T cells expressing CD5/CD7 bispecific chimeric antigen receptors with fully human heavy-chain-only domains mitigate tumor antigen escape.

Bispecific chimeric antigen receptor T-cell (CAR-T) therapies have shown promising results in clinical trials for advanced B-cell malignancies. However, it is challenging to broaden the success of bispecific CAR-T therapies to treat refractory/relapse (r/r) T-cell leukemia/lymphoma because targeting multiple T-cell-expressing antigens leads to exacerbated CAR-T cell fratricide and potential safety concerns. Fully human heavy chain variable (FHVH) antibodies that specifically target CD5 or CD7 were screened and constructed to CD5/CD7 bispecific CARs. A truncated Epidermal growth factor receptor were integrated into CAR constructs to address safety concerns. To tackle the fratricidal issue of CAR-T cells targeting T-cell-pan marker(s), CRISPR/Cas9-based CD5 and CD7 genes knockout were performed before lentiviral transduction of bispecific CARs. Functional comparison between different bispecific CAR structures: tandem CARs and dual CAR were performed in vitro and in vivo to determine the optimal construct suitable for addressing T-cell malignancy antigen escape in clinical setting. Knockout of CD5 and CD7 prevents fratricide of CD5/CD7 bispecific CAR-T cells, and FHVH-derived CD5/CD7 bispecific CAR-T cells demonstrate potent antitumor activity in vitro and in vivo. The fratricide-resistant FHVH-derived CD5/CD7 bispecific CAR-T cells have potent antitumor activity against T-cell malignancies, and tandem CARs are more effective than dual CAR in preventing tumor escape in heterogeneous leukemic cells. The meaningful clinical efficacy and safety of tandem CD5/CD7 CAR-T cells deserve to be explored urgently.

In vivo acquired sorafenib-resistant patient-derived tumor model displays alternative angiogenic pathways, multi-drug resistance and chromosome instability.

Acquired resistance to targeted therapies is an important clinical challenge. Research focusing on acquired resistance is hindered by the lack of relevant model systems. In the present study, the generation and characterization of an in vivo acquired sorafenib-resistant hepatocellular carcinoma (HCC) xenograft model derived from a patient tumor is reported. A cancer cell line (LIXC-004SR) was generated from a tumor that had developed following ~100 days of sorafenib treatment of a HCC patient-derived xenograft (PDX) model (LIX004). The xenograft tumors derived from this cell line demonstrated sorafenib-resistance in vivo. By contrast, a cell line (LIXC-004NA) generated from a vehicle-treated LIX004 PDX model remained sensitive to sorafenib in vivo. Following treatment with sorafenib in vivo, angiogenesis was significantly elevated in the LIXC-004SR tumors when compared with that in the LIXC-004NA tumors. The LIXC-004SR cell culture supernatant stimulated human umbilical vein endothelial cell proliferation and extracellular-signal-regulated kinase and protein kinase B phosphorylation, which can only be inhibited by the combination of sorafenib and a fibroblast growth factor receptor 1 (FGFR1) inhibitor, AZD4547. The tumor growth of the sorafenib-resistant LIXC-004SR xenograft was inhibited by the FGFR1 inhibitor in vivo, suggesting that one of the underlying mechanisms of the acquired resistance is likely due to activation of alternative angiogenic pathways. The LIXC-004SR cell line also exhibited signs of multi-drug resistance and genetic instability. Taken together, these data suggest that this in vivo model of acquired resistance from a PDX model may reflect sorafenib-resistance in certain patients and may facilitate drug resistance research, as well as contributing to the clinical prevention and management of drug resistance.

Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts.

Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.

HZ08 reverse the aneuploidy-induced cisplatin-resistance in Gastric cancer by modulating the p53 pathway.

We evaluated the influence of DNA aneuploidy on chemotherapy-resistance in human Gastric cancer cell MKN45; we also evaluated the reversal effects of HZ08 on these cells and then preliminary investigated the possible involved pathway. We made use of a pair of human Gastric cancer cell dip-MKN45 (diploid MKN45) and aneu-MKN45 (aneuploid MKN45). Growth inhibition in response to chemotherapeutic drugs was evaluated by CellTiter-Glo Luminescent Cell Viability assay and clone formation assay. Flow cytometry and immuno-assay were applied to evaluate apoptosis and the expression of relative signaling molecules. MKN45 xenograft was generated to evaluate in vivo action. Aneu-MKN45 developed a resistance to cisplatin which could be reversed by HZ08; Flow cytometry and western-blot indicates that HZ08-combination could induce apoptosis and increase the expression of apoptosis-related biomarkers on aneu-MKN45; in vivo study also reflect the same correlation between aneuploidy and cisplatin-resistance, which could be antagonized by HZ08 combination; When investigating the involved pathway, in anue-MKN45, the expression of molecules in p53 pathway was decreased; HZ08 could increase the expression of p53 down-stream molecules as well as elevate the activity of p53, while inhibiting Mdm2, the major negative regulator of p53; p53 inhibitor Pifithrin-α could completely abrogate HZ08's synergism effects, and mimic cisplatin-resistance on dip-MKN45.Lower p53 pathway expression that attenuates cisplatin-induced apoptosis might be at least partly the reason of cisplatin-resistance occurred in aneuploid MKN45 both in vitro and in vivo; Combination of HZ08 could sensitize cisplatin-induced apoptosis through the activation of the p53 pathway, therefore represented a synergism effect on aneuploid MKN45 cells.

Identification of NME5 as a contributor to innate resistance to gemcitabine in pancreatic cancer cells.

The limited therapeutic effect of gemcitabine on pancreatic cancer is largely attributed to pre-existing or acquired resistance of the tumor cells. This study was aimed at screening for candidate resistance-related gene(s) and elucidating the underlying mechanisms. NME5 was found to be highly expressed in an innate gemcitabine-resistant human pancreatic cancer sample and the cell line PAXC002 derived from the sample. Downregulation of NME5 significantly reversed gemcitabine resistance in PAXC002 cells, whereas NME5 overexpression induced gemcitabine resistance in the pancreatic cancer cell line BxPC-3. NME5 attenuated the induction of apoptosis and cell cycle arrest induced by gemcitabine, probably accounting for the blunted sensitivity to gemcitabine. Furthermore, NME5 was demonstrated to play its role in a nuclear factor kappaB (NF-κB)-dependent manner. NME5 was capable of directly binding NF-κB, and possibly regulated its expression level in PAXC002 cells. Our results also suggest that NF-κB is a key executor of NME5 in regulating apoptosis and cell cycle. All of these data suggest that NME5 is a promising target for relieving innate gemcitabine resistance in pancreatic cancer cells.

Intrinsic gemcitabine resistance in a novel pancreatic cancer cell line is associated with cancer stem cell-like phenotype.

Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal malignancies in the world, often diagnosed at an advanced stage, resistant to conventional chemotherapy and having high invasive and metastatic potential. The mechanism of drug resistance of PDA is still not clear. In the present study, we established two novel pancreatic cancer cell lines PAXC-002 and PAXC-003 from human primary xenograft models. The cell lines were characterized by morphology, karyotype, pancreatic cancer marker and short tandem repeat (STR) analysis, and growth kinetics and tumorigenicity. The in vitro anti-proliferation test revealed that PAXC-002 cell was intrinsically resistant to the standard of care chemotherapy-gemcitabine, compared with that of PAXC-003 and other widely used pancreatic cancer cell lines. Interestingly, the gemcitabine resistant PAXC-002 cell line was more potent in forming colonies in 3-Dimensional matrigel culture conditions and had a higher percentage of CD133 positive cells, which is recognized as a cancer stem cell marker, compared to the gemcitabine-sensitive PAXC-003 cell line. In this study, we present two novel pancreatic cancer cell lines which could be used for gemcitabine resistance investigation, mechanism identification of pancreatic cancer and anticancer drug screening. The preliminary data indicate that the drug resistance of pancreatic carcinoma cells is associated with a cancer stem cell-like phenotype.

Strong humoral response elicited by a DNA vaccine targeting gastrin-releasing peptide with optimized adjuvants inhibits murine prostate carcinoma growth in vivo.

Previous studies demonstrated that the elevated expression and receptor binding of gastrin-releasing peptide (GRP) in various types of cancer suggest that GRP might be a putative target for immunotherapy in neoplastic diseases. DNA vaccine for hormone/growth factor immune deprivation represents a feasible and attractive approach for cancer treatment; nevertheless, there is still a need to increase the potency of the DNA vaccine. Here, based on six copies of the B cell epitope GRP(18-27) in a linear alignment as an immunogen, we designed several anti-GRP DNA vaccines containing different combinations of immunoadjuvants, such as HSP65, tetanus toxoid(830-844) (T), pan HLA-DR-binding epitope (PADRE) (P), and mycobacterial HSP70(407-426) (M), on a backbone of pCR3.1 plasmid vector with eight 5'-GACGTT-3' CpG motifs and the VEGF183 signal peptide (VS). The effects of these immunoadjuvants in enhancing GRP-specific humoral immune response were then evaluated by comparing the respective immunogenicity and antitumor effects. Immunization of mice with pCR3.1-VS-HSP65-TP-GRP6-M2 elicited much higher levels of specific anti-GRP antibodies and more effectively inhibited the growth of a GRP-dependent tumor RM-1 in vivo. Interestingly, plasmids encoding for 2HSP70(407-426), but not the one with 1 or 3HSP70(407-426) showed stronger immune stimulatory potential as well as impressive antitumor activity, suggesting that 2HSP70(407-426) is an efficient molecular adjuvant for developing self-epitope vaccines. The highly immunogenic, potent anti-tumorigenic and antiangiogenesis activities of the anti-GRP DNA vaccine offered a novel immunotherapeutic approach in the treatment of GRP-dependent tumors and their complications.

Improved efficacy of DNA vaccination against prostate carcinoma by boosting with recombinant protein vaccine and by introduction of a novel adjuvant epitope.

DNA vaccine represents an attractive approach for cancer treatment by inducing active immune-deprivation of gastrin-releasing peptide (GRP) from tumor cells, the growth of which is dependent on the stimulation of GRP. In this study, we developed a DNA vaccine using a plasmid vector to deliver the immunogen of six copies of the B cell epitope GRP(18-27) (GRP6). In order to increase the potency of this DNA vaccine, multiple strategies have been applied including DNA-prime protein-boost immunization and introduction of a foreign T-helper epitope into DNA vaccine. Mice vaccinated DNA vaccine boosting with HSP65-GRP6 protein induced high titer and relatively high avidity of anti-GRP antibodies as well as inhibition effect on the growth of murine prostate carcinoma, superior to the treatment using DNA alone or BCG priming HSP65-GRP6 protein boosting. Furthermore, the introduction of a novel foreign T-helper epitope into the GRP DNA vaccine showed a markedly stronger humoral immune response against GRP and tumor rejection even than the DNA-prime protein-boost strategy. No further stronger immunogenicity of this foreign T-helper epitope modified DNA vaccine was observed even using the strategy of modified DNA vaccine-priming and HSP65-GRP6 boosting method. The data presented demonstrate that improvement of potency of anti-GRP DNA vaccine with the above two feasible approaches should offer useful methods in the development of new DNA vaccine against growth factors for cancer immunotherapy.

Specific antibodies elicited by a novel DNA vaccine targeting gastrin-releasing peptide inhibit murine melanoma growth in vivo.

The elevated expression and receptor binding of gastrin-releasing peptide (GRP) in various types of cancer, especially in malignant melanoma of the skin, suggest that GRP might be a putative target for immunotherapy in neoplastic diseases. We have therefore constructed a novel DNA vaccine coding for six tandem repeats of a fragment of GRP from amino acids 18 to 27 (GRP6) flanked by helper T-cell epitopes for increased immunogenicity, including HSP65, a tetanus toxoid fragment from amino acids 830 to 844 (T), pan-HLA-DR-binding epitope (PADRE) (P), and two repeats of a mycobacterial HSP70 fragment from amino acids 407 to 426 (M). The anti-GRP DNA vaccine (pCR3.1-VS-HSP65-TP-GRP6-M2) was constructed on a backbone of a pCR3.1 plasmid vector with eight 5'-GACGTT-3' CpG motifs and the VEGF183 signal peptide (VS). Intramuscular (IM) injections of anti-GRP vaccine in mice stimulated the production of high titers of specific antibodies against GRP and suppressed the growth of subcutaneous tumors of B16-F10 melanoma cells. Parallel results were obtained in vitro, showing inhibition of B16-F10 cell proliferation by GRP antisera. IM injections of the DNA vaccine also significantly attenuated tumor-induced angiogenesis associated with intradermal tumors of B16-F10 cells. In addition, lung invasion of intravenously injected cells was highly diminished, suggesting potent antimetastatic activity of the DNA vaccine. These findings support the highly immunogenic and potent antitumorigenic activity of specific anti-GRP antibodies elicited by the anti-GRP DNA vaccine.