Dongmei Fan

Efficient inhibition of human B-cell lymphoma xenografts with an anti-CD20×anti-CD3 bispecific diabody

Bispecific antibodies have been exploited both as cancer immunodiagnostics and as cancer therapeutics, and have shown promise in several clinical trials in cancer imaging and therapy. A number of bispecific antibodies against B-cell markers have been shown to be effective in vitro in mediating tumor cell lysis and in vivo in inhibiting tumor growth in animal models. We have constructed a bispecific diabody from the variable genes encoding two hybridoma-derived monoclonal antibodies directed against human CD20 on B cells and CD3 on T cells. The anti-CD20 x anti-CD3 diabody was expressed in a single Escherichia coli host and purified by a one-step affinity chromatography. The bispecific diabody bound as efficiently to both CD20- and CD3-positive cells as the respective parental antibodies, and was capable of cross-linking CD20-positive tumor cells and human T lymphocytes as shown by cellular rosetting. The diabody effectively lysed human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Further, when combined with human PBL and interleukin-2, the diabody significantly prolonged the survival of nude mice inoculated with human B-lymphoma cells. Taken together, our results suggest that an anti-CD20 x anti-CD3 diabody may have significant clinical application in the treatment of human CD20-positive B-cell malignancies.

Human Umbilical Cord Mesenchymal Stem Cells as Vehicles of CD20-Specific TRAIL Fusion Protein Delivery: A Double-Target Therapy against Non-Hodgkin’s Lymphoma

Mesenchymal stem cells (MSCs) are an attractive candidate for cell-based therapy. We have designed a promising double-target therapeutic system for non-Hodgkins lymphoma (NHL) therapy. The system is based on MSC homing capacity and scFvCD20 antigen-restriction to NHL. In this system, a novel secreted fusion protein scFvCD20-sTRAIL, which contains a CD20-specific single chain Fv antibody fragment (scFv) and a soluble tumor necrosis factor related apoptosis-inducing ligand (sTRAIL, aa residues 114-281) with an isoleucine zipper (ISZ) added to the N-terminal (ISZ-sTRAIL), was expressed in human umbilical cord derived mesenchymal stem cells (HUMSCs). When compared with ISZ-sTRAIL protein, the scFvCD20-sTRAIL fusion protein demonstrated a potent inhibition of cell proliferation in CD20-positive BJAB cells, moderate inhibition in Raji cells, weak inhibition in CD20-negative Jurkat cells, and no effect on normal human peripheral blood mononuclear cells (PBMCs). The scFvCD20-sTRAIL fusion protein also caused significant increase of cellular apoptosis through both extrinsic and intrinsic apoptosis signaling pathways. Using a NOD/SCID mouse subcutaneous BJAB lymphoma xenograft model, the tropism of the firefly luciferase (fLuc) labeled MSC was monitored by bioluminescent imaging (BLI) for fLuc activity. Our study indicated that HUMSCs selectively migrated to the tumor site after 24 h of intravenous injection and mice injected with the MSC.scFvCD20-sTRAIL significantly inhibited the tumor growth when compared with those treated with MSC.ISZ-sTRAIL. The treatment was tolerated well in mice, as no obvious toxicities were observed. Our study has suggested that scFvCD20-sTRAIL secreting HUMSCs is a novel and efficient therapeutic approach for the treatment of non-Hodgkins lymphoma.

Co-expression of cytokeratin 8 and breast cancer resistant protein indicates a multifactorial drug-resistant phenotype in human breast cancer cell line.

AIMS The aim was to determine whether increased CK8 and BCRP expression cooperatively contribute to multidrug resistance (MDR) in MCF-7/MX cells. Accumulating evidence suggests that the development and maintenance of cancer MDR involves complex multimodal mechanisms that interact concomitantly and complementarily. In this report, we observed elevated expression of cytokeratin 8 (CK8) in MCF-7/MX, a mitoxantrone (MX)-selected human breast tumor cell line with the MDR phenotype known as overexpression of breast cancer resistant protein (BCRP). MAIN METHODS Gene transfection methods were used to express CK8 and BCRP in NIH3T3 fibroblasts, individually or in combination. KEY FINDINGS Taken together, our present study suggests that CK8 together with BCRP may play significant roles in conferring the multifactorial MDR phenotype of MCF-7/MX cells, but may act independently via potentially different mechanisms. Although expressing either CK8 or BCRP alone was able to confer resistance to mitoxantrone, cells co-expressing both proteins demonstrated significantly increased drug resistance. Furthermore, RNAi knockdown of CK8 and BCRP, alone and in combination, in MCF-7/MX cells significantly attenuated their resistance to chemotherapeutic agents. Interestingly, in contrast to inhibition of BCRP expression via anti-BCRP shRNA vector transfection, reversal of mitoxantrone resistance by transfection with anti-CK8 shRNA was not accompanied by an increase in intracellular drug accumulation. SIGNIFICANCE Combinational approaches that target multiple drug-resistance-related molecules/pathways in cancer cells may represent more efficacious strategies to overcome MDR.

Suppression of orthotopically implanted hepatocarcinoma in mice by umbilical cord-derived mesenchymal stem cells with sTRAIL gene expression driven by AFP promoter.

Mesenchymal stem cells (MSCs) are promising vehicles for delivering therapeutic agents in tumor therapy. Human umbilical cord-derived mesenchymal stem cells (HUMSCs) resemble bone marrow-derived MSCs with respect to hepatic differentiation potential in injured livers in animals, while their hepatic differentiation under the hepatocarcinoma microenvironment is unclear. In this study, HUMSCs were isolated and transduced by lentiviral vectors coding the soluble human tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) gene driven by alpha-fetoprotein (AFP) promoter to investigate the therapeutic effects of these HUMSC against orthotopically implanted hepatocarcinoma in mice. We showed that HUMSCs can be transduced by lentivirus efficiently. HUMSCs developed cuboidal morphology, and expressed AFP and albumin in a two-step protocol. HUMSCs were capable of migrating to hepatocarcinoma in vitro as well as in vivo. In the orthotopical hepatocarcinoma microenvironment, the AFP promoter was activated during the early hepatic differentiation of HUMSCs. After intravenous injected, MSC.AFPILZ-sTRAIL expressed sTRAIL exclusively at the tumor site, and exhibited significant antitumor activity. This effect was stronger when in combination with 5-FU. The treatment was tolerated well in mice. Collectively, our results provide a potential strategy for targeted tumor therapy relying on the use of the tumor tropism and specific differentiation of HUMSCs as vehicles.

The development of bispecific antibodies and their applications in tumor immune escape

During the past two decades, a great evolution of bispecific antibodies (BsAbs) for therapeutic applications has been made. BsAbs can bind simultaneously two different antigens or epitopes, which leads to a wide range of applications including redirecting T cells or NK cells to tumor cells, blocking two different signaling pathways, dual targeting of different disease mediators, and delivering payloads to targeted sites. Aside from approved catumaxomab (anti-CD3 and anti-EpCAM) and blinatumomab (anti-CD3 and anti-CD19), many more BsAbs are now in various phases of clinical development. Here, this review focus on the development of bispecific antibodies and their applications in tumor immune escape.

AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells

BackgroundLeukemic stem cells (LSCs) are frequently seen as a cause of treatment failure and relapse in patients with acute myeloid leukemia (AML). Thus, successful new therapeutic strategies for the treatment of AML should aim at eradicating LSCs. The identification of targets on the cell surface of LSCs is getting more and more attention. Among these, CD123, also known as the interleukin-3 (IL3)-receptor α chain, has been identified as a potential immunotherapeutic target due to its overexpression on LSCs in AML as well as on AML blasts, rather than normal hematopoietic stem cells.MethodsWe constructed a CD123-targeted fusion protein antiCD3Fv-⊿IL3, with one binding site for T cell antigen receptor (TCRCD3) and the other for CD123, by recombinant gene-engineering technology. Cysteine residues were introduced into the V domains of the antiCD3Fv segment to enhance its stability by locking the two chains of Fv together with disulfide covalent bonds. The stability and cytotoxicity of the two fusion proteins were detected in vitro and in vivo.ResultsBoth fusion proteins were produced and purified from Escherichia coli 16C9 cells with excellent yields in fully active forms. High-binding capability was observed between these two fusion proteins and human IL3R, leading to the specific lysis of CD123-expressing cell lines KG1a; also, mononuclear cells from primary AML patients were inhibited in a colony forming assay in vitro, presumably by redirecting T lymphocytes in vitro. In addition, they displayed an antileukemic activity against KG1a xenografts in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, especially disulfide-stabilized (ds)-antiCD3Fv-⊿IL3 for its improved stability.ConclusionsThese results suggest that both fusion proteins display the antileukemic activity against CD123-expressing cell lines as well as leukemic progenitors in vitro and in vivo, especially ds-antiCD3Fv-⊿IL3. They could be the promising candidates for future immunotherapy of AML.

Down‐regulation of c‐fos by shRNA sensitizes adriamycin‐resistant MCF‐7/ADR cells to chemotherapeutic agents via P‐glycoprotein inhibition and apoptosis augmentation

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer. Research indicated that the main mechanisms of most cancers included so‐called “pump” (P‐glycoprotein, P‐gp) and “non‐pump” (apoptosis) resistance. Identification of novel signaling molecules associated with both P‐gp and apoptosis will facilitate the development of more effective strategies to overcome MDR in tumor cells. Since the proto‐oncogene c‐fos has been implicated in cell adaptation to environmental changes, we analyzed its role in mediating “pump” and “non‐pump” resistance in MCF‐7/ADR, an adriamycin (ADR)‐selected human breast cancer cell line with the MDR phenotype. Elevated expression of c‐fos in MCF‐7/ADR cells and induction of c‐fos by ADR in the parental drug‐sensitive MCF‐7 cells suggested a link between c‐fos and MDR phenotype. Down‐regulation of c‐fos expression via shRNA resulted in sensitization of MCF‐7/ADR cells to chemotherapeutic agents, including both P‐gp and non‐P‐gp substrates. Further results proved that c‐fos down‐regulation in MCF‐7/ADR cells resulted in decreased P‐gp expression and activity, enhanced apoptosis, and altered expression of apoptosis‐associated proteins (i.e., Bax, Bcl‐2, p53, and PUMA). All above facts indicate that c‐fos is involved in both P‐gp‐ and anti‐apoptosis‐mediated MDR of MCF‐7/ADR cells. Based on these results, we propose that c‐fos may represent a potential molecular target for resistant cancer therapy, and suppressing c‐fos gene expression may therefore be an effective means to temper breast cancer cells MDR to cytotoxic chemotherapy. J. Cell. Biochem. 114: 1890–1900, 2013.

Expression of ABCB5 gene in hematological malignances and its significance

Abstract We examined ABCB5 gene expression using real-time polymerase chain reaction (PCR) in leukemia cells from 29 patients with acute lymphoblastic leukemia (ALL), 24 patients with chronic lymphocytic leukemia (CLL), 42 with acute myeloid leukemia (AML), 22 with chronic myeloid leukemia (CML), 17 with lymphoma and 10 with multiple myeloma (MM). It was confirmed that expression of the ABCB5 gene is highly increased in B-precursor ALL and French–American–British (FAB) M1 and M2 types of AML and lymphoma. The ABCB5 gene is expressed more highly in patients with relapsed or refractory disease than in patients with drug sensitive acute leukemia. Furthermore, there was an evident positive correlation between ABCB5 mRNA expression and MDR1 mRNA expression, but no correlation with MRP mRNA expression or BCRP mRNA expression. Quantification of the ABCB5 gene by real-time PCR offers particular promise as a prognostic marker and a marker for drug resistance in acute leukemia. Our findings raise the possibility that ABCB5 may be responsible for both the progression and chemotherapeutic refractoriness of advanced acute leukemia, and that ABCB5-targeted approaches might therefore represent novel and translationally relevant therapeutic strategies for drug resistance in leukemia.

Efficient inhibition of human B-cell lymphoma in SCID mice by synergistic antitumor effect of human 4-1BB ligand/anti-CD20 fusion proteins and anti-CD3/anti-CD20 diabodies.

Here we constructed and produced a recombinant human 4-1BB ligand (4-1BBL)/anti-CD20 fusion protein and examined its antitumor activity, alone and in combination with an anti-CD3/anti-CD20 bispecific diabody. The 4-1BBL/anti-CD20 fusion protein retained both the costimulatory activity of 4-1BBL on T cells and the tumor targeting ability of CD20 antibody on B cells. The fusion protein bound as efficiently to 4-1BB- and CD20-positive cells as its respective parental antibodies, and was capable of cross-linking human T lymphocytes and CD20-positive tumor cells. Combination treatment with 4-1BBL/anti-CD20 fusion protein and anti-CD3/anti-CD20 diabody led to significantly increased T-cell cytotoxicity to human B-lymphoma cells in vitro and drastically more potent tumor inhibitory activity in vivo in xenografted B-cell lymphoma in severe combined immunodeficiency disease mice. Mechanistic studies revealed that the combination treatment remarkably inhibited apoptosis of human peripheral blood lymphocytes, accompanied by upregulation of Bcl-XL and Bf1-1, perforin and granzyme B mRNA, and increased interleukin-2 production. Taken together, these results suggest that targeted delivery of 4-1BBL to the tumor site, when combined with anti-CD3/anti-CD20 diabody, could strongly potentiate the antitumor activity of the diabody, thus may have significant clinical application in the treatment of human CD20-positive B-cell malignancies.

Mesenchymal stromal cells as vehicles of tetravalent bispecific Tandab (CD3/CD19) for the treatment of B cell lymphoma combined with IDO pathway inhibitor D-1-methyl-tryptophan.

BackgroundAlthough blinatumomab, a bispecific T cell engaging antibody, exhibits high clinical response rates in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia (B-ALL) and B cell non-Hodgkin’s lymphoma (B-NHL), it still has some limitations because of its short half-life. Mesenchymal stromal cells (MSCs) represent an attractive approach for delivery of therapeutic agents to cancer sites owing to their tropism towards tumors, but their immunosuppression capabilities, especially induced by indoleamine 2,3-dioxygenase (IDO), should also be taken into consideration.MethodsHuman umbilical cord-derived MSCs (UC-MSCs) were genetically modified to secrete Tandab (CD3/CD19), a tetravalent bispecific tandem diabody with two binding sites for CD3 and two for CD19. The tropism of MSCs towards Raji cells in vitro was determined by migration assays, and the homing property of MSCs in vivo was analyzed with firefly luciferase-labeled MSCs (MSC-Luc) by bioluminescent imaging (BLI). The cytotoxicity of T cells induced by MSC-secreting Tandab (CD3/CD19) was detected in vitro and in vivo in combination with d-1-methyl-tryptophan (D-1MT), an IDO pathway inhibitor.ResultsThe purified Tandab (CD3/CD19) was functional with high-binding capability both for CD3-positive cells and CD19-positive cells and was able to induce specific lysis of CD19-positive cell lines (Raji, Daudi, and BJAB) in the presence of T cells. Additionally, results from co-culture killing experiments demonstrated that Tandab (CD3/CD19) secreted from MSCs was also effective. Then, we confirmed that D-1MT could enhance the cytotoxicity of T cells triggered by MSC-Tandab through reversing T cell anergy with down-regulation of CD98 and Jumonji and restoring the proliferation capacity of T cells. Furthermore, MSC-Luc could selectively migrate to tumor site in a BALB/c nude mouse model with Raji cells. And mice injected with MSC-Tandab in combination with D-1MT significantly inhibited the tumor growth.ConclusionsThese results suggest that UC-MSCs releasing Tandab (CD3/CD19) is an efficient therapeutic tool for the treatment of B cell lymphoma when combined with D-1MT.