyzed by using an extensive set of state-of-the-art and orthogonal analytical technologies to provide a comprehensive characterization of the innovative product rituximab and two biosimilar candidates. The similarity study showed that biosimilar candidate 1 (BC1) and the reference product (RP) MabThera had an identical protein amino acid sequences and highly similar primary structures along with similar purity, heterogeneity profiles, antigen binding activity, Fc receptor binding affinity, and Fc-effector functions. Biosimilar candidate 2 (BC2), which had an amino acid replacement at a constant region, a different N-glycosylation profiling, and purity, was not analytically similar to RP. Although BC2 showed improvement such as an increased level of afucose, another IgG1 allotype, and similar biological activities, it was not recommended to be applied as a biosimilar compound in drug registration because the biosimilar manufacturer must first show that its primary structure was identical to that of RP. Our physicochemical characterizations and bioassay comparability study provided a deepened understanding of the structure-function relationship of quality attributes.

Physicochemical characterization and phase I study of CMAB008, an infliximab biosimilar produced by a different expression system.

BACKGROUND: Infliximab (Remicade), a chimeric monoclonal antibody against human TNFα, will inevitably face competition from biosimilar products, because of its effectiveness in autoimmune diseases and rapidly increasing market demand. According to guidelines for biosimilar development, the "biosimilar-expression system" may differ from that of the innovator, but more appropriate studies should be carried out to demonstrate the comparability between biosimilar and innovator. CMAB008 is an infliximab biosimilar candidate developed by the State Key Laboratory of Antibody Medicine and Targeted Therapy of China. Infliximab was expressed in SP2/0 cells, while CMAB008 was produced in a CHO-expression system. METHODS: In this study, infliximab and CMAB008 were compared on physicochemical and biological characterizations, including protein content, activity, physiochemical integrity, impurities, additives, and immunogenicity. RESULTS: The results showed that they were highly similar and comparable, except some differences in glycosylation. As glycosylation profiles can influence immunogenicity and occurrence of allergy or other adverse reactions of antibody therapeutics, primary tolerability and pharmacokinetics of CMAB008 were evaluated. In the phase I clinical trial, plasma concentration of CMAB008 and antidrug antibodies were also measured using ELISA and bridging ELISA, respectively. CMAB008 exhibited favorable clinical tolerability, no adverse events in the 3 mg/kg single-dose group (recommended therapeutic dosage), and no serious adverse events in the multiple-dose group. Also, no injection-site reactions were observed in the experiment. CONCLUSION: In summary, CMAB008 might have the potential to be an effective drug compared with infliximab.

A novel glycosylated anti-CD20 monoclonal antibody from transgenic cattle.

The monoclonal antibody (mAb) against CD20 known as Rituxan is widely used to treat autoimmune diseases and lymphomas. However, further application of Rituxan faces challenges of high production cost, which limits its availability in developing countries. Here, we report a new approach for large production of a recombinant anti-CD20 mAb in the milk of transgenic cattle (at a yield of up to ~6.8 mg/mL), with ~80% recovery rate and >99% purity. Crystallography study showed that our recombinant mAb is structurally nearly identical to Rituxan with only minor differences in N-linked glycosylation pattern. Functional study showed that, while our mAb shared similar target-cell binding capacities and complement-dependent cytotoxicity with Rituxan, our product exhibited a higher binding affinity for FcγRIIIα and a greater antibody-dependent cellular cytotoxicity. Accordingly, our recombinant mAb demonstrated a superior efficacy over Rituxan against B-cell lymphomas in severe combined immunodeficiency mice. Taken together, our data supports transgenic cattle as a novel model for cost-competitive, large-scale production of therapeutic antibodies.

Elimination of tumor by CD47/PD-L1 dual-targeting fusion protein that engages innate and adaptive immune responses.

The host immune system generally serves as a barrier against tumor formation. Programmed death-ligand 1 (PD-L1) is a critical "don't find me" signal to the adaptive immune system, whereas CD47 transmits an anti-phagocytic signal, known as the "don't eat me" signal, to the innate immune system. These and similar immune checkpoints are often overexpressed on human tumors. Thus, dual targeting both innate and adaptive immune checkpoints would likely maximize anti-tumor therapeutic effect and elicit more durable responses. Herein, based on the variable region of atezolizumab and consensus variant 1 (CV1) monomer, we constructed a dual-targeting fusion protein targeting both CD47 and PD-L1 using "Knobs-into-holes" technology, denoted as IAB. It was effective in inducing phagocytosis of tumor cells, stimulating T-cell activation and mediating antibody-dependent cell-mediated cytotoxicity in vitro. No obvious sign of hematological toxicity was observed in mice administered IAB at a dose of 100 mg/kg, and IAB exhibited potent antitumor activity in an immune-competent mouse model of MC38. Additionally, the anti-tumor effect of IAB was impaired by anti-CD8 antibody or clodronate liposomes, which implied that both CD8+ T cells and macrophages were required for the anti-tumor efficacy of IAB and IAB plays an essential role in the engagement of innate and adaptive immune responses. Collectively, these results demonstrate the capacity of an elicited endogenous immune response against tumors and elucidate essential characteristics of synergistic innate and adaptive immune response, and indicate dual blockade of CD47 and PD-L1 by IAB may be a synergistic therapy that activates both innate and adaptive immune response against tumors.

Alterations in expressed prostate secretion-urine PSA N-glycosylation discriminate prostate cancer from benign prostate hyperplasia.

The prostate specific antigen (PSA) test is widely used for early diagnosis of prostate cancer (PCa). However, its limited sensitivity has led to over-diagnosis and over-treatment of PCa. Glycosylation alteration is a common phenomenon in cancer development. Different PSA glycan subforms have been proposed as diagnostic markers to better differentiate PCa from benign prostate hyperplasia (BPH). In this study, we purified PSA from expressed prostate secretions (EPS)-urine samples from 32 BPH and 30 PCa patients and provided detailed PSA glycan profiles in Chinese population. We found that most of the PSA glycans from EPS-urine were complex type biantennary glycans. We observed two major patterns in PSA glycan profiles. Overall there was no distinct separation of PSA glycan profiles between BPH and PCa patients. However, we detected a significant increase of glycan FA2 and FM5A2G2S1 in PCa when compared with BPH patients. Furthermore, we observed that the composition of FA2 glycan increased significantly in advanced PCa with Gleason score ≥8, which potentially could be translated to clinic as a marker for aggressive PCa.

[Characterization of N-glycosylation in an anti-EGFR monoclonal antibody produced by different expression systems].

The use of mammalian expression systems results in a remarkable heterogeneity of mAb products, generally due to post-translational modifications, and glycosylation is a critical post-translation modification because it has a profound impact on the safety and efficacy of mAbs. The present study was designed to explore the impact of a different expression system on mAb N-glycosylation. The detailed structures of individual glycans between anti-EGFR monoclonal antibodies produced by different expression systems were successfully characterized at the level of free oligosaccharides using liquid chromatography electrospray ionization quadrupole time-of-fight mass spectrometry (LC-ESI-QTof MS). An alternating low and elevated collision energy scan, in source collision-induced dissociation and MS/MS in combination with exoglycosidase digestion method was also adopted. The combined data revealed that the Fab region of anti-EGFR antibody produced by CHO cell expression system had a pattern of glycosylation differing from that of the SP2/0 cell expression system whereas the Fc region remained basically unchanged. We confirmed that anti-EGFR antibody produced by SP2/0 cell expression system had a much more diverse mixture of glycans with α-Gal and an undesired, aberrant form of sialylation N-glycolylneuraminic acid (NGNA). The α-Gal was absent in mAb produced by CHO cell expression system containing sialic acid predominantly N-acetyl neuraminic acid (NANA) which is the desired, normal human-type sialylation. This study theoretically predicts that anti-EGFR antibody produced by CHO cell expression system may show better clinical tolerance, and very low potential for active hypersensitivity reactions, CHO cell lines can be the preferred expression system for producing anti-EGFR biobetter.

Therapeutic efficacy of an anti-PD-L1 antibody based immunocytokine in a metastatic mouse model of colorectal cancer.

Immunocytokines (antibody-cytokine fusions) have been proved to be a promising class of therapeutic agents for tumors. Anti-PD-L1 antibodies or IL-2 have been used to treat a variety of cancers. Here, in order to remove T cell inhibition and increasing the IL-2 concentration in the tumor microenvironment, we engineered a novel anti-PD-L1 antibody based immunocytokine by fusing hIL-2 to the C-Term of atezolizumab, denoted as BIPI. Our results revealed that BIPI was effective in stimulating T cell activation in vitro and could selectively localize to the tumor. Furthermore, tumor regression and prolonged survival were also observed in the metastatic colorectal cancer mouse model. The obviously longer survival mice in BIPI treatment group turned out depending on the function of CD8+ T cells. The IFN- secreted from CD8+ T cells in the spleen also contributed to the better tumor inhibition profile in BIPI treatment group than in anti-PD-L1 or IL-2 treatment alone. Taken together, our data evidenced the enhanced antitumor potency of BIPI, suggesting its potential use for cancers with a low response to the anti-PD-L1 or IL-2 treatment.

In-Depth Characterization of a Pro-Antibody-Drug Conjugate by LC-MS.

Pro-antibody-drug conjugate (PDC) is a hybrid structural format of immunoconjugate, where the structural complexity of pro-antibody and intrinsic heterogeneity of ADCs impose a prominent analytical challenge to the in-depth characterization of PDCs. In the present study, we successfully prepared and characterized PanP-DM1 as a model of PDCs, which is an anti-EGFR pro-antibody following conjugation with DM1 at lysine residues. The drug-to-antibody ratio (DAR) of PanP-DM1 was determined by LC-MS after deglycosylation, and verified by UV/vis spectroscopy. Following reduction or IdeS digestion, the pro-antibody fragments linked with DM1 were investigated by middle-down mass spectrometry. Furthermore, more than 20 modified lysine conjugation sites were determined by peptide mapping after trypsin digestion. Additionally, more than ten glycoforms of PanP-DM1 were also identified and quantified. In summary, critical quality attributes (CQAs) of PDCs including DAR, drug load distribution, and conjugation sites were fully characterized, which would contribute to the development of other PDCs for cancer treatment.

Acid-induced aggregation propensity of nivolumab is dependent on the Fc.

Nivolumab, an anti-programmed death (PD)1 IgG4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG1), panitumumab (IgG2) and atezolizumab (aglyco-IgG1) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG1 < aglyco-IgG1 < IgG2 < IgG4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG4 backbone.