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.

A human-specific insertion promotes cell proliferation and migration by enhancing TBC1D8B expression.

Human-specific insertions play important roles in human phenotypes and diseases. Here we reported a 446-bp insertion (Insert-446) in intron 11 of the TBC1D8B gene, located on chromosome X, and traced its origin to a portion of intron 6 of the EBF1 gene on chromosome 5. Interestingly, Insert-446 was present in the human Neanderthal and Denisovans genomes, and was fixed in humans after human-chimpanzee divergence. We have demonstrated that Insert-446 acts as an enhancer through binding transcript factors that promotes a higher expression of human TBC1D8B gene as compared with orthologs in macaques. In addition, over-expression TBC1D8B promoted cell proliferation and migration through "a dual finger" catalytic mechanism (Arg538 and Gln573) in the TBC domain in vitro and knockdown of TBC1D8B attenuated tumorigenesis in vivo. Knockout of Insert-446 prevented cell proliferation and migration in cancer and normal cells. Our results reveal that the human-specific Insert-446 promotes cell proliferation and migration by upregulating the expression of TBC1D8B gene. These findings provide a significant insight into the effects of human-specific insertions on evolution.

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.

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.

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.

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.

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells.

Nivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC- UV- MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products.

Preclinical studies of a Pro-antibody-drug conjugate designed to selectively target EGFR-overexpressing tumors with improved therapeutic efficacy.

Antibody-drug conjugates (ADCs) have exhibited potent clinical benefits in cancer therapy. However, development of ADCs against epidermal growth factor receptor (EGFR) has limitations because of wide expression of EGFR in both normal and tumor tissues. Previously, we developed an anti-EGFR protease-activated antibody (pro-antibody), termed as PanP, which remains inert against EGFR until activated by tumor-specific protease. Herein, we for the first time report a new class of pro-antibody-drug conjugate (PDC) against EGFR, denoted as PanP-DM1. It has been designed to selectively target the EGFR-overexpressing tumor cells and exert greater anti-tumor activity compared with PanP. Our data showed that PanP-DM1 also could be selectively activated by tumor-specific protease 'uPA'. Furthermore, activated PanP-DM1 was potently cytotoxic against EGFR-overexpressing tumor cell lines in vitro. Crucially, our data indicated that PanP-DM1 was significantly more effective in eradicating EGFR-overexpressing tumors in vivo. Additionally, toxicity was preliminarily evaluated in mice as measured by body weight loss. In summary, our study suggests that PanP-DM1, a novel pro-antibody-drug conjugate, has cancer-selectivity, efficacy and safety profile that supports its potential use for EGFR-overexpressing tumors.

CD20 Antibody-Conjugated Immunoliposomes for Targeted Chemotherapy of Melanoma Cancer Initiating Cells.

Cancer initiating cells (CIC) are tumorigenic cancer cells that have properties similar to normal stem cells. CD20 is a phenotype of melanoma CIC that is responsible for melanoma drug resistance. Vincristine (VCR) is commonly used in melanoma therapy; however, it has been found ineffective against CIC. To target CD20+ melanoma CIC, we prepared VCR-containing immunoliposomes that were conjugated to CD20 antibodies (VCR-Lip-CD20). The drug release profile and the antibody-mediated targeting of the immunoliposomes were optimized to target CD20+ melanoma CIC. The immunoliposomes had desirable particle size (163 nm), drug encapsulation efficiency (91.8%), and drug release profile. We demonstrated that these immunoliposomes could successfully target more than 55% of CD20+ Chinese Hamster Ovary cells (CHO-CD20) even when the CHO-CD20 cells accounted for only 0.1% of a mixed population of CHO-CD20 and CHO cells. After treating WM266-4 melanoma mammospheres for 96 h, the ICo values of the drug delivered in VCR-Lip-CD20, VCR-Lip (VCR liposomes), and VCR were found to be 53.42, 98.99, and 99.09 µg/mL, respectively, suggesting that VCR-Lip-CD20 was 1.85 times more effective than VCR-Lip and VCR. VCR-Lip-CD20 could almost completely remove the tumorigenic ability of WM266-4 mammospheres in vivo, and showed the best therapeutic effect in WM266-4 melanoma xenograft mice. Significantly, VCR-Lip-CD20 could selectively kill CD20+ melanoma CIC in populations of WM266-4 cells both in vitro and in vivo. We demonstrated that VCR-Lip-CD20 has the potential to efficiently target and kill CD20+ melanoma CIC.

Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency.

Panitumumab, as a commercially available antibody, is an effective anticancer therapeutic against epidermal growth factor receptor (EGFR), although it exerts weak antibody-dependent cell-mediated cytotoxicity (ADCC) activity owing to its IgG2 nature. Here, we firstly engineered panitumumab by grafting its variable region into an IgG1 backbone. The engineered panitumumab (denoted as Pan) retained binding activity identical to the parental antibody while exhibiting stronger ADCC activity in vitro and more potent antitumor effect in vivo. To further enhance the target selectivity of Pan, we generated Pan-P by tethering an epitope-blocking peptide to Pan via a tumor-specific protease selective linker. Pan-P showed almost 40-fold weaker affinity compared with Pan, but functional activity was restored to a similar extent as Pan when Pan-P was selectively activated by urokinase-type plasminogen activator (uPA). More importantly, targeted localization of Pan-P was observed in tumor samples from colorectal cancer (CRC) patients and tumor-bearing nude mice, strongly indicating that specific activation also existed ex vivo and in vivo. Furthermore, Pan-P also exhibited effective in vivo antitumor potency similar to Pan. Taken together, our data evidence the enhanced antitumor potency and excellent target selectivity of Pan-P, suggesting its potential use for minimizing on-target toxicity in anti-EGFR therapy.

Molecular architecture of the ErbB2 extracellular domain homodimer.

Human epidermal growth factor receptors (HERs or ErbBs) play crucial roles in numerous cellular processes. ErbB2 is a key member of ErbB family, and its overexpression is recognized as a frequent molecular abnormality. In cancer, this overexpression correlates with aggressive disease and poor patient outcomes. Dimer-dependent phosphorylation is a key event for the signal transduction of ErbBs. However, the molecular mechanism of the dimerization of ErbB2 remains elusive. In the present work, we report the homodimer architecture of the ErbB2 extracellular domain (ECD) which is unique compared with other dimer-models of ErbBs. The structure of the ErbB2 ECD homodimer represents a "back to head" interaction, in which a protruding ß-hairpin arm in domain II of one ErbB2 protomer is inserted into a C-shaped pocket created by domains I-III of the adjacent ErbB2 protomer. This dimerized architecture and its impact on the phosphorylation of ErbB2 intracellular domain were further verified by a mutagenesis study. We also elucidated the different impacts of two clinically administered therapeutic antibodies, trastuzumab and pertuzumab, on ErbB2 dimerization. This information not only provides an understanding of the molecular mechanism of ErbBs dimerization but also elucidates ErbB2-targeted therapy at the molecular level.