RESUMEN
BACKGROUND: Resistance to immune checkpoint inhibitor (ICI) therapy narrows the efficacy of cancer immunotherapy. Although 4-1BB is a promising drug target as a costimulatory molecule of immune cells, no 4-1BB agonist has been given clinical approval because of severe liver toxicity or limited efficacy. Therefore, a safe and efficient immunostimulatory molecule is urgently needed for cancer immunotherapy. METHODS: HK010 was generated by antibody engineering, and the Fab/antigen complex structure was analyzed using crystallography. The affinity and activity of HK010 were detected by multiple in vitro bioassays, including enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry, and luciferase-reporter assays. Humanized mice bearing human PD-L1-expressing MC38 (MC38/hPDL1) or CT26 (CT26/hPDL1) tumor transplants were established to assess the in vivo antitumor activity of HK010. The pharmacokinetics (PK) and toxicity of HK010 were evaluated in cynomolgus monkeys. RESULTS: HK010 was generated as an Fc-muted immunoglobulin (Ig)G4 PD-L1x4-1BB bispecific antibody (BsAb) with a distinguished Fab/antigen complex structure, and maintained a high affinity for human PD-L1 (KD: 2.27 nM) and low affinity for human 4-1BB (KD: 493 nM) to achieve potent PD-1/PD-L1 blockade and appropriate 4-1BB agonism. HK010 exhibited synergistic antitumor activity by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously, and being strictly dependent on the PD-L1 receptor in vitro and in vivo. In particular, when the dose was decreased to 0.3 mg/kg, HK010 still showed a strong antitumor effect in a humanized mouse model bearing MC38/hPDL1 tumors. Strikingly, HK010 treatment enhanced antitumor immunity and induced durable antigen-specific immune memory to prevent rechallenged tumor growth by recruiting CD8+ T cells and other lymphocytes into tumor tissue and activating tumor-infiltrating lymphocytes. Moreover, HK010 not only did not induce nonspecific production of proinflammatory cytokines but was also observed to be well tolerated in cynomolgus monkeys in 5 week repeated-dose (5, 15, or 50 mg/kg) and single-dose (75 or 150 mg/kg) toxicity studies. CONCLUSION: We generated an Fc-muted anti-PD-L1x4-1BB BsAb, HK010, with a distinguished structural interaction with PD-L1 and 4-1BB that exhibits a synergistic antitumor effect by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously. It is strictly dependent on the PD-L1 receptor with no systemic toxicity, which may offer a new option for cancer immunotherapy.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias Colorrectales , Receptor de Muerte Celular Programada 1 , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Inmunoterapia , Macaca fascicularis , Anticuerpos Biespecíficos/farmacologíaRESUMEN
BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies and the patient survival rate remains unacceptably low. The anti-programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) antibody-based immune checkpoint inhibitors have been added to CRC treatment regimens, however, only a fraction of patients benefits. As an important co-stimulatory molecule, 4-1BB/CD137 is mainly expressed on the surface of immune cells including T and natural killer (NK) cells. Several agonistic molecules targeting 4-1BB have been clinically unsuccessful due to systemic toxicity or weak antitumor effects. We generated a humanized anti-4-1BB IgG4 antibody, HuB6, directed against a unique epitope and hypothesized that it would promote antitumor immunity with high safety. METHODS: The antigen binding specificity, affinity and activity of HuB6 were determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), biolayer interferometry (BLI) and flow cytometry. The antitumor effects were evaluated in humanized mice bearing syngeneic tumors, and possible toxicity was evaluated in humanized mice and cynomolgus monkeys. RESULTS: HuB6 showed high specificity and affinity for a binding epitope distinct from those of other known 4-1BB agonists, including utomilumab and urelumab, and induced CD8 + T, CD4 + T and NK cell stimulation dependent on Fcγ receptor (FcγR) crosslinking. HuB6 inhibited CRC tumor growth in a dose-dependent manner, and the antitumor effect was similar with urelumab and utomilumab in humanized mouse models of syngeneic CRC. Furthermore, HuB6 combined with an anti-PD-L1 antibody significantly inhibited CRC growth in vivo. Additionally, HuB6 induced antitumor immune memory in tumor model mice rechallenged with 4 × 106 tumor cells. Toxicology data for humanized 4-1BB mice and cynomolgus monkeys showed that HuB6 could be tolerated up to a 180 mg/kg dose without systemic toxicity. CONCLUSIONS: This study demonstrated that HuB6 should be a suitable candidate for further clinical development and a potential agent for CRC immunotherapy.
Asunto(s)
Neoplasias Colorrectales , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Neoplasias Colorrectales/tratamiento farmacológico , Epítopos , Inmunoterapia , Macaca fascicularis , Ratones , Receptores de IgGRESUMEN
The c-erbB-2 proto-oncogene encodes a 185kDa protein p185, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p185. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.
Asunto(s)
Anticuerpos Monoclonales/genética , Receptor ErbB-2/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Secuencia de Bases , Sitios de Unión , Cricetinae , Citometría de Flujo , Expresión Génica , Vectores Genéticos/genética , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Ratones , Datos de Secuencia Molecular , Unión Proteica , Ingeniería de Proteínas , Proto-Oncogenes Mas , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única , Células Tumorales CultivadasRESUMEN
AIM: To explore the apoptotic effect of the combined treatment of anti-p185(c-erbB-2/neu) engineered antibody with paclitaxel on p185-overexpressing human malignant breast cancer cell lines BT474 and to study its emerging mechanism. METHODS: The prohibitory effect of engineeded antibody plus paclitaxel on BT474 cells was assessed by MTS assay. The number of apoptotic cells was detected by Annexin V-FITC/PI. DNA content and cell cycle distribution were determined by FCM; DNA-binding activity of NF-kappaB was demonstrated by EMSA. RESULTS: Anti-p185(c-erbB-2/neu) engineered antibody plus paclitaxel resulted in synergistic effect on proliferative inhibiton of BT474 cells, which was mediated via apoptotic induction and caused cell cycle to arrest at G1 phase remarkably. Furthermore, the combined treatment of the engineered antibody with paclitaxel effectively suppressed the activation of NF-kappaB in BT474 cells. CONCLUSION: The combined treatment of anti-p185(c-erbB-2/neu) engineered antibody with paclitaxel rendered p185-overexpressing human malignant breast cancer cells BT474 more susceptible to paclitaxel-induced apoptosis by the effective suppression of NF-kappaB activation.
Asunto(s)
Anticuerpos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Paclitaxel/farmacología , Anticuerpos/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Fase G1/efectos de los fármacos , Humanos , FN-kappa B/metabolismo , Receptor ErbB-2/inmunologíaRESUMEN
AIM: To explore the possibility of RNA interference (RNAi)-based gene therapy against HER2-overexpressing tumors using adenovirus-mediated vector. METHODS: A plasmid named pHER2-GFP containing HER2 and green fluorescent protein (GFP) fusion was constructed and cotransfected into CHO-K1 cells respectively with nine small interference RNA (siRNA)-expressing plasmids targeting different regions of HER2. The siRNA-expressing plasmids with best interference effect were screened out and then used to identify the gene silence effect in HER2-overexpressing SKBR3 breast cancer cells. Subsequently, the siRNA-expressing cassettes were subcloned into adenoviral vectors. Downregulation of HER2 by adenovirus-mediated RNAi and its effect on SKBR3 cell proliferation were identified again. RESULTS: Two siRNA-expressing plasmids with best interference effect were screened out and HER2 was also efficiently downregulated in SKBR3 cells infected with the adenovirus containing these siRNA-expressing cassettes. Downregulation of HER2 resulted in the increase of cells in G1 phase and the induction of apoptosis. Furthermore, infection of adenovirus inhibited SKBR3 cell growth, which was confirmed by MTT and cell long-term proliferation assays. CONCLUSION: The adenovirus-mediated RNAi could downregulate the HER2 expression efficiently and exert an inhibitory effect on growth of HER2-overexpressing breast cancer cell.
Asunto(s)
Regulación hacia Abajo , Interferencia de ARN/fisiología , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Animales , Western Blotting , Células CHO , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Cricetinae , Cricetulus , Terapia Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
AIM: To prepare and identify the antibodies against snake venom C-type lectin like protein Agkisacutacin. METHODS: A BALB/c mouse was immunized with Agkisacutacin and the cells were fused by standard hybridoma technique to prepare mAbs. The stability of the hybridomas secreting mAbs was detected by indirect ELISA. The nuclear type of the hybridomas was analyzed by fluorescent staining and the specificity of mAbs was detected by Western blot. RESULTS: Three cell strains of hybridomas that could steadily secrete anti-Agkisacutacin mAb were obtained. CONCLUSION: The successful preparation of anti-Agkisacutacin mAbs provides an important tool for studying the in vivo metabolism of new anti-thrombus drugs by detecting Agkisacutacin and investigating the mechanism of anti-thrombus of the drugs.
Asunto(s)
Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Venenos de Crotálidos/inmunología , Venenos de Serpiente/química , Animales , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Venenos de Crotálidos/biosíntesis , Venenos de Crotálidos/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Femenino , Hibridomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Venenos de Serpiente/inmunologíaRESUMEN
AIM: To explore the effects of anti-HER-2 chimeric antibody chA21 on proliferation and apoptosis of ovarian cancer cell lines SKOV3. METHODS: MTT colorometric assay, HE staining, transmission electron microscopy, flow cytometry and TUNEL staining were used to study the proliferative inhibition and apoptotic induction of SKOV3 cells by chA21 in vitro. RESULTS: Proliferative inhibition rate and apoptotic rate of SKOV3 cells were increased with dose and action time by chA21 (0.2 mg/L-5.4 mg/L). CONCLUSION: chA21 can remarkably inhibit proliferation of SKOV3 cells in vitro and induction of apoptosis may be a principal way.
Asunto(s)
Anticuerpos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/farmacología , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Etiquetado Corte-Fin in Situ , Microscopía Electrónica de Transmisión , Neoplasias Ováricas/patología , Neoplasias Ováricas/ultraestructuraRESUMEN
Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain I-II and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E. coli Origami B (DE3) pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10-15 mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain I-II and domain IV of p185 ECD and this method may also be used to express other disulfide bond rich proteins.
Asunto(s)
Disulfuros/inmunología , Escherichia coli/metabolismo , Receptor ErbB-2/biosíntesis , Anticuerpos Monoclonales/inmunología , Escherichia coli/genética , Vectores Genéticos/genética , Humanos , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Solubilidad , TransfecciónRESUMEN
AIM: To study the cellular metabolism(RIA) in vitro and the biodistribution in vivo of (125)I labeled anti-p185 antibodies(murine mAb A21, mouse-human chimeric antibody A21scFv-Fc and single chain antibody A21 scFv), to provide the basis for clinical application in the diagnosis and treatment of tumor overexpressing p185. METHODS: The specificity of binding of these antibodies to SKOV(3) known to highly express surface antigen p185 was assessd by FACS. The metabolism of the antibodies in SKOV(3) was assayed by cellular RIA. The biodistribution of radiolabeled antibodies was evaluated in nude mice bearing SKOV(3) tumor. RESULTS: Cellular RIA demonstrated these antibodies were internalized after binding to cells, followed by degradation and deiodination in cells, (125)I was then exocytosed. In vivo test showed that these antibodies were concentrated in tumor, but no concentration of control antibody in tumor was found. CONCLUSION: mAb A21, A21 scFv-Fc and A21 scFv can target human ovarian carcinoma cells (SKOV(3)) overexpressing p185 in vitro and in vivo and may be used in the diagnosis and treatment of tumors overexpressing p185.