RUNX-3-expressing CAR T cells targeting glypican-3 in patients with heavily pretreated advanced hepatocellular carcinoma: a phase I trial.

Background: Glypican-3 (GPC3) is a well-characterized hepatocellular carcinoma (HCC)-associated antigen and a promising target for HCC treatment. CT017 CAR T cells were engineered to co-express CAR-GPC3 and runt-related transcription factor 3 (RUNX3), which triggers CD8+ T-cell infiltration into the cancer microenvironment. Methods: This single-center, single-arm, open-label, phase I clinical study enrolled heavily pretreated patients with GPC3-positive HCC between August 2019 and December 2020 (NCT03980288). Patients were treated with CT017 CAR T cells at a dose of 250 × 106 cells. The primary objective was to assess the safety and tolerability of this first-in-human product. Findings: Six patients received 7 infusions (one patient received 2 infusions) at the 250 × 106 cells dose. Three patients received CT017 monotherapy, and three patients received CT017-tyrosine kinase inhibitor (TKI) combination therapy at the first infusion. One patient received CT017-TKI combination therapy at the second infusion after CT017 monotherapy. All patients experienced cytokine release syndrome (CRS), with 50% (3/6) at Grade 2, 50% (3/6) at Grade 3, and all events resolved after treatment. No immune effector cell-associated neurotoxicity syndrome was observed. Dose escalation was not performed due to the investigator's decision regarding safety. Of six evaluable patients, one achieved partial response and two had stable disease for a 16.7% objective response rate, 50% disease control rate, 3.5-month median progression-free survival, 3.2-month median duration of disease control, and 7.9-month median overall survival (OS) with 7.87-month median follow-up. The longest OS was 18.2 months after CT017 infusion. Interpretation: Current preliminary phase I data showed a manageable safety profile and promising antitumor activities of CT017 for patients with advanced HCC. These results need to be confirmed in a robust clinical trial. Funding: This study was funded by CARsgen Therapeutics Co., Ltd.

Long term complete response of advanced hepatocellular carcinoma to glypican-3 specific chimeric antigen receptor T-Cells plus sorafenib, a case report.

The clinical efficacy of current therapies for Hepatocellular carcinoma (HCC) are unsatisfactory. In recent years, chimeric antigen receptor (CAR) T-cell therapies have been developed for solid tumors including advanced HCC (aHCC), but limited progress has been made. Glypican-3 is a promising immunotherapeutic target for HCC since it is specifically highly expressed in HCC. A previous study indicated that GPC3-targeted CAR T-(CAR-GPC3) cells were well-tolerated and had prolonged survival for HCC patients and that Sorafenib could increase the antitumor activities of CAR-GPC3 T-cells against HCC in mouse models. Here, we report a patient with aHCC who achieved a complete response (CR) and a long survival period after the combination therapy of CAR-GPC3 T-cell plus sorafenib. A 60-year-old Asian male diagnosed with hepatitis B virus (HBV) related HCC developed liver recurrence and lung metastasis after liver tumor resection and trans-arterial chemoembolization therapy. The patient also previously received microwave ablation therapy for lung metastasis. After the enrollment, the patient underwent leukapheresis for CAR-GPC3 T-cells manufacturing. Seven days after leukapheresis, the patient started to receive 400 mg of Sorafenib twice daily. The patient received 4 cycles of CAR-GPC3 T cells (CT011) treatment and each cycle was divided into two infusions. Prior to each cycle of CT011 treatment, lymphodepletion was performed. The lymphodepletion regimen was cyclophosphamide 500 mg/m2/day for 2 to 3 days, and fludarabine 20-25 mg/m2/day for 3 to 4 days. A total of 4×109 CAR-GPC3 T cells were infused. The CT011 plus Sorafenib combination therapy was well tolerated. All the ≥ grade 3 AEs were hematological toxicities which were deemed an expected event caused by the preconditioning regimen. This patient obtained partial responses from the 3rd month and achieved CR in the 12th month after the first cycle of CT011 infusion according to the RECIST1.1 assessment. The tumor had no progression for more than 36 months and maintained the CR status for more than 24 months after the first infusion.

Claudin18.2-specific CAR T cells in gastrointestinal cancers: phase 1 trial interim results.

Despite success in hematologic malignancies, the treatment landscape of chimeric antigen receptor (CAR) T cell therapy for solid tumors remains limited. Claudin18.2 (CLDN18.2)-redirected CAR T cells showed promising efficacy against gastric cancer (GC) in a preclinical study. Here we report the interim analysis results of an ongoing, open-label, single-arm, phase 1 clinical trial of CLDN18.2-targeted CAR T cells (CT041) in patients with previously treated, CLDN18.2-positive digestive system cancers ( NCT03874897 ). The primary objective was safety after CT041 infusion; secondary objectives included CT041 efficacy, pharmacokinetics and immunogenicity. We treated 37 patients with one of three CT041 doses: 2.5 × 108, 3.75 × 108 or 5.0 × 108 cells. All patients experienced a grade 3 or higher hematologic toxicity. Grade 1 or 2 cytokine release syndrome (CRS) occurred in 94.6% of patients. No grade 3 or higher CRS or neurotoxicities, treatment-related deaths or dose-limiting toxicities were reported. The overall response rate (ORR) and disease control rate (DCR) reached 48.6% and 73.0%, respectively. The 6-month duration of response rate was 44.8%. In patients with GC, the ORR and DCR reached 57.1% and 75.0%, respectively, and the 6-month overall survival rate was 81.2%. These initial results suggest that CT041 has promising efficacy with an acceptable safety profile in patients with heavily pretreated, CLDN18.2-positive digestive system cancers, particularly in those with GC.

Chimeric Antigen Receptor-Glypican-3 T-Cell Therapy for Advanced Hepatocellular Carcinoma: Results of Phase I Trials.

PURPOSE: Our preclinical studies demonstrated the potential of chimeric antigen receptor (CAR)-glypican-3 (GPC3) T-cell therapy for hepatocellular carcinoma (HCC). We report herein the first published results of CAR-GPC3 T-cell therapy for HCC. PATIENTS AND METHODS: In two prospective phase I studies, adult patients with advanced GPC3+ HCC (Child-Pugh A) received autologous CAR-GPC3 T-cell therapy following cyclophosphamide- and fludarabine-induced lymphodepletion. The primary objective was to assess the treatment's safety. Adverse events were graded using the Common Terminology Criteria for Adverse Events (version 4.03). Tumor responses were evaluated using the RECIST (version 1.1). RESULTS: A total of 13 patients received a median of 19.9 × 108 CAR-GPC3 T cells by a data cutoff date of July 24, 2019. We observed pyrexia, decreased lymphocyte count, and cytokine release syndrome (CRS) in 13, 12, and nine patients, respectively. CRS (grade 1/2) was reversible in eight patients. One patient experienced grade 5 CRS. No patients had grade 3/4 neurotoxicity. The overall survival rates at 3 years, 1 year, and 6 months were 10.5%, 42.0%, and 50.3%, respectively, according to the Kaplan-Meier method. We confirmed two partial responses. One patient with sustained stable disease was alive after 44.2 months. CAR T-cell expansion tended to be positively associated with tumor response. CONCLUSIONS: This report demonstrated the initial safety profile of CAR-GPC3 T-cell therapy. We observed early signs of antitumor activity of CAR-GPC3 T cells in patients with advanced HCC.

Armored Inducible Expression of IL-12 Enhances Antitumor Activity of Glypican-3-Targeted Chimeric Antigen Receptor-Engineered T Cells in Hepatocellular Carcinoma.

Adoptive immunotherapy based on chimeric antigen receptor-modified T (CAR-T) cells has been demonstrated as one of the most promising therapeutic strategies in the treatment of malignancies. However, CAR-T cell therapy has shown limited efficacy for the treatment of solid tumors. This is, in part, because of tumor heterogeneity and a hostile tumor microenvironment, which could suppress adoptively transferred T cell activity. In this study, we, respectively, engineered human- or murine-derived-armored glypican-3 (GPC3)-specific CAR-T cells capable of inducibly expressing IL-12 (GPC3-28Z-NFAT-IL-12) T cells. The results showed that GPC3-28Z-NFAT-IL-12 T cells could lyse GPC3+ tumor cells specifically and increase cytokine secretion compared with GPC3-28Z T cells in vitro. In vivo, GPC3-28Z-NFAT-IL-12 T cells augmented the antitumor effect when encountering GPC3+ large tumor burdens, which could be attributed to IL-12 increasing IFN-γ production, favoring T cells infiltration and persistence. Furthermore, in immunocompetent hosts, low doses of GPC3-m28Z-mNFAT-mIL-12 T cells exerted superior antitumor efficacy without prior conditioning in comparison with GPC3-m28Z T cells. Also, mIL-12 secretion decreased regulatory T cell infiltration in established tumors. In conclusion, these findings demonstrated that the inducible expression of IL-12 could boost CAR-T function with less potential side effects, both in immunodeficient and immunocompetent hosts. The inducibly expressed IL-12-armored GPC3-CAR-T cells could broaden the application of CAR-T-based immunotherapy to patients intolerant of lymphodepletion chemotherapy and might provide an alternative therapeutic strategy for patients with GPC3+ cancers.

Claudin18.2-Specific Chimeric Antigen Receptor Engineered T Cells for the Treatment of Gastric Cancer.

BACKGROUND: Claudin18.2 (CLDN18.2), a gastric-specific membrane protein, has been regarded as a potential therapeutic target for gastric cancer and other cancer types. The aim of our study was to elucidate whether chimeric antigen receptor T (CAR T) cells redirected to CLDN18.2 have the potential to be used in the treatment of this deadly disease. METHODS: CLDN18.2-specific humanized antibodies were developed using hybridoma and humanization technology. CLDN18.2-specific CAR T cells were prepared by lentiviral vector transduction. In vitro antitumor activities and cytokine production of the CAR T cells to gastric cancer cell lines were examined by cytotoxicity and ELISA assay. In vivo antitumor activities of CAR T cells were evaluated in mice bearing gastric cancer cell line and patient-derived tumor xenograft (PDX) models (n ≥ 6 mice per group). All statistical tests were two-sided. RESULTS: Humanized CLDN18.2-specific hu8E5 and hu8E5-2I single-chain fragment variables (scFv) were successfully developed. CLDN18.2-specific CAR T cells were developed using hu8E5 or hu8E5-2I scFv as targeting moieties. Both hu8E5-28Z and hu8E5-2I-28Z CAR T cells comprising the CD28 costimulatory domain potently suppressed tumor growth in a cancer cell line xenograft mouse model (mean [SD] tumor volume: hu8E5-28Z = 118.0 [108.6] mm3 and hu8E5-2I-28Z group = 75.5 [118.7] mm3 vs untransduced T cell group = 731.8 [206.3] mm3 at day 29 after tumor inoculation, P < .001). Partial or complete tumor elimination was observed in CLDN18.2-positive gastric cancer PDX models treated with the hu8E5-2I-28Z CAR T cells (P < .001), which persist well in vivo and infiltrate efficiently into the tumor tissues. Although the CLDN18.2 CAR T cells could lyse target cells expressing murine CLDN18.2 (mCLDN18.2), no obvious deleterious effect on the normal organs including the gastric tissues was observed in the mice. CONCLUSIONS: CLDN18.2-specific CAR T cells could be a promising treatment strategy for gastric cancer and potentially other CLDN18.2-positive tumors.

Selective Targeting of Glioblastoma with EGFRvIII/EGFR Bitargeted Chimeric Antigen Receptor T Cell.

The heterogeneous expression of EGFRvIII [variant III mutant of epidermal growth factor receptor (EGFR)] in glioblastoma has significant impact on the clinical response to the treatment of EGFRvIII-specific chimeric antigen receptor-engineered T (CAR T) cells. We hypothesized that CAR T cells that could target both EGFRvIII and the form of EGFR expressed on tumor cells, but not EGFR on normal cells, would greatly improve efficacy without inducing on-target, off-tumor toxicity. Therefore, we developed a humanized single-chain antibody, M27, with a single specificity that binds to an epitope found both on wild-type EGFR- and EGFRvIII-overexpressing tumor cells, but not EGFR-expressing normal cells, including primary keratinocytes, on which wild-type EGFR is highly expressed. M27-derived CAR T cells effectively lysed EGFRvIII- or EGFR-overexpressing tumor cells, but showed no observable toxicity on normal cells. Inclusion of the CD137 (4-1BB) costimulatory intracellular domain in the M27-28BBZ CAR provided CAR T cells with higher tumor lysis activity than when not included (as in the M27-28Z CAR). The growth of established EGFR- or EGFRvIII-overexpressing glioma xenografts was suppressed by M27-28BBZ CAR T cells as well. The growth of heterogeneic xenograft tumors, created by mixing EGFR- and EGFR-overexpressing glioblastoma cells, was also effectively inhibited by M27-28BBZ CAR T cells. The survival of mice in the orthotopic models was significantly prolonged after M27-28BBZ CAR T-cell infusion. These results suggested that tumor-selective, bitargeted anti-EGFR/EGFRvIII CAR T cells may be a promising modality for the treatment of patients with EGFR/EGFRvIII-overexpressing glioblastoma. Cancer Immunol Res; 6(11); 1314-26. ©2018 AACR.

Species-Specific Involvement of Integrin αIIbß3 in a Monoclonal Antibody CH12 Triggers Off-Target Thrombocytopenia in Cynomolgus Monkeys.

CH12 is a novel humanized monoclonal antibody against epidermal growth factor receptor variant III (EGFRvIII) for cancer treatment. Unfortunately, in pre-clinical safety evaluation studies, acute thrombocytopenia was observed after administration of CH12 in cynomolgus monkeys, but not rats. More importantly, in vitro experiments found that CH12 can bind and activate platelets in cynomolgus monkey, but not human peripheral blood samples. Cynomolgus monkey-specific thrombocytopenia has been reported previously; however, the underlying mechanism remains unclear. Here, we first showed that CH12 induced thrombocytopenia in cynomolgus monkeys through off-target platelet binding and activation, resulting in platelet destruction. We subsequently found that integrin αIIbß3 (which is expressed on platelets) contributed to this off-target toxicity. Furthermore, three-dimensional structural modeling of the αIIbß3 molecules in cynomolgus monkeys, humans, and rats suggested that an additional unique loop exists in the ligand-binding pocket of the αIIb subunit in cynomolgus monkeys, which may explain why CH12 binds to platelets only in cynomolgus monkeys. Moreover, this study supported the hypothesis that the minor differences between cynomolgus monkeys and humans can confuse human risk assessments and suggests that species differences can help the prediction of human risks and avoid losses in drug development.

Treatment of hepatocellular carcinoma with a GPC3-targeted bispecific T cell engager.

There are limited strategies for the treatment of hepatocellular carcinoma (HCC). In this study, we prepared a Bispecific T cell engager (BiTE) targeting Glypican 3 (GPC3) and CD3. The GPC3/CD3 BiTE was prepared by fusing the single-chain variable fragment (scFv) of the humanized anti-GPC3 antibody (9F2) with the scFv of the anti-CD3 antibody (OKT3). The in vitro and in vivo cytotoxic activities of the GPC3/CD3 BiTE were evaluated against various HCC cell lines. The GPC3/CD3 BiTE could efficiently mediate the T cell killing of GPC3-positive HCC in vitro, which was dependent on GPC3 expression on the surface of HCC cells. Moreover, our study indicates that, in the presence of the GPC3/CD3 BiTE, T cells could efficiently destroy GPC3-positive human HCC cells in vitro and in vivo. Additionally, our study further proved that GPC3 is not expressed in normal tissues. Thus, GPC3 may be a cancer-specific antigen. Collectively, these findings suggest that this anti-GPC3 BiTE might be a promising anti-tumor reagent for patients with GPC3-positive HCC.

EGFR modulates monounsaturated fatty acid synthesis through phosphorylation of SCD1 in lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR), a well-known oncogenic driver, contributes to the initiation and progression of a wide range of cancer types. Aberrant lipid metabolism including highly produced monounsaturated fatty acids (MUFA) is recognized as a hallmark of cancer. However, how EGFR regulates MUFA synthesis in cancer remains elusive. This is the focus of our study. METHODS: The interaction between EGFR and stearoyl-CoA desaturase-1 (SCD1) was detected byco-immunoprecipitation. SCD1 protein expression, stability and phosphorylation were tested by western blot. The synthesis of MUFA was determined by liquid chromatography-mass spectrometry. The growth of lung cancer was detected by CCK-8 assay, Annexin V/PI staining, colony formation assay and subcutaneous xenograft assay. The expression of activated EGFR, phosphorylated and total SCD1 was tested by immunohistochemistry in 90 non-small cell lung cancersamples. The clinical correlations were analyzed by Chi-square test, Kaplan-Meier survival curve analysis and Cox regression. RESULTS: EGFR binds to and phosphorylates SCD1 at Y55. Phosphorylation of Y55 is required for maintaining SCD1 protein stability and thus increases MUFA level to facilitate lung cancer growth. Moreover, EGFR-stimulated cancer growth depends on SCD1 activity. Evaluation of non-small cell lung cancersamples reveals a positive correlation among EGFR activation, SCD1 Y55 phosphorylation and SCD1 protein expression. Furthermore, phospho-SCD1 Y55 can serve as an independent prognostic factor for poor patient survival. CONCLUSIONS: Ourstudy demonstrates that EGFR stabilizes SCD1 through Y55 phosphorylation, thereby up-regulating MUFA synthesis to promote lung cancer growth. Thus, we provide the first evidence that SCD1 can be subtly controlled by tyrosine phosphorylation and uncover a previously unknown direct linkage between oncogenic receptor tyrosine kinase and lipid metabolism in lung cancer. We also propose SCD1 Y55 phosphorylation as a potential diagnostic marker for lung cancer.

Anti-angiogenesis through noninvasive to minimally invasive intraocular delivery of the peptide CC12 identified by in vivo-directed evolution.

Anti-vascular endothelial growth factor (VEGF) therapies are widely used for the treatment of neovascular fundus diseases such as diabetic retinopathy. However, these agents need to be injected intravitreally, because their strong hydrophilicity and high molecular weight prevent them from penetrating cell membranes and complex tissue barriers. Moreover, the repeated injections that are required can cause infection and tissue injury. In this study, we used in vivo-directed evolution phage display technology to identify a novel dodecapeptide, named CC12, with the ability to penetrate the ocular barrier in a noninvasive (via conjunctival sac instillation) or minimally invasive (via retrobulbar injection) manner. KV11, an antiangiogenesis peptide previously demonstrated to inhibit pathological neovascularization in the retina, was then used as a model antiangiogenesis cargo for CC12. We found that conjugation of KV11 peptide with CC12 peptide facilitated the delivery of KV11 to the retina, resulting in significant inhibition of retinal neovascularization development via topical application without tissue toxicity. Collectively, our data of multilevel evaluations demonstrate that CC12 may enable the noninvasive to minimally invasive intraocular delivery of antiangiogenic therapeutics.

Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma.

Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1+GPC3- tumor cells, but they exhibited a similar cytotoxicity against GPC3+ASGR1- and GPC3+ASGR1+ HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3+ASGR1+ HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3+ASGR1+ HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3+ASGR1+ HCC.

The monoclonal antibody CH12 augments 5-fluorouracil-induced growth suppression of hepatocellular carcinoma xenografts expressing epidermal growth factor receptor variant III.

5-Fluorouracil (5-FU) is one of the most common chemotherapeutic agents used for the treatment of hepatocellular carcinoma (HCC). However, chemoresistance has precluded the use of 5-FU alone in clinical regimens. Combination therapies with 5-FU and other anticancer agents are considered to be a therapeutic option for patients with HCC. We previously reported that the expression of epidermal growth factor receptor variant III (EGFRvIII) can decrease the sensitivity of HCC cells to 5-FU. To overcome this problem, in this study, we elucidated the mechanism underlying EGFRvIII-mediated 5-FU resistance. We observed that EGFRvIII expression can induce miR-520d-3p downregulation and the ensuing upregulation of the transcription factor E2F-1 and the enzyme thymidylate synthase (TS), which may lead to drug resistance. Intriguingly, we found that CH12, a monoclonal antibody directed against EGFRvIII, and 5-FU together had an additive antitumor effect on EGFRvIII-positive HCC xenografts and significantly improved survival in all mice with established tumors when compared with either 5-FU or CH12 alone. Mechanistically, compared with 5-FU alone, the combination more noticeably downregulated EGFR phosphorylation and Akt phosphorylation as well as the expression of the apoptotic protector Bcl-xL and the cell cycle regulator cyclin D1. Additionally, the combination upregulated the expression of the cell cycle inhibitor p27 in in vivo treatment. More interestingly, CH12 treatment upregulated miR-520-3p and downregulated E2F-1 and TS at the mRNA and protein levels. Collectively, these observations suggest that the combination of 5-FU with mAb CH12 is a potential means of circumventing EGFRvIII-mediated 5-FU resistance in HCC.

A novel EGFR isoform confers increased invasiveness to cancer cells.

As a validated therapeutic target in several human cancers, the EGF receptor (EGFR) provides a focus to gain deeper insights into cancer pathophysiology. In this study, we report the identification of a naturally occurring and widely expressed EGFR isoform termed EGFRvA, which substitutes a Ser/Thr-rich peptide for part of the carboxyl-terminal regulatory domain of the receptor. Intriguingly, EGFRvA expression relates more closely to histopathologic grade and poor prognosis in patients with glioma. Ectopic expression of EGFRvA in cancer cells conferred a higher invasive capacity than EGFR in vitro and in vivo. Mechanistically, EGFRvA stimulated expression of STAT3, which upregulated heparin-binding EGF (HB-EGF). Reciprocally, HB-EGF stimulated phosphorylation of EGFRvA at Y845 along with STAT3, generating a positive feedback loop that may reinforce invasive function. The significance of EGFRvA expression was reinforced by findings that it is attenuated by miR-542-5p, a microRNA that is a known tumor suppressor. Taken together, our findings define this newfound EGFR isoform as a key theranostic molecule.

Inhibition of hepatocellular carcinoma cell growth by an anti-insulin-like growth factor-I receptor monoclonal antibody.

Hepatocellular carcinoma (HCC) overexpresses insulin-like growth factor-I receptor (IGF-IR), as compared with normal hepatocytes. Since IGF-1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for treating HCC. Here, we have generated a murine anti-IGF-1R antibody, 4F2, that recognizes the IGF-IRα subunit and blocks in vitro IGF-I and IGF-II-induced cell proliferation of SMMC-7721 and Bel-7402 HCC cell lines. 4F2 can inhibit IGF-IR autophosphorylation, IRS-1 phosphorylation and the activation of the major downstream signaling molecules AKT and mitogen-activated protein kinase. Additionally, we observed a moderate increase in apoptosis as demonstrated by detection of changes in the expression of the pro-apoptotic and anti-apoptotic proteins Bax and Bcl-2 after 4F2 treatment. Combined treatment with 4F2 and doxorubicin was more effective in reducing cell proliferation and promoting apoptosis than either agent alone. These data support that therapeutic anti-IGF-IR antibodies are potential new agents for treating HCC.

Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells.

The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 10(5) cells/mL. Then, the basic metabolic characteristics of CHO-C12 cells were studied in stirred bioreactor batch cultures. The results showed that the limiting concentrations of glucose and glutamine were 6 and 1 mM, respectively. The culture process consumed significant amounts of aspartate, glutamate, asparagine, serine, isoleucine, leucine, and lysine. Aspartate, glutamate, asparagine, and serine were particularly exhausted in the early growth stage, thus limiting cell growth and antibody synthesis. Based on these findings, fed-batch and perfusion processes in the bioreactor were successfully developed with a balanced amino acid feed strategy. Fed-batch and especially perfusion culture effectively maintained high cell viability to prolong the culture process. Furthermore, perfusion cultures maximized the efficiency of nutrient utilization; the mean yield coefficient of antibody to consumed glucose was 44.72 mg/g and the mean yield coefficient of glutamine to antibody was 721.40 mg/g. Finally, in small-scale bioreactor culture, the highest total amount of C12 antibody (1,854 mg) was realized in perfusion cultures. Therefore, perfusion culture appears to be the optimal process for small-scale production of C12 antibody by rCHO-C12 cells.

Growth suppression of human hepatocellular carcinoma xenografts by a monoclonal antibody CH12 directed to epidermal growth factor receptor variant III.

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (K(d)) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-x(L), Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression.

A novel gene delivery system targeting Tie2 for cancer gene therapy.

UNLABELLED: The aim of this study was to explore a novel gene vector for targeting gene therapy. MATERIALS AND METHODS: We conjugated a peptide ligand (named GA3) for endothelial TEK tyrosine kinase (Tie2) with polyethylenimine (PEI) to construct a GA3-PEI complex and used the vector to transfer reporter and therapeutic gene in vitro and in vivo respectively. RESULTS: The results demonstrated the vehicle was able to transfer reporter genes specifically into lung cancer SPC-A1 cells and SPC-A1 xenografts highly expressing Tie2 and epithelial cells of bronchus, but not in heart, liver, spleen, kidney, lung alveolar and vascular tissues. In the gene therapy study, tumor growth was significantly inhibited in SPC-A1 xenograft-bearing mice treated with GA3-PEI/p53 complexes compared with control groups (p<0.05). CONCLUSION: Our results indicated that GA3-PEI is an efficient gene delivery system targeting Tie2.

Identification and characterization of Ch806 mimotopes.

The chimeric antibody 806 (Ch806) is a promising antitumor agent that recognizes both the epidermal growth factor receptor variant III (EGFRvIII) and the overexpressed epidermal growth factor receptor (EGFR) in cancer tissues but does not recognize the wild type EGFR in normal tissues. However, passive antibody immunization could not produce effective antitumor titers unless the immunization was administered repeatedly over long periods. To overcome this limitation, we generated epitope mimics that bind to Ch806 and tested whether the peptide mimics could induce the production of similar antibodies when actively immunizing mice with the peptides. We used the PH.D-12 phage display peptide library to identify peptides that bind to the monoclonal antibody (mAb) 12H23, which also recognizes similar epitopes of Ch806. Two mimotopes (WHTEILKSYPHE and LPAFFVTNQTQD) were shown to mimic the mAb 12H23 and Ch806 epitope using immunoassays. The mimotopes were conjugated to immunogenic carrier proteins and used to intraperitoneally immunize BALB/c mice. Interestingly, sera from the mice immunized with the isolated mimotopes not only recognize the recombinant or synthetic 806 eptitope, but can also recognize EGFR that is overexpressed in A431 cells and EGFRvIII expressed in Huh7-EGFRvIII cells, whereas sera from mice immunized with the control peptide-KLH (keyhole limpet hemocyanin) and carrier KLH alone failed to show a similar reactivity. Furthermore, in an antibody-dependent cellular cytotoxicity assay (ADCC), the mimotope-induced antibodies specifically lysed human Huh-7-EGFRvIII cells. Our data indicate that the isolated mimotopes reported here may potentially be used as new alternative agents for treating cancer with EGFRvIII expression or EGFR overexpression.