This phase I clinical trial (NCT01935843) is to evaluate the safety, feasibility, and activity of chimeric antigen receptor-engineered T cell (CART) immunotherapy targeting human epidermal growth factor receptor 2 (HER2) in patients with advanced biliary tract cancers (BTCs) and pancreatic cancers (PCs). Eligible patients with HER2-positive (>50%) BTCs and PCs were enrolled in the trial. Well cultured CART-HER2 cells were infused following the conditioning treatment composed of nab-paclitaxel (100-200 mg/m) and cyclophosphamide (15-35 mg/kg). CAR transgene copy number in the peripheral blood was serially measured to monitor the expansion and persistence of CART-HER2 cells in vivo. Eleven enrolled patients received 1 to 2-cycle CART-HER2 cell infusion (median CAR T cell 2.1 × 10/kg). The conditioning treatment resulted in mild-to-moderate fatigue, nausea/vomiting, myalgia/arthralgia, and lymphopenia. Except one grade-3 acute febrile syndrome and one abnormal elevation of transaminase (>9 ULN), adverse events related to the infusion of CART-HER2 cells were mild-to-moderate. Post-infusion toxicities included one case of reversible severe upper gastrointestinal hemorrhage which occurred in a patient with gastric antrum invaded by metastasis 11 days after the CART-HER2 cell infusion, and 2 cases of grade 1-2 delayed fever, accompanied by the release of C-reactive protein and interleukin-6. All patients were evaluable for assessment of clinical response, among which 1 obtained a 4.5-months partial response and 5 achieved stable disease. The median progression free survival was 4.8 months (range, 1.5-8.3 months). Finally, data from this study demonstrated the safety and feasibility of CART-HER2 immunotherapy, and showed encouraging signals of clinical activity.
Aged ; Biliary Tract Neoplasms ; immunology ; therapy ; Female ; Humans ; Immunotherapy, Adoptive ; Male ; Middle Aged ; Pancreatic Neoplasms ; immunology ; therapy ; Receptor, ErbB-2 ; immunology ; Receptors, Chimeric Antigen ; immunology ; T-Lymphocytes ; immunology

Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 and CD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.
Animals ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasms, Experimental ; immunology ; pathology ; therapy ; Receptor, ErbB-2 ; immunology ; Receptors, Antigen, T-Cell ; immunology ; Stomach Neoplasms ; immunology ; pathology ; therapy ; Tumor Cells, Cultured

Tumor surface antigen human epidermal growth factor receptor 2 (Her2) is a type I receptor tyrosine kinase, which belongs to human epidermal growth factor receptor family. Her2-overexpression is associated with tumorigenesis and metastasis. Due to significant clinical effects, Her2-targeted cancer therapy especially therapeutic antibody has become the hot spot in the field of cancer treatment. Anti-Her2 antibody drugs include monoclonal antibodies, antibody-drug conjugates, bispecific antibodies and emerging "two in one" antibody. Based on structure and function of Her2, this review focuses on recent advances in active mechanisms and clinical researches of these antibodies.
Antibodies, Bispecific ; therapeutic use ; Antibodies, Monoclonal ; therapeutic use ; Humans ; Immunoconjugates ; therapeutic use ; Neoplasms ; drug therapy ; Receptor, ErbB-2 ; immunology

OBJECTIVETo construct human phage single-chain antibody (scFv) library against breast cancer, and to identify anti-HER2 specific antibodies from the human phage display scFv library to offer a stronger affinity sequence targeting HER2 for fusion protein targeting HER2 and CXCR4.

METHODSTotal RNA was extracted from the adjacent lymphatic tissue harvested from breast cancer patients. The variable regions of the whole antibody were amplified by using RT-PCR and were cloned into the vector pCANTAB-5E through a linker. The products were electroporated into competent E.coli TG1 cells. Recombinant phages specific for breast cancer cells were enriched in SKBR-3 after four rounds. The antigen-positive clones were selected by ELISA and immunohistochemistry.

RESULTSThe fragment of VH and VL were about 375 and 330 bp and were linked in vitro to form scFv of 750 bp that was resistant to the breast cancer HER2 single strand. A fusion phage display library that contained total of 2.48×10(8) pfu /ml was established. ELISA and immunohistochemical results confirmed that the antibody has a strong affinity with HER2 antigen in breast cancer tissue. Compared to human IgG antibody, a scFv phage library against human breast cancer was successfully constructed with high capacity. The scFv was highly specific to HER2 antigen and the sequencing results indicated that VL and VH genes were highly homologous with the variable region of human antibody.

CONCLUSIONThis strategy may achieve new targeted antibody resistant to the breast cancer for clinical treatment and provide a carrier that uses HER2 as a target of the fusion protein for anti-tumor therapy.

Breast Neoplasms ; genetics ; immunology ; Female ; Humans ; Peptide Library ; Receptor, ErbB-2 ; immunology ; Single-Chain Antibodies ; immunology

OBJECTIVE: To synthesize mesoporous silica-core-shell magnetic nanoparticles (MNPs) encapsulated by liposomes (Lipo [MNP@m-SiO2]) in order to enhance their stability, allow them to be used in any buffer solution, and to produce trastuzumab-conjugated (Lipo[MNP@m-SiO2]-Her2Ab) nanoparticles to be utilized in vitro for the targeting of breast cancer. MATERIALS AND METHODS: The physiochemical characteristics of Lipo[MNP@m-SiO2] were assessed in terms of size, morphological features, and in vitro safety. The multimodal imaging properties of the organic dye incorporated into Lipo[MNP@m-SiO2] were assessed with both in vitro fluorescence and MR imaging. The specific targeting ability of trastuzumab (Her2/neu antibody, Herceptin(R))-conjugated Lipo[MNP@m-SiO2] for Her2/neu-positive breast cancer cells was also evaluated with fluorescence and MR imaging. RESULTS: We obtained uniformly-sized and evenly distributed Lipo[MNP@m-SiO2] that demonstrated biological stability, while not disrupting cell viability. Her2/neu-positive breast cancer cell targeting by trastuzumab-conjugated Lipo[MNP@m-SiO2] was observed by in vitro fluorescence and MR imaging. CONCLUSION: Trastuzumab-conjugated Lipo[MNP@m-SiO2] is a potential treatment tool for targeted drug delivery in Her2/neu-positive breast cancer.
3T3 Cells ; Animals ; Antibodies, Monoclonal, Humanized/*administration & dosage ; Antineoplastic Agents/*administration & dosage ; Breast Neoplasms/chemistry/*drug therapy ; Cell Line, Tumor ; Drug Delivery Systems/methods ; Female ; Ferric Compounds/chemistry ; Humans ; Liposomes ; Magnetite Nanoparticles/administration & dosage/*chemistry ; Mice ; Molecular Targeted Therapy/methods ; Nanoconjugates/administration & dosage/*chemistry ; Nanoparticles/chemistry ; *Receptor, erbB-2/immunology ; Silicon Dioxide/administration & dosage/*chemical synthesis/chemistry

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Cell Line ; Chimera ; Cloning, Molecular ; Humans ; Lentivirus ; genetics ; metabolism ; Mice ; Receptor, ErbB-2 ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection

OBJECTIVETo explore the cytotoxic responses of spleen T lymphocytes (CTL) in BALB/c mice induced by recombinant HSP110-HER2/neu ICD complex.

METHODSTumor-bearing mouse model was immunized by HSP110-HER2/neu ICD complex. The IFN-γ level secreted by activated spleen T lymphocytes was detected by enzyme linked immunospot assay (ELISPOT). The corresponding CTL activity was measured by granzyme release assay.

RESULTSThe BALB/c mouse model of human mammary tumor highly expressing HER2/neu was established. HSP110-HER2/neu ICD complex immunization led to a significantly higher level of INF-γ than that in HSP110-P(789-797) immunized and HER2/neu ICD immunized mice. HSP110-HER2/neu ICD complex immunized animals also show significant CTL activity. The results of immunohistochemical staining showed that the number of blue spots in the PBS group was 4.57 ± 1.33, HSP110 group 6.83 ± 2.08, HER2/neu ICD group 16.17 ± 2.86, HSP110-P(789-797) group 43.67 ± 4.78, and SP110-HER2/neu ICD group 76.51 ± 8.17. The number of IFN-γ-secreting spleen lymphocytes in the HSP110-HER2/neu ICD group was significantly higher than that in the HSP110-P(789-797) group, and that of HSP110-P(789-797) group was significantly higher than that of HER2/neu ICD group (P < 0.01). The target cell-killing rate of the PBS group was (8.15 ± 1.27)%, HSP110 group (9.51 ± 1.51)%, HER2/neu ICD group (14.03 ± 2.45)%, HSP110-P(789-797) group (25.99 ± 3.04)% and HSP110-HER2/neu ICD group (38.15 ± 3.95)% (all P < 0.01).

CONCLUSIONSHSP110-HER2/neu ICD complex can promote the proliferation and maturation of T lymphocytes into CTLs, and might be used as anti-tumor vaccine to induce potent cytotoxic T lymophocyte immunoresponse against specific tumor cells.

Animals ; Breast Neoplasms ; metabolism ; pathology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Female ; HSP110 Heat-Shock Proteins ; immunology ; Humans ; Interferon-gamma ; metabolism ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Receptor, ErbB-2 ; immunology ; metabolism ; Recombinant Proteins ; immunology ; Spleen ; cytology ; immunology ; T-Lymphocytes ; cytology ; immunology ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, Synthetic ; immunology

It has been well established that immune surveillance plays critical roles in preventing the occurrence and progression of tumor. More and more evidence in recent years showed the host anti-tumor immune responses also play important roles in the chemotherapy and radiotherapy of cancers. Our previous study found that tumor- targeting therapy of anti-HER2/neu mAb is mediated by CD8(+) T cell responses. However, we found here that enhancement of CD8(+) T cell responses by combination therapy with IL-15R/IL-15 fusion protein or anti-CD40, which are strong stimultors for T cell responses, failed to promote the tumor therapeutic effects of anti-HER2/neu mAb. Analysis of tumor microenviornment showed that tumor tissues were heavily infiltrated with the immunosuppressive macrophages and most tumor infiltrating T cells, especially CD8(+) T cells, expressed high level of inhibitory co-signaling receptor PD-1. These data suggest that tumor microenvironment is dominated by the immunosuppressive strategies, which thwart anti-tumor immune responses. Therefore, the successful tumor therapy should be the removal of inhibitory signals in the tumor microenvironment in combination with other therapeutic strategies.
Animals ; Antibodies, Monoclonal ; immunology ; therapeutic use ; Breast Neoplasms ; drug therapy ; immunology ; pathology ; Cell Line, Tumor ; Female ; Humans ; Immune Tolerance ; immunology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Receptor, ErbB-2 ; immunology ; Tumor Microenvironment ; immunology

Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective alternatives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-based regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.
Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Antigens, CD20 ; immunology ; Humans ; Immunoconjugates ; therapeutic use ; Neoplasms ; drug therapy ; immunology ; Protein Engineering ; RANK Ligand ; immunology ; Receptor, Epidermal Growth Factor ; immunology ; Receptor, ErbB-2 ; immunology ; Vascular Endothelial Growth Factor A ; immunology

A recombinant plasmid pET28a-HBcAg-delta n was constructed, in which three mimic B-epitopes of HER family were inserted into the truncated HBc vector. The fusion protein expressed was purified and used to immunize BALB/c mice to induce antibody against the epitopes. Three mimic epitope genes were inserted into the sequences of amino acid residues 78 and 79 of HBcAg by overlap PCR. The PCR product was then cloned into pET28a to construct recombinant expression plasmid which was transformed to E. coli BL21 (DE3) and induced by IPTG. After purification, the fused protein designed HBHE was used to immunize BALB/c mice to detect humoral immunoresponse. The recombinant plasmid was successfully constructed by DNA sequencing analysis. A fusion protein with correct molecular mass was expressed and confirmed by SDS-PAGE. High titre antibody was elicited in the mice immunized with HBHE by indirect ELISA and Western blotting. The HBc particle vector containing three B-epitopes of HER family had been successfully prepared, purified and high titre antibody against HBHE was detected. All these data are helpful in further research of the broad-spectrum anti-tumour effect of combine polypeptide epi-position vaccine of EGFR and HER2.
Animals ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Epitopes ; immunology ; Genetic Vectors ; Hepatitis B Core Antigens ; genetics ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; Random Allocation ; Receptor, Epidermal Growth Factor ; genetics ; immunology ; Receptor, ErbB-2 ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccination ; methods ; Vaccines, Combined ; immunology