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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.

Supplementary Figures S1-S2 from Genetically Targeted T Cells Eradicate Established Breast Cancer in Syngeneic Mice

Purpose: CD11c is an antigen receptor predominantly expressed on dendritic cells (DC), to which antigen targeting has been shown to induce robust antigen-specific immune responses. To facilitate targeted delivery of tumor antigens to DCs, we generated fusion proteins consisting of the extracellular domain of human HER or its rat homologue neu, fused to the single-chain fragment variable specific for CD11c (scFvCD11c-HER2/neu).Experimental Design: Induction of cellular and humoral immune responses and antitumoral activity of the fusion proteins admixed with DC-activating CpG oligonucleotides (scFvCD11c-HER2/neuCpG) were tested in transplantable HER2/neu-expressing murine tumor models and in transgenic BALB-neuT mice developing spontaneous neu-driven mammary carcinomas.Results: Vaccination of BALB/c mice with scFvCD11c-HER2CpG protected mice from subsequent challenge with HER2-positive, but not HER2-negative, murine breast tumor cells, accompanied by induction of strong HER2-specific T-cell and antibody responses. In a therapeutic setting, injection of scFvCD11c-HER2CpG caused rejection of established HER2-positive tumors. Importantly, antitumoral activity of such a fusion protein vaccine could be reproduced in immunotolerant BALB-neuT mice, where scFvCD11c-neuCpG vaccination significantly protected against a subsequent challenge with neu-expressing murine breast tumor cells and markedly delayed the onset of spontaneous mammary carcinomas.Conclusions: CD11c-targeted protein vaccines for in vivo delivery of tumor antigens to DCs induce potent immune responses and antitumoral activities and provide a rationale for further development of this approach for cancer immunotherapy.

We reported previously the development of SMFv-PE38KDEL type I mutant (PE38KDEL-I; Mut-I), a recombinant immunotoxin in which a single-chain antibody derived from mouse SM5-1 monoclonal antibody is genetically fused to PE38KDEL-I. In comparison with the SMFv-PE38KDEL wild-type, Mut-I showed improved therapeutic efficacy and reduced toxicity. To overcome the problems associated with the immune response to the Pseudomonas exotoxin A (PE) component of Mut-I, we have constructed PE38KDEL-I-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with F(ab′) fragments of a humanized SM5-1 monoclonal antibody (PE-NP-S). PE-NP-S specifically bound to SM5-1 binding protein-expressing hepatocellular carcinoma cell lines and was then internalized by these cells, resulting in significant cytotoxic effect. In SM5-1 binding protein-overexpressing tumor xenograft model, administration of PE-NP-S significantly inhibited tumor development and induced tumor regression. Moreover, PE-NP-S was shown to be much weaker in inducing vascular leakage syndrome in mice than Mut-I. The LD50 of PE-NP-S was about 4-fold higher than that of Mut-I. Remarkably, PE-NP-S was of low immunogenicity in development of anti-PE neutralizing antibodies in vivo and was less susceptible to inactivation by anti-PE neutralizing antibodies compared with Mut-I. In conclusion, the resultant PE-NP-S possessed increased cancer therapeutic efficacy and had reduced nonspecific toxicity and immunogenicity, suggesting that it is a potential candidate in cancer therapy. [Mol Cancer Ther 2008;7(10):3399–407]

Supplementary Data from Targeted Delivery of Tumor Antigens to Activated Dendritic Cells via CD11c Molecules Induces Potent Antitumor Immunity in Mice

Supplementary Fig. S1 from Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F(ab′) fragments

Purpose: The purpose of the present study was to evaluate the capacity and mechanisms of genetically modified erbB2-specific T cells to eradicate erbB2+ tumors in syngeneic mice.Experimental Design: Primary mouse T cells were modified to target the breast tumor–associated antigen erbB2 through retroviral-mediated transfer of a chimeric antigen receptor, termed single-chain antibody (scFv)–CD28–ζ. Antitumor efficacy of scFv-CD28-ζ–modified T cells was analyzed in mice bearing D2F2/E2 breast tumors.Results: The scFv-CD28-ζ–modified T cells were shown to specifically secrete T cytotoxic-1 cytokines and lyse erbB2+ breast tumor cells following receptor stimulation in vitro. Treatment with scFv-CD28-ζ–modified T cells was able to lead to long-term, tumor-free survival in mice bearing erbB2+ D2F2/E2 breast tumors. Importantly, the surviving mice developed a host memory response to D2F2/E2 tumor cells, and this host response was able to protect against a rechallenge with erbB2+ D2F2/E2 tumor cells and parental erbB2- D2F2 tumor cells. In addition, scFv-CD28-ζ T-cell expression of perforin and interferon-γ were essential for complete antitumor efficacy.Conclusions: Treatment with scFv-CD28-ζ–modified T cells was able to induce a host antitumor immunity in syngeneic mice. Complete tumor elimination by scFv-CD28-ζ–modified T cells required T cell–derived interferon-γ and perforin, indicating that cytotoxicity and cytokine secretion play a role in the in vivo response.

Supplementary Methods and Materials from Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

Supplementary Figure Legends 1-4 from Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

Supplementary Figure 4 from Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

Supplementary Figure 3 from Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

Supplementary Materials and Methods, Figure Legends 1-2 from Suppression of Tumor Growth and Metastasis by Simultaneously Blocking Vascular Endothelial Growth Factor (VEGF)–A and VEGF-C with a Receptor-Immunoglobulin Fusion Protein

To develop more effective anti-CD20 reagents for B-cell lymphoma, we designed and constructed a bispecific tetravalent anti-CD20 antibody, 11B8/2F2(ScFvHL)4-Fc, derived from two fully human monoclonal antibodies (mAb), 2F2 and 11B8. 2F2 is a type I CD20 mAb, which is potent in complement-dependent cytotoxicity (CDC) assays but poor at inducing apoptosis, whereas 11B8 is a type II CD20 mAb, which is effective in induction of apoptosis but ineffective in CDC. Our results showed that 11B8/2F2(ScFvHL)4-Fc possessed apoptosis-inducing activity markedly superior to that of 2F2, and even 11B8, 11B8 plus 2F2, and 2F2(ScFvHL)4-Fc, a 2F2-derived monospecific tetravalent antibody developed previously. Interestingly, 11B8/2F2(ScFvHL)4-Fc displayed a similar ability to mediate CDC as 2F2(ScFvHL)4-Fc, although two of its four antigen-binding arms originated from 11B8. To explore why 11B8/2F2(ScFvHL)4-Fc was so potent in both CDC and apoptotic activity, a bispecific divalent antibody composed of 2F2 and 11B8, denoted as 11B8/2F2-ScFvFc, was constructed and characterized. Our results partially explained the reason for the potent CDC and apoptosis-inducing activity of 11B8/2F2(ScFvHL)4-Fc. Further in vivo therapy studies showed that 11B8/2F2(ScFvHL)4-Fc had a significantly more potent antitumor activity compared with 2F2, 11B8, 2F2 plus 11B8, and 2F2(ScFvHL)4-Fc. These data suggest that 11B8/2F2(ScFvHL)4-Fc may serve as a potential therapeutic agent for B-cell lymphoma. Cancer Res; 70(15); 6293–302. ©2010 AACR.

Supplementary Figure 1 from Construction and Characterization of a Bispecific Anti-CD20 Antibody with Potent Antitumor Activity against B-Cell Lymphoma

Supplementary Methods, Figure Legends 1-2 from Construction and Characterization of a Bispecific Anti-CD20 Antibody with Potent Antitumor Activity against B-Cell Lymphoma

Supplementary Figure 2 from Construction and Characterization of a Bispecific Anti-CD20 Antibody with Potent Antitumor Activity against B-Cell Lymphoma

Supplementary Figure 2 from Suppression of Tumor Growth and Metastasis by Simultaneously Blocking Vascular Endothelial Growth Factor (VEGF)–A and VEGF-C with a Receptor-Immunoglobulin Fusion Protein

PDF file - 150K, Relative binding affinity of anti-ErbB2 antibodies for the trastuzumab epitope on ErbB2 in BT-474 cells (S1); Relative binding affinity of anti-ErbB2 antibodies for the pertuzumab epitope on ErbB2 in BT-474 cells (S2); Binding affinities of recombinant anti-ErbB2 antibodies for ErbB2-ECD (S3); Pharmacokinetic parameters of recombinant anti-ErbB2 antibodies in mice (S4); ErbB receptor expression on breast cancer cell lines (S5).

PDF file - 362K, Competitive binding assay (S1); TPL inhibits ErbB2 signaling in breast cancer cell lines (S2); ErbB2 knockdown inhibits ErbB2 signaling and results in growth inhibition in the MDA-MB-231 cell line (S3); TPL inhibited MDA-MB-231 breast cancer cell growth in a dose-dependent manner both in vitro and in vivo (S4); The effects of siRNA knockdown of EGFR or ErbB3 on the BT-474TraR cell line (S5).

Supplementary Figure 1 from Suppression of Tumor Growth and Metastasis by Simultaneously Blocking Vascular Endothelial Growth Factor (VEGF)–A and VEGF-C with a Receptor-Immunoglobulin Fusion Protein

The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. However, resistance to trastuzumab is common. Heterodimerization between ErbB2 and other ErbBs may redundantly trigger cell proliferation signals and confer trastuzumab resistance. Here, we developed a bispecific anti-ErbB2 antibody using trastuzumab and pertuzumab, another ErbB2-specific humanized antibody that binds to a distinct epitope from trastuzumab. This bispecific antibody, denoted as TPL, retained the full binding activities of both parental antibodies and exhibited pharmacokinetic properties similar to those of a conventional immunoglobulin G molecule. Unexpectedly, TPL showed superior ErbB2 heterodimerization-blocking activity over the combination of both parental monoclonal antibodies, possibly through steric hindrance and/or inducing ErbB2 conformational change. Further data indicated that TPL potently abrogated ErbB2 signaling in trastuzumab-resistant breast cancer cell lines. In addition, we showed that TPL was far more effective than trastuzumab plus pertuzumab in inhibiting the growth of trastuzumab-resistant breast cancer cell lines, both in vitro and in vivo. Importantly, TPL treatment eradicated established trastuzumab-resistant tumors in tumor-bearing nude mice. Our results suggest that trastuzumab-resistant breast tumors remain dependent on ErbB2 signaling and that comprehensive blockade of ErbB2 heterodimerization may be an effective therapeutic avenue. The unique potential of TPL to overcome trastuzumab resistance warrants its consideration as a promising treatment in the clinic. Cancer Res; 73(21); 6471–83. ©2013 AACR.

Modest clinical outcomes of dendritic cell (DC) vaccine trials call for novel strategies. In this study, we have created a chimeric CD40 molecule that incorporates a single chain Fv (scFv) molecule specific for human ErbB2 antigen and fusing to the membrane spanning and cytosolic domains of murine CD40. After adenoviral transfer to bone marrow–derived DC, this chimeric receptor (CR) induced nuclear factor-κB (NF-κB)–dependent DC activation and effector function when cultured with immobilized ErbB2 protein or ErbB2-positive tumor cells in vitro. In vivo migration assays showed that ∼40% injected CR-modified DC (scFv-CD40-DC) effectively migrated to ErbB2-positive tumors, where they were activated after ErbB2 antigen stimulation, and sequentially homed into the draining lymph nodes. In murine ErbB2-positive D2F2/E2 breast tumor (BALB/c) and EL4/E2 thymoma (C57BL/6) models, i.v. injection of 1 × 106 scFv-CD40-DC significantly inhibited tumor growth and cured established tumors. Importantly, the cured mice treated by injection of scFv-CD40-DC were effective in preventing both ErbB2-positive and parental ErbB2-negative tumor rechallenge. Analysis of the underlying mechanism revealed that i.v. infusion of scFv-CD40-DC elicited tumor-specific CTL responses, and the transfer of CTLs from scFv-CD40-DC–treated mice protected naive mice against a subsequent tumor challenge. These results support the concept that genetic modification of DC with tumor-associated antigen-specific CD40 chimeric receptor might be a useful strategy for treatment of human cancers. [Cancer Res 2008;68(10):3854–62]

Supplementary Figure 2 from Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

Predicting smartphone users location with WiFi fingerprints has been a popular research topic recently. In this work, we propose two novel deep learning-based models, the convolutional mixture density recurrent neural network and the VAE-based semi-supervised learning model. The convolutional mixture density recurrent neural network is designed for path prediction, in which the advantages of convolutional neural networks, recurrent neural networks and mixture density networks are combined. Further, since most of real-world datasets are not labeled, we devise the VAE-based model for the semi-supervised learning tasks. In order to test the proposed models, we conduct the validation experiments on the real-world datasets. The final results verify the effectiveness of our approaches and show the superiority over other existing methods., Comment: 11 pages, 10 figures

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.

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

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.

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.

Recognizing user location with WiFi fingerprints is a popular approach for accurate indoor positioning problems. In this work, our goal is to interpret WiFi fingerprints into actual user locations. However, WiFi fingerprint data can be very high dimensional in some cases, we need to find a good representation of the input data for the learning task first. Otherwise, using neural networks will suffer from severe overfitting. In this work, we solve this issue by combining the Information Bottleneck method and Variational Inference. Based on these two approaches, we propose a Variational Information Bottleneck model for accurate indoor positioning. The proposed model consists of an encoder structure and a predictor structure. The encoder is to find a good representation in the input data for the learning task. The predictor is to use the latent representation to predict the final output. To enhance the generalization of our model, we also adopt the Dropout technique for each hidden layer of the decoder. We conduct the validation experiments on a real-world dataset. We also compare the proposed model to other existing methods so as to quantify the performances of our method.

Analyzing human mobility with geo-location data collected from smartphones has been a hot research topic in recent years. In this paper, we attempt to discover daily mobile patterns using the GPS data. In particular, we view this problem from a probabilistic perspective. A non-parametric Bayesian modeling method, the Infinite Gaussian Mixture Model (IGMM) is used to estimate the probability density of the daily mobility. We also utilize the Kullback-Leibler (KL) divergence as the metrics to measure the similarity of different probability distributions. Combining the IGMM and the KL divergence, we propose an automatic clustering algorithm to discover mobility patterns for each individual user. Finally, the effectiveness of our method is validated on the real user data collected from different real users.

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.

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.

Energy consumption and power management are key issues problem when using smartphone handsets or any other connected devices. This is linked to two opposite users’ expectations: having a better autonomy on one side and having devices with more computer power and bigger screens on the other side. In literature, many previous works focused on modeling the smartphone power drain at the hardware or software levels. As opposed to these approaches, we present, in this paper the construction of a handset energy model at user level based on coarse grain collected data. To that way, we adopt a crowd-sourcing approach, based on data collected by the Device Analyzer project. This data is covering all the activities/events of the user relatively to his/her smartphone. However, the data is collected within a fixed time period which involved imprecisions in the time stamp associated to each event. So as to model the energy drain and to improve events time location, we propose a top-down method based on reactive multi-agent systems paradigm. In this approach, each event is represented by an agent in a virtual world which can move along the time axis according to the behavior of its mates and which influences the battery discharging curve. As soon as the multi-agent system is stabilized, the behavioral parameters of the agents are used to build up the power model. The value of the multi-agent approach is tied to its ability to incorporate implicit information about battery drain from operating system events such as network usage. Finally, the proposed modeling method is tested on real users data and several coordination strategies are compared. Index Terms—Smartphone, energy model, multi-agent model, crowd-sourcing, user behavior

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.

The rapid growth of biotherapeutics (monoclonal antibodies and antibody derivatives) demands improved techniques for their quality control and batch-to-batch study. Traditionally, the top-down and bottom-up mass spectrometric approaches may be good options, but they have their share of drawbacks. The middle-down technology has unique superiorities for fast characterization of very large proteins. In this article, we report a systematic approach to characterize Fc-containing proteins (antibody, Fc fusion protein, and antibody–drug conjugate) by middle-down mass spectrometry following IdeS proteolytic digestion. We characterized the main protein modifications including glycosylation, glycation, and other modifications with a relatively small proportion, and results were consistent with free glycan profiling analysis. Meanwhile, the O-acetylated sialic acid modification of cetuximab was reported for the first time; its potential roles in biotherapeutics need further study. The middle-down technology following IdeS proteolytic digestion could be used not only for analyzing the modifications of Fc-containing proteins at the subunit level but also for characterizing the potential modifications at early development stages.

During antibody drug research and development, it is important to make sure the protein therapeutics are biosynthesized correctly, and peptide mapping works as a powerful analytical approach to confirm the identity of the monoclonal antibodies (mAbs). In this study, a novel quality control method combining tryptic peptide mapping with QDa detector for characterization of biotherapeutics was developed and validated. The peptides derived from complementarity determining regions (CDRs) of adalimumab were used as surrogates of adalimumab. Ten other antibody drugs were used to assess the method specificity. The results showed that this method has high specificity due to the high UV–MS selectivity; the limit of detection was 20 μg mL−1; carry-over was about 1.69 %. Additionally, other validation parameters, like stability, were also evaluated. This novel LC–UV–QDa method may be a simple, cost-effective and robust peptide-mapping method for other recombinant monoclonal antibodies during routine quality control analysis.

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.

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 β-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.