Regulation of cell growth and apoptosis through lactate dehydrogenase C over-expression in Chinese hamster ovary cells.

Lactate has long been credited as a by-product, which jeopardizes cell growth and productivity when accumulated over a certain concentration during the manufacturing process of therapeutic recombinant proteins by Chinese hamster ovary (CHO) cells. A number of efforts to decrease the lactate concentration have been developed; however, the accumulation of lactate is still a critical issue by the late stage of fed-batch culture. Therefore, a lactate-tolerant cell line was developed through over-expression of lactate dehydrogenase C (LDH-C). In fed-batch culture, sodium lactate or sodium pyruvate was supplemented into the culture medium to simulate the environment of lactate accumulation, and LDH-C over-expression increased the highest viable cell density by over 30 and 50 %, respectively, on day 5, meanwhile the viability was also improved significantly since day 5 compared with that of the control. The percentages of cells suffering early and late apoptosis decreased by 3.2 to 12.5 and 2.0 to 4.3 %, respectively, from day 6 onwards in the fed-batch culture when 40 mM sodium pyruvate was added compared to the control. The results were confirmed by mitochondrial membrane potential assay. In addition, the expression of cleaved caspases 3 and 7 decreased in cells over-expressing LDH-C, suggesting the mitochondrial pathway was involved in the LDH-C regulated anti-apoptosis. In conclusion, a novel cell line with higher lactate tolerance, lowered lactate production, and alleviated apoptosis response was developed by over-expression of LDH-C, which may potentially represent an efficient and labor-saving approach in generating recombinant proteins.

Development and characterization of a novel anti-IgE monoclonal antibody.

IgE is the central macromolecular mediator responsible for the progression of allergic reactions. Omalizumab (Xolair) is a humanized monoclonal anti-IgE antibody directed at the FcepsilonRI-binding domain of human IgE, which represents a novel therapeutic approach in the management of asthma. In this study, we developed a monoclonal antibody (7A5) against human IgE via hybridoma technique. Our data showed that 7A5 could inhibit free IgE molecules to bind to receptors without affecting IgE already bound to cellular receptors. Importantly, 7A5 was able to inhibit IgE-induced histamine release of basophilic leukemia cells. Next, the phage display peptide library technology was employed to select peptides binding to 7A5 and a striking peptide sequence motif was recovered, which is homologous to the sequence (391)KQR(393) within the Cepsilon3 domain of IgE-Fc, Our results further indicated that 7A5 specifically bound to the synthesized peptide "(388)KEEKQRN(394)" containing the (391)KQR(393) motif in IgE-Fc. The epitope of 7A5 was found to be spatially close to the FcepsilonRI-binding site, suggesting that 7A5 binding to IgE might block IgE binding to receptors via steric hindrance. The anti-IgE monoclonal antibody 7A5 may have the potential to be developed as a therapeutic agent for the treatment of allergic diseases.

PE38KDEL-loaded anti-HER2 nanoparticles inhibit breast tumor progression with reduced toxicity and immunogenicity.

The clinical use of Pseudomonas exotoxin A (PE)-based immunotoxins is limited by the toxicity and immunogenicity of PE. To overcome the limitations, we have developed PE38KDEL-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles conjugated with Fab' fragments of a humanized anti-HER2 monoclonal antibody (rhuMAbHER2). The PE38KDEL-loaded nanoparticles-anti-HER2 Fab' bioconjugates (PE-NP-HER) were constructed modularly with Fab' fragments of rhuMAbHER2 covalently linked to PLGA nanoparticles containing PE38KDEL. Compared with nontargeted nanoparticles that lack anti-HER2 Fab', PE-NP-HER specifically bound to and were sequentially internalized into HER2 overexpressing breast cancer cells, which result in significant cytotoxicity in vitro. In HER2 overexpressing tumor xenograft model system, administration of PE-NP-HER showed a superior efficacy in inhibiting tumor growth compared with PE-HER referring to PE38KDEL conjugated directly to rhuMAbHER2. Moreover, PE-NP-HER was well tolerated in mice with a higher LD(50) (LD(50) of 6.86 +/- 0.47 mg/kg vs. 2.21 +/- 0.32 mg/kg for PE-NP-HER vs. PE-HER (mean +/- SD); n = 3), and had no influence on the plasma level of plasma alanine aminotransferase (ALT) of animals when injected at a dose of 1 mg/kg where PE-HER caused significant increase of serum ALT in the treated mice. Notably, PE-NP-HER was of low immunogenicity in development of anti-PE38KDEL neutralizing antibodies and was less susceptible to inactivation by anti-PE38KDEL antibodies compared with PE-HER. This novel bioconjugate, PE-NP-HER, may represent a useful strategy for cancer treatment.

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.

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 LD(50) 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.

Development of SM5-1-conjugated ultrasmall superparamagnetic iron oxide nanoparticles for hepatoma detection.

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are maghemite or magnetite nanoparticles currently used as contrast agent in magnetic resonance imaging (MRI). In this study, a targeted contrast agent (SM-USPIO) was prepared by conjugating coprecipitated USPIO to a humanized SM5-1 antibody which can specifically react with human hepatocellular carcinoma (HCC) cells. The binding and internalization of SM-USPIO to the HCC cell line ch-hep-3 was confirmed by flow cytometry and confocal microscopy. Furthermore, SM-USPIO was demonstrated to be able to selectively accumulate in the tumor cells, resulting in a marked decrease of MRI T2-weighted signal intensity. Biodistribution studies demonstrated the efficient accumulation of SM-USPIO in the ch-hep-3 tumor in nude mice. The in vivo study in the ch-hep-3 tumor-bearing nude mice indicated that MRI using the SM-USPIO as contrast agent possessed good diagnostic ability, suggesting that SM-USPIO had the potential to be a promising targeted contrast agent for diagnosis of HCC.

Blockade of LIGHT/HVEM and B7/CD28 signaling facilitates long-term islet graft survival with development of allospecific tolerance.

BACKGROUND: Previous studies have shown that blockade of LIGHT, a T-cell costimulatory molecule belonging to the tumor necrosis factor (TNF) superfamily, by soluble lymphotoxin beta receptor-Ig (LTbetaR-Ig) inhibited the development of graft-versus-host disease. The cardiac allografts were significantly prolonged in LIGHT deficient mice. No data are yet available regarding the role of the LIGHT/HVEM pathway in more stringent fully allogeneic models such as skin and islet transplantation models. METHODS: Streptozotocin-induced chemical diabetic BALB/C mice underwent transplantation with allogeneic C57BL/6 islets and were treated with LTbetaR-Ig, CTLA4-Ig or a combination of both in the early peritransplant period. RESULTS: Administration of CTLA4-Ig or LTbeta R-Ig alone only increased graft survival to 55 days and 27 days respectively, whereas simultaneous blockade of both pathways significantly prolonged the islet allograft survival for more than 100 days. Long-term survivors were retransplanted with donor-specific (C57BL/6) islets and the grafted islets remained functional for more than 100 days. All of islet allografts were protected against rejection when the mixtures of 1x10(6) CD4+ T cells from tolerant mice and islet allografts were cotransplanted under the renal capsule of the naïve BALB/c recipients. CONCLUSIONS: These data indicate that: 1) a synergistic effect for prolonged graft survival can be obtained by simultaneously blocking LIGHT and CD28 signaling in the stringent model of islet allotransplantation; 2) development of donor-specific immunological tolerance is associated with the presence of regulatory T-cell activity; and 3) local cotransplantation of the allografts with the regulatory T cells can effectively prevent allograft rejection and induce donor-specific tolerance in lymphocytes-sufficient recipients.

A chimeric SM5-1 antibody inhibits hepatocellular carcinoma cell growth and induces caspase-dependent apoptosis.

cSM5-1 is a mouse-human chimeric antibody which has a high specificity for hepatocellular carcinoma, melanoma and breast cancer. In this study, cSM5-1 was found to be able to inhibit cell growth and induce apoptosis in hepatocellular carcinoma cells. The antitumor activity of cSM5-1 was closely correlated with the expression level of the SM5-1 binding protein in the cancer cells. The role of caspases in cSM5-1-induced apoptosis was also investigated, indicating that cSM5-1-induced apoptosis was partially caspase-dependent and caspase-10 played a critical role. These in vitro data indicate that cSM5-1 has the potential to be a promising candidate for cancer treatment.

Preparation and Characterization of Paclitaxel-loaded PLGA nanoparticles coated with cationic SM5-1 single-chain antibody.

The purpose of this study was to develop paclitaxel-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles coated with cationic SM5-1 single-chain antibody (scFv) containing a polylysine (SMFv-polylys). SM5-1 scFv (SMFv) is derived from SM5-1 monoclonal antibody, which binds to a 230 kDa membrane protein specifically expressed on melanoma, hepatocellular carcinoma and breast cancer cells. SMFv-polylys was expressed in Escherichia coli and purified by cation-exchange chromatography. Purified SMFv-polylys was fixed to paclitaxel-loaded PLGA nanoparticles to form paclitaxel-loaded PLGA nanoparticles coated with SMFv-polylys (Ptx-NP-S). Ptx-NP-S was shown to retain the specific antigen-binding affinity of SMFv-polylys to SM5-1 binding protein-positive Ch-hep-3 cells. Finally, the cytotoxicity of Ptx-NP-S was evaluated by a non-radioactive cell proliferation assay. It was demonstrated that Ptx-NP-S had significantly enhanced in vitro cytotoxicity against Ch-hep-3 cells as compared with non-targeted paclitaxel-loaded PLGA nanoparticles. In conclusion, our results suggest that cationic SMFv-polylys has been successfully generated and may be used as targeted ligand for preparing cancer-targeted nanoparticles.

Combination of dextran sulfate and recombinant trypsin on aggregation of Chinese hamster ovary cells.

In laboratory scale therapeutical protein production, cell clumps form typically in shake flasks, which hinders cell growth and decreases protein yield. To minimize clumps during the culture of Chinese hamster ovary cells, we employed the combination of two reagents, dextran sulfate (DS) and recombinant trypsin (r-trypsin). Our results showed that both DS and r-trypsin could diminish cell aggregation when adding them respectively, but clumps were still noticed obviously. In order to further mitigate cell agglomerate, a combination of 1.2 g/L DS and 8.0 mg/L r-trypsin was employed and no clumps were found under the bright field microscope. Strikingly, the highest viable cell density of combination group was increased from 5.12 × 10(6) to 7.13 × 10(6) cells/mL, while the integral of viable cells concentration was raised from 35.13 × 10(6) to 60.87 × 10(6) cells·days/mL, and the culture period was prolonged by 4 days. In addition, the antibody integrity was maintained in the combination group compared with that of the control.

Co-delivery of doxorubicin and PEGylated C16-ceramide by nanoliposomes for enhanced therapy against multidrug resistance.

AIM: To develop novel nanoliposomes (Lip-ADR-Cer) codelivering doxorubicin (ADR) and PEGylated C16 ceramide (PEG-ceramide C16) to overcome multidrug resistance. MATERIALS & METHODS: The antitumor activity and mechanism of Lip-ADR-Cer were evaluated. RESULTS & CONCLUSION: The IC50 of Lip-ADR-Cer after 48-h treatment with the MCF-7/ADR and HL-60/ADR cancer cells, both being ADR resistant, was 2.2- and 1.4-fold effective respectively versus the general nanoliposomes with no PEG-ceramide C16 (Lip-ADR). The antitumor assay in mice bearing MCF-7/ADR or HL-60/ADR xenograft tumors confirmed the superior antitumor activity of Lip-ADR-Cer over Lip-ADR. We found that the improved therapeutic effect of Lip-ADR-Cer may be attributed to both of the cytotoxic effect of PEG-ceramide C16 and glucosylceramide synthase overexpression in multidrug resistance cells.

TGF-ß1 induces epigenetic silence of TIP30 to promote tumor metastasis in esophageal carcinoma.

TGF-ß1, a potent EMT (epithelial-mesenchymal transition) inducer present in the tumor microenvironment, is involved in the metastasis and progression of various carcinomas, including esophageal squamous cell carcinoma (ESCC). TIP30 (30kDa HIV-1 Tat interacting protein) is a putative tumor metastasis suppressor. Here, we found TIP30 was decreased in cells undergoing EMT induced by TGF-ß1, an occurrence that was related to promoter hypermethylation. TGF-ß1 induced TIP30 hypermethylation via increasing DNMT1 and DNMT3A expression, which could be restored by TGF-ß antibodies. In our in vitro and in vivo studies, we showed that silence of TIP30 led to EMT, enhanced migrative and invasive abilities of ESCC cells, promoted tumor metastasis in xenografted mice; alternatively, overexpression of TIP30 inhibited TGF-ß1-induced EMT, and metastatic abilities of ESCC cells. Mechanically, TIP30 silencing induced the nuclear translocation and transcriptional activation of ß-catenin in an AKT-dependent manner, which further resulted in the initiation of EMT. Consistently, TIP30 was frequently methylated and downregulated in ESCC patients. Loss of TIP30 correlated with nuclear ß-catenin and aberrant E-cadherin expression. TIP30 was a powerful marker in predicting the prognosis of ESCC. Taken together, our results suggest a novel and critical role of TIP30 involved in TGF-ß1-induced activation of AKT/ß-catenin signaling and ESCC metastasis.

SOX10, a novel HMG-box-containing tumor suppressor, inhibits growth and metastasis of digestive cancers by suppressing the Wnt/ß-catenin pathway.

SOX10 was identified as a methylated gene in our previous cancer methylome study. Here we further analyzed its epigenetic inactivation, biological functions and related cell signaling in digestive cancers (colorectal, gastric and esophageal cancers) in detail. SOX10 expression was decreased in multiple digestive cancer cell lines as well as primary tumors due to its promoter methylation. Pharmacologic or genetic demethylation reversed SOX10 silencing. Ectopic expression of SOX10 in SOX10-deficient cancer cells inhibits their proliferation, tumorigenicity, and metastatic potentials in vitro and in vivo. SOX10 also suppressed the epithelial to mesenchymal transition (EMT) and stemness properties of digestive tumor cells. Mechanistically, SOX10 competes with TCF4 to bind ß-catenin and transrepresses its downstream target genes via its own DNA-binding property. SOX10 mutations that disrupt the SOX10-ß-catenin interaction partially prevented tumor suppression. SOX10is thus a commonly inactivated tumor suppressor that antagonizes Wnt/ß-catenin signaling in cancer cells from different digestive tissues.

The finely regulating well-defined functional polymeric nanocarriers for anti-tumor immunotherapy.

Cancer is the second leading cause of death around the world. Cancer may be induced by viral infection (EBV,HBV and HPV), bacterial infection (Helicobacter pylori), carcinogen, ultraviolet (UV) radiation exposure, and genetic mutation. Tumor can be suppressed by traditional surgery, radiotherapy, and/or chemotherapy with devastating side effects and very poor quality of postoperative life. The therapeutic index has been further promoted by the newly developed nanomedicine. However, the disseminated tumor cells can result in micrometastases. So the cancer can just be supresssed but not cured by these ways. Fortunately, the developments of immunology have successfully improved many disciplines with special effort on oncology. Various immune cells including B cells, T-lymphocytes (TL), natural killer(NK) cells, dendritic cells (DCs), macrophages, and polymorphonuclear leukocytes are recruited to the tumor. These immune cells can recognize, eliminate, and protect the body from viral, bacterial infections, and the transformed cells(pre-cancer cell) extension. The modification of host immune system, and/or the utilization of components of the immune system for cancer treatment are called immunotherapy. The immunotherapy is not only to target and kill tumor cells in aspecific manner, but also to alert the immune system to eradicate the disseminated tumor cells present in the blood circulation and micro-metastases in remote organs. Herein, the development of immunology, cancer immunotherapy,tumor immuno escape was introduced firstly. Then the correlations between host, the tumor and the nano particulates were proposed. And how to improve the cancer immunotherapy by finely nanocarrier's engineering (nanoimmunotherapy) was systematically illustrated with special focus on the unique pathology of tumor microenviroments and properties of immuno cells.

EGFR-specific PEGylated immunoliposomes for active siRNA delivery in hepatocellular carcinoma.

The development of immunoliposomes for systemic siRNA (small interfering RNA) delivery is highly desired. We reported previously the development of targeted LPD (liposome-polycation-DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab' (TLPD-FCC) for siRNA delivery, which showed superior gene silencing activity in EGFR-overexpressing breast cancers. However, TLPD-FCC did not achieve satisfactory gene silencing activity in EGFR-overexpressing hepatocellular carcinoma (HCC). In this study, some modifications including increased antibody conjugation efficiency and reduced PEGylation degree were made to TLPD-FCC to increase gene silencing activity in HCC. The resultant optimized liposomes denoted as TLPD-FP75 efficiently bound and delivered to EGFR-overexpressing HCC, resulting in enhanced gene silencing activity compared to untargeted LPD (NTLPD-FP75). Tissue distribution in vivo revealed that the accumulation of TLPD-FP75 was higher than NTLPD-FP75 in orthotopic HCC model of mice. The promoted uptake of TLPD-FP75 in HCC cells was confirmed by confocal microscopy. To investigate the in vivo gene silencing activity, we administered TLPD-FP75 by intravenous injections into mice bearing orthotopic HCC. The results showed TLPD-FP75 potently suppressed luciferase expression, while little silencing was observed in NTLPD-FP75. TLPD-FP75 was demonstrated to possess potent gene silencing activity in HCC and will potentially increase the feasibility of HCC gene therapy.

Tolerability, pharmacokinetics and pharmacodynamics of CMAB007, a humanized anti-immunoglobulin E monoclonal antibody, in healthy Chinese subjects.

The goal of the studies presented here was to determine the tolerability, pharmacokinetic and pharmacodynamic profiles of CMAB007, a biosimilar of omalizumab (Xolair; a humanized anti-immunoglobulin E monoclonal antibody), in healthy, male Chinese subjects. Thirty-six healthy Chinese men participated in two open-label, dose-escalation studies: 27 in a single-dose study (150, 300 or 600 mg) and 9 in a multiple-dose study (150 or 300 mg every 4 weeks for 20 weeks). The safety profiles of both studies were generally unremarkable. No drug-related adverse event was observed. CMAB007 exhibited a linear PK profile over the dose range of 150-600 mg. In the single-dose study, maximum concentration (Cmax) was reached within 6-8 d, and Cmax and area under concentration-time curve (AUC) increased linearly with the dose. In the multiple-dose study, steady-state appeared to have been achieved after the third dose. Css-max and AUCτ also showed dose-linearity. A dose-dependent suppression of free IgE was observed during treatment, as a median percentage change from baseline, 91.9-98.8%, in the three single-dose groups. No anti-CMAB007 antibodies were detected after dosing in any subject. Subcutaneous administration of CMAB007 was well-tolerated and seemed to be effective in reducing free IgE in healthy Chinese volunteers, which provides important information for further clinical studies.

Chemotherapy for gastric cancer by finely tailoring anti-Her2 anchored dual targeting immunomicelles.

Micelles with high in vivo serum stability and intratumor accumulation post intravenous (i.v.) injection are highly desired for promoting chemotherapy. Herein, we finely synthesized and tailored well-defined anti-Her2 antibody Fab fragment conjugated immunomicelles (FCIMs), which showed interesting dual targeting function. The thermosensitive poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)(118) (PID(118)) shell with volume phase transition temperature (VPTT: 39 °C) and the anchored anti-Her2 Fab moiety contributed to the passive and active targeting, respectively. The doxorubicin (DOX) loading capacity of such FCIMs was successfully increased about 2 times by physically enhanced hydrophobicity of inner reservoir without structural deformation. The cellular uptake and intracellular accumulation of DOX by temperature regulated passive and antibody navigated active targeting was 4 times of Doxil. The cytotoxicity assay against Her2 overexpression gastric cancer cells (N87s) showed that the IC50 of the FCIMs was ≈ 9 times lower than that of Doxil under cooperatively targeting by Fab at T > VPTT. FCIMs showed high serum stability by increasing the corona PID(118) chain density (S(corona)/N(agg)). In vivo tissue distribution was evaluated in Balb/c nude mice bearing gastric cancer. As observed by the IVIS(®) imaging system, the intratumor accumulation of such finely tailored FCIMs system was obviously promoted 24 h post i.v. administration. Due to the high stability and super-targeting, the in vivo xenografted gastric tumor growth was significantly inhibited with relative tumor volume <2 which was much smaller than ≈ 5 of the control. Consequently, such finely tailored FCIMs with anti-Her2 active and temperature regulated passive dual tumor-targeting function show high potent in chemotherapy.

A bispecific antibody effectively inhibits tumor growth and metastasis by simultaneous blocking vascular endothelial growth factor A and osteopontin.

Both vascular endothelial growth factor A (VEGF) and osteopontin (OPN) can directly induce tumor angiogenesis, which is essential for the growth and metastasis of solid tumors. Here we engineered a bispecific antibody (VEGF/OPN-BsAb) using the anti-VEGF-A antibody bevacizumab and the anti-OPN antibody hu1A12. Compared with hu1A12 alone and bevacizumab alone, VEGF/OPN-BsAb was significantly more effective in inhibiting tumor angiogenesis in a highly metastatic human hepatocellular carcinoma nude mouse model. Further study demonstrated that VEGF/OPN-BsAb could effectively suppress primary tumor growth and metastasis to lungs, suggesting that it might be a promising therapeutic agent for treatment of metastatic cancer.

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 major cause of cancer mortality is the metastatic spread of tumor cells that can occur via multiple routes, including the vascular system and the lymphatic system. In this study, we developed an IgG-like fusion protein molecule [vascular endothelial growth factor (VEGF) receptor 31-immunoglobulin (VEGFR31-Ig)] which could simultaneously bind the angiogenic growth factor VEGF-A and the lymphangiogenic growth factor VEGF-C. Importantly, VEGFR31-Ig exhibited VEGF-A-binding affinity similar to that of VEGFTrap, the most potent VEGF-A binder, and VEGF-C-binding affinity comparable with that of the soluble fusion protein VEGFR3-Ig (sVEGFR3). Pharmacokinetic analysis in mice showed that VEGFR31-Ig had improved pharmacokinetic properties compared with either VEGFTrap or sVEGFR3. In a highly metastatic human hepatocellular carcinoma (HCCLM3) model in severe combined immunodeficient mice, VEGFR31-Ig potently blocked both tumor angiogenesis and lymphangiogenesis, effectively inhibiting primary tumor growth and metastasis to lungs and lymph nodes. In contrast, VEGFTrap only suppressed primary tumor growth and metastasis to lungs by inhibiting tumor angiogenesis, whereas VEGFR3 was only effective in suppressing tumor metastasis to lymph nodes by blocking tumor lymphangiogenesis. Although a combination of VEGFTrap (25 mg/kg twice weekly) and sVEGFR3 (25 mg/kg twice weekly) can achieve the same therapeutic effect as VEGFR31-Ig (25 mg/kg twice weekly) in the HCCLM3 xenograft mouse model, developing two separate receptor-Ig fusion proteins for clinical use as combination therapy is impractical, mainly owing to regulatory hurdles and cost. Taken together, the VEGFR31-Ig fusion protein presented here has been suggested to have great potential for the treatment of metastatic cancer.

Targeted delivery of tumor antigens to activated dendritic cells via CD11c molecules induces potent antitumor immunity in 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 (scFv(CD11c)-HER2/neu). EXPERIMENTAL DESIGN: Induction of cellular and humoral immune responses and antitumoral activity of the fusion proteins admixed with DC-activating CpG oligonucleotides (scFv(CD11c)-HER2/neu(CpG)) 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 scFv(CD11c)-HER2(CpG) 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 scFv(CD11c)-HER2(CpG) 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 scFv(CD11c)-neu(CpG) 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.

Development and characterization of a fully functional small anti-HER2 antibody.

The penetrating of monoclonal antibodies (mAbs) into solid tumor may be hampered by their large size. The antibody mimetics, composed of two complementarity-determining regions (CDRs) through a cognate framework region (FR), have been demonstrated to have the capacity to penetrate tumors superior to its parental intact IgG. In this study, we used CDR and FR sequences from the humanized anti-HER2 monoclonal antibody trastuzumab to design four antibody mimetics. Then these antibody mimetics were fused to human IgG Fc to generate mimetics-Fc small antibodies. One of the four mimetics-Fc antibodies binds well to HER2-overexpressing SK-BR3 cells and effectively inhibits the binding of trastuzumab. This mimetics-Fc, denoted as HMTI-Fc, was shown to be effective in mediating antibody-dependent cellular cytotoxicity and exhibit an antiproliferative effect in SK-BR3 cells. To our knowledge, the HMTI-Fc antibody shown here is the smallest fully functional antibody and may have a potential for treatment of cancer.