Preparation of Anti-HER2 Monoclonal Antibody-paclitaxel Immunoconjugate and Its Biological Evaluation

Anti-HER2 monoclonal antibody （Sc7301）-paclitaxel （TAX） immunoconjugate was pre- pared and its specific binding to tumor cells was investigated in this study. Sc7301 was conjugated to TAX by the active ester method and then the TAX-Sc7301 immunoconjugate was obtained. After purification and labeling by Cyano-fluorescein isothiocyanate （FITC）, the specific binding of TAX-Sc7301 to HER2-positive tumor cells （SKOV3） and HER2-negative tumor cells （HepG2） was evaluated respectively. TAX-Sc7301 （20 nmol/L） showed distinct specific binding to SKOV3 cells rather than HepG2 cells. And the uptake of the immunoconjugate by SKOV3 cells was increased with the TAX-So7301 concentration （3-48 nmol/L） and the incubation time （P〈0.05）. It was concluded that the TAX-Sc7301 immunoconjugate is ootentially applicable as a targeted agent against HER2-10ositive tumor cells.

Epirubicin-[Anti-HER2/neu] Synthesized with an Epirubicin-(C13-imino)-EMCS Analog: Anti-Neoplastic Activity against Chemotherapeutic-Resistant SKBr-3 Mammary Carcinoma in Combination with Organic Selenium

Purpose: Discover the anti-neoplastic efficacy of epirubicin-(C13-imino)-[anti-HER2/neu] against chemotherapeutic- resistant SKBr-3 mammary carcinoma and delineate the capacity of selenium to enhance it’s cytotoxic anti-neoplastic potency. Methods: In molar excess, EMCH was combined with epirubicin to create a covalent epirubicin-(C13-imino)-EMCH-maleimide intermediate with sulfhydryl-reactive properties. Monoclonal immunoglobulin selective for HER2/neu was then thiolated with 2-iminothiolane at the terminal ε-amine group of lysine residues. The sulfhydryl-reactive epirubicin-(C13-imino)-EMCH intermediate was then combined with thiolated anti-HER2/neu monoclonal immunoglobulin. Western-blot analysis was utilized to characterize the molecular weight profiles while binding of epirubicin-(C13-imino)-[anti-HER2/neu] to membrane receptors was determined by cell-ELISA utilizing populations of SKBr-3 mammary carcinoma that highly over-expresses HER2/neu complexes. Anti-neoplastic potency of epirubicin-(C13-imino)-[anti-HER2/ neu] between the epirubicin-equivalent concentrations of 10–12 M and 10–7 M was determined by vitality staining analysis with and without the presence of selenium (5 μM). Results: Epiribucin-(C13-imino)-[anti-HER2/neu] between epirubicin-equivalent concentrations of 10–8 M to 10–7 M consistently evoked higher anti-neoplastic potency than “free” non- conjugated epirubicin which corresponded with previous investigations utilizing epirubicin-(C3-amide)-[anti-HER2/neu] and epirubicin-(C3-amide)-[anti-EGFR]. Selenium at 5 mM consistently enhanced the cytotoxic anti-neoplastic potency of epirubicin-(C13-imino)-[anti-HER2/neu] at epirubicin equivalent concentrations (10–12 to 10–7 M). Conclusions: Epirubicin-(C13-imino)-[anti-HER2/neu] is more potent than epirubicin against chemotherapeutic-resistant SKBr-3 mammary carcinoma and selenium enhances epirubicin-(C13-imino)-[anti-HER2/neu] potency. The methodology applied for synthesizing epirubicin-(C13-imino)-[anti-HER2/neu] is relatively time convenient and has low instrumentation requirements.

Assessment of the expression pattern of HER2 and its correlation with HER2-targeting antibody-drug conjugate therapy in urothelial cancer

Background:Human epidermal growth factor receptor 2(HER2)overexpression is related to anti-HER2 therapy in many tumors.RC48-antibody-drug conjugate(ADC)has shown promising efficacy in patients with HER2-positive locally advanced or metastatic urothelial carcinoma(UC).The characteristic expression and scoring systems of HER2 are nonexistent in UC.We aimed to explore HER2 status and its correlation with the efficacy of HER2-targeting ADC therapy in UC.Methods:A total of 137 and 43 patients were enrolled in cohort 1 and cohort 2,respectively,from March 2009 to December 2018.The patients in cohort 2 were enrolled in a phase II study of RC48-ADC.UC samples were tested for HER2 status using immunohistochemistry(IHC)and/or fluorescence in situ hybridization(FISH).The 2018 ASCO/CAP HER2 scoring system was adopted and modified to score HER2 expression in UC.Results:The HER2-positive(IHC 2+or 3+)rate was 24.1%(33/137).In HER2 IHC 2+or 3+patients,the HER2 gene amplification rate was 31%(13/42).The objective response rates(ORRs)in RC48-ADC-treated patients with IHC 3+,IHC 2+and FISH+,IHC 2+and FISH-were 58.8%,66.7%and 40%,respectively.The ORR showed a trend toward a better benefit for RC48-ADC therapy in patients with HER2 amplification than in those without amplification(61.5%vs.44.8%,P=0.059).The heterogeneity of HER2 expression in the primary tumor was 55.5%(15/27),and the ORR was not significantly different between patients with tumor heterogeneity and homogeneity.Conclusions:IHC testing should be performed to assess the HER2 status before the initiation of HER2-ADC therapy.There was a trend toward a better benefit for patients with HER2 amplification,and tumor heterogeneity did not influence the drug efficacy.

Hematological and pathological toxicity of anti-HER2/neu peptide mimetic modified paclitaxel bovine serum albumin nanoparticles

Nanoparticles have been widely applied in diagnosis and therapy due to the high loading of insoluble drug, increased target accumulation and interaction with biological tissues. Recently, severe side effects of nanoparticles have been reported, but the underlying mechanism remains largely unknown. In our study, we aim to understand the safety of paclitaxel （PTX） loaded bovine albumin nanoparticles （BNPs） and active targeted PTX loaded BNPs to normal vital organ or tissue in vivo. The anti-human epidermal growth factor receptor 2 （HER2/neu） peptide mimetic （AHNP） was covalent bound to surface of BNPs （AHNP-BNPs） to exert selected delivery to HER2＋ cells. In HER2＋ tumor xenographs, saline （control）, PTX traditional formula （medium of Cremophor EL-ethanol）, BNPs, and AHNP-BNPs were administrated to evaluate the toxicity. There is no severe neutropenia or anemia with treatment of BNPs and AHNP-BNPs compared with traditional PTX injection. We also evaluated their damage on normal organs, including liver, kidney, spleen, lung and heart to fully estimate the safety of AHNP-BNPs and BNPs delivery systems. We observed similar toxicity in liver and lung in mice treated with BNPs or PTX injection, but decreased liver damage in mice treated with AHNP-BNPs. Further studies are rcouired to confirm our conclusion.

The tumor immunosuppressive microenvironment impairs the therapy of anti-HER2/neu antibody

It has been well established that immune surveillance plays critical roles in preventing the occurrence and prog-ression of tumor.More and more evidence in recent years showed the host anti-tumor immune responses also play important roles in the chemotherapy and radiotherapy of cancers.Our previous study found that tumor-targeting therapy of anti-HER2/neu mAb is mediated by CD8+T cell responses.However,we found here that enhancement of CD8+T cell responses by combination therapy with IL-15R/IL-15 fusion protein or anti-CD40,which are strong stimultors for T cell responses,failed to promote the tumor therapeutic effects of anti-HER2/neu mAb.Analysis of tumor microenviornment showed that tumor tissues were heavily infiltrated with the immunosuppressive macrophages and most tumor infiltrating T cells,especially CD8^(+)T cells,expressed high level of inhibitory co-signaling receptor PD-1.These data suggest that tumor microenvironment is dominated by the immunosuppressive strategies,which thwart anti-tumor immune responses.Therefore,the successful tumor therapy should be the removal of inhibitory signals in the tumor microenvir-onment in combination with other therapeutic strategies.

Corrosion Behavior of X52 Anti-H_2S Pipeline Steel Exposed to High H_2S Concentration Solutions at 90℃

Initial corrosion kinetics of X52 anti-H2S pipeline steel exposed to 90 ℃/1.61 MPa H2S solutions was investigated through high temperature and high pressure immersion tests. Corrosion rates were obtained based on weight loss calculation. The corrosion products were analyzed by scanning electron microscopy （SEM）, transmission electron microscopy （TEM）, X-ray diffraction （XRD） and electron probe micro-analysis （EPMA）. The initial corrosion kinetics was found to obey the exponential law. With increasing immersion time, the main corrosion products changed from iron-rich mackinawite to sulfur-rich pyrrhotite. The corrosion films had two layers： an inner fine-grained layer rich in iron and an outer columnar-grained layer rich in sulfur. The corrosion film formed through the combination of outward diffusion of Fe2＋ ions and inward diffusion of HS^- ions. The variation of the corrosion products and compaction of the corrosion layer resulted in a decrease in the diffusion coefficient with increasing immersion time. The double-layered corrosion film formed after long time immersion acted as an effective barrier against diffusion.

Simultaneous Dual Selective Targeted Delivery of Two Covalent Gemcitabine Immunochemotherapeutics and Complementary Anti-Neoplastic Potency of [Se]-Methylselenocysteine

The anti-metabolite chemotherapeutic, gemcitabine is relatively effective for a spectrum of neoplastic conditions that include various forms of leukemia and adenocarcinoma/carcinoma. Rapid systemic deamination of gemcitabine accounts for a brief plasma half-life but its sustained administration is often curtailed by sequelae and chemotherapeutic-resistance. A molecular strategy that diminishes these limitations is the molecular design and synthetic production of covalent gemcitabine immunoche-motherapeutics that possess properties of selective “targeted” delivery. The simultaneous dual selective “targeted” delivery of gemcitabine at two separate sites on the external surface membrane of a single cancer cell types represents a therapeutic approach that can increase cytosol chemotherapeutic deposition;prolong chemotherapeutic plasma half-life (reduces administration frequency);minimize innocent exposure of normal tissues and healthy organ systems;and ultimately enhance more rapid and thorough resolution of neoplastic cell populations. Materials and Methods: A light-reactive gemcitabine intermediate synthesized utilizing succinimidyl 4,4-azipentanoate was covalently bound to anti-EGFR or anti-HER2/neu IgG by exposure to UV light (354-nm) resulting in the synthesis of covalent immunoche-motherapeutics, gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu]. Cytotoxic anti-neoplastic potency of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] between?gemcitabine-equivalent concentrations of 10-12 M and 10-6 M was determined utilizing chemotherapeutic-resistant mammary adenocarcinoma (SKRr-3). The organoselenium compound, [Se]-methylselenocysteine was evaluated to determine if it complemented the anti-neoplastic potency of the covalent gemcitabine immunoche-motherapeutics. Results: Gemcitabine-(C4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] and the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] all had anti-neoplastic cytotoxic potency against mammary adenocarcinoma. Gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] produced progressive increases in anti-neoplastic cytotoxicity that were greatest between gemcitabine-equivalent concentrations of 10-9 M and 10-6 M. Dual simultaneous combinations of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] produced levels of anti-neoplastic cytotoxicity intermediate between each of the individual covalent gemcitabine immunochemotherapeutics. Total anti-neoplastic cytotoxicity of the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] against chemothe-rapeutic-resistant mammary adenocarcinoma (SKBr-3) was substantially higher when formulated with [Se]-methylsele-no-cysteine.

Demethylation of FANCF gene may be a potential treatment through inhibiting the proliferation of cervical cancer

Objective: The aim of the study was to explore the effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on expression of Fanconi anemia complementation group F (FANCF) gene and the proliferation of cervical cancer cells, to observe cell's sensitivity to chemotherapeutic drug taxol, and to explore the antitumor effect of 5-ADC as well as the new treatment of cervical cancer. Methods: Cervical cancer cell lines SiHa (FANCF gene full-methylated) and Hela (unmethylated) were treated with 5-ADC. We used the methylation-specific PCR (MSP), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to detect the FANCF methylation, mRNA and protein respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of cells. The cytotoxicity of taxol was measured by flow cytometer. The nude mice bearing SiHa was used to observe the effect of 5-ADC in vivo. Results: Inhibition of DNA promoter methylation by 5-ADC reactivated the expression of FANCF mRNA and protein in SiHa cells, consistent with decreased growth speed and increased taxol resistance. These results were proven in experiments in vivo. Conclusion: The 5-ADC probably become a potential treatment drug through inhibiting the proliferation of cervical cancer cells in taxol-resistant patients.

Synthesis of Gemcitabine-(C₄-<i>amide</i>)-[anti-HER2/<i>neu</i>] Utilizing a UV-Photoactivated Gemcitabine Intermediate: Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3

Gemcitabine is a pyrimidine nucleoside analog that becomes triphosphorylated intracellularly where it competitively inhibits cytidine incorporation into DNA strands. Another mechanism-of-action of gemcitabine (diphosphorylated form) involves irreversible inhibition of the enzyme ribonucleotide reductase thereby preventing deoxyribonucleotide synthesis. Functioning as a potent chemotherapeutic gemcitabine promote decreases in neoplastic cell proliferation and apoptosis which is frequently found to be effective for the treatment of several leukemias and a wide spectrum of carcinomas. A brief plasma half-life in part due to rapid deamination and chemotherapeutic-resistance restricts the utility of gemcitabine in clinical oncology. Selective “targeted” delivery of gemcitabine represents a potential molecular strategy for simultaneously prolonging its plasma half-life and minimizing innocient tissues and organ systems exposure to chemotherapy. The molecular design and an organic chemistry based synthesis reaction is described that initially generates a UV-photoactivated gemcitabine intermediate. In a subsequent phase of the synthesis method the UV-photoactivated gemcitabine intermediate is covalently bonded to a monoclonal immunoglobulin yielding an end-product in the form of gemcitabine-(C4-amide)-[anti-HER2/neu]. Analysis by SDS-PAGE/chemiluminescent auto-radiography did not detect evidence of gemcitabine-(C4-amide)-[anti-HER2/neu] polymerization or degradative fragmentation while cell-ELISA demonstrated retained binding-avidity for HER2/neu trophic membrane receptor complexes highly over-expressed by chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Compared to chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3), the covalent immunochemotherapeutic, gemcitabine-(C4-amide)-[anti-HER2/neu] is anticipated to exert greater levels of cytotoxic anti-neoplastic potency against other neoplastic cell types like pancreatic carcinoma, small-cell lung carcinoma, neuroblastoma, glioblastoma, oral squamous cell carcinoma, cervical epitheliod carcinoma, or leukemia/lymphoid neoplastic cell types based on their reported sensitivity to gemcitabine and gemcitabine covalent conjugates.

Her2抗体与MMAE偶联物的制备及生物活性研究

目的:制备Her2抗体与海兔毒素MMAE的偶联物,检测该抗体-药物偶联药物(ADC)对于乳腺癌细胞的抑制作用。方法:将Her2抗体通过一个可降解的linker与小分子毒素(MMAE)连接起来,形成抗体药物偶联物(ADC)。通过细胞学试验检测Her2抗体-MMAE偶联物对于肿瘤细胞抑制、细胞凋亡和细胞周期的作用。结果:通过优化偶联条件,ADC偶联率达80%以上。肿瘤细胞生长抑制的实验显示ADC药物的IC50比单克隆抗体的IC50明显降低,作用效果更加明显。细胞凋亡和细胞周期实验结果表明,ADC药物72h诱导细胞凋亡率高达40%,单一抗体药物则仅为20%。结论:该ADC药物具有很好的抑制乳腺癌细胞的作用。