Weizhu Qian

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 (scFvCD11c-HER2/neu). Experimental Design: Induction of cellular and humoral immune responses and antitumoral activity of the fusion proteins admixed with DC-activating CpG oligonucleotides (scFvCD11c-HER2/neuCpG) 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 scFvCD11c-HER2CpG 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 scFvCD11c-HER2CpG 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 scFvCD11c-neuCpG 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.

Characterization of a rituximab variant with potent antitumor activity against rituximab-resistant B-cell lymphoma.

Despite widespread use of the anti-CD20 monoclonal antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. A better understanding of rituximab-mediated killing mechanisms is essential to develop more effective therapeutic agents. In this study, we modulated the binding property of rituximab by introducing several point mutations in its complementarity-determining regions. The data showed that changing the binding avidity of rituximab in the range from 10(-8) to 10(-10) M could regulate its antibody-dependent cellular cytotoxicity but not affect its complement-dependent cytotoxicity and apoptosis-inducing activity in B-lymphoma cells. Contradictory to previous findings, we found that the complement-dependent cytotoxicity potency of CD20 mAb was independent of the off-rate. Despite still being a type I CD20 mAb, a rituximab triple mutant (H57DE/H102YK/L93NR), which had a similar binding avidity to a double mutant (H57DE/H102YK), was unexpectedly found to have extremely potent apoptosis-inducing activity. Moreover, this triple mutant, which was demonstrated to efficiently initiate both caspase-dependent and -independent apoptosis, exhibited potent in vivo therapeutic efficacy, even in the rituximab-resistant lymphoma model, suggesting that it might be a promising therapeutic agent for B-cell lymphomas.

Comparison of the inhibition mechanisms of Adalimumab and Infliximab in treating tumor necrosis factor α-associated diseases from a molecular view.

Background: The epitope and the TNFα inhabitation mechanism of Adalimumab remain unclear. Results: The crystal structure of the TNFα in complex with Adalimumab is reported at a resolution of 3.1 Å. Conclusion: The epitope of Adalimumab provided information that Adalimumab may have clinical advantage compared with Infliximab. Significance: These data reveal the Adalimumabs mechanism of TNFα inhibition and its advantages compared with other TNF inhibitors in clinical practice. TNFα-targeting therapy with the use of the drugs Etanercept, Infliximab, and Adalimumab is used in the clinical treatment of various inflammatory and immune diseases. Although all of these reagents function to disrupt the interaction between TNFα and its receptors, clinical investigations showed the advantages of Adalimumab treatment compared with Etanercept and Infliximab. However, the underlying molecular mechanism of action of Adalimumab remains unclear. In our previous work, we presented structural data on how Infliximab binds with the E-F loop of TNFα and functions as a TNFα receptor-binding blocker. To further elucidate the variations between TNFα inhibitors, we solved the crystal structure of TNFα in complex with Adalimumab Fab. The structural observation and the mutagenesis analysis provided direct evidence for identifying the Adalimumab epitope on TNFα and revealed the mechanism of Adalimumab inhibition of TNFα by occupying the TNFα receptor-binding site. The larger antigen-antibody interface in TNFα Adalimumab also provided information at a molecular level for further understanding the clinical advantages of Adalimumab therapy compared with Infliximab.

Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin.

There is an urgent need for improved therapy for advanced ovarian carcinoma, which may be met by administering immune-modulatory monoclonal antibodies (mAbs) to generate a tumor-destructive immune response. Using the ID8 mouse ovarian cancer model, we investigated the therapeutic efficacy of various mAb combinations in mice with intraperitoneal (i.p.) tumor established by transplanting 3 × 106 ID8 cells 10 days previously. While most of the tested mAbs were ineffective when given individually or together, the data confirm our previous finding that 2 i.p. injections of a combination of anti-CD137 with anti-PD-1 mAbs doubles overall survival. Mice treated with this mAb combination have a significantly increased frequency and total number of CD8+ T cells both in the peritoneal lavage and spleens, and these cells are functional as demonstrated by antigen-specific cytolytic activity and IFN-γ production. While administration of anti-CD137 mAb as a single agent similarly increases CD8+ T cells, these have no functional activity, which may be attributed to up-regulation of co-inhibitory PD-1 and TIM-3 molecules induced by CD137. Addition of the anti-cancer drug cisplatin to the 2 mAb combination increased overall survival >90 days (and was probably curative) by a mechanism which included a systemic CD8+ T cell response with tumor specificity and immunological memory. Strikingly, combined treatment of cisplatin and CD137/PD-1 mAb also gave rise to the long-term survival of mice with established TC1 lung tumors. A similar combination of the 2 mAbs and cisplatin should be considered for clinical ‘translation’.

Development of Novel Tetravalent Anti-CD20 Antibodies with Potent Antitumor Activity

Despite the effectiveness of the anti-CD20 monoclonal antibody (mAb) Rituximab (C2B8) in the treatment of B-cell lymphoma, its efficacy remains variable and often modest. It seems likely that a combination of multiple mechanisms, such as complement-dependent cytotoxicity (CDC) and apoptotic signaling, underlies the therapeutic success of anti-CD20 mAbs. Unfortunately, all the current anti-CD20 mAbs effective in CDC are relatively inactive in signaling cell death and vice versa. In this study, we developed two genetically engineered tetravalent antibodies (TetraMcAb) respectively derived from the anti-CD20 mAbs C2B8 and 2F2. TetraMcAbs, with a molecular mass only 25 kDa higher than native divalent antibodies (DiMcAb), were shown not only to be as effective in mediating CDC and antibody-dependent cellular cytotoxicity against B-lymphoma cells as DiMcAbs but also to have antiproliferative and apoptosis-inducing activity markedly superior to that of DiMcAbs. Interestingly, whereas 2F2 and C2B8 were equally effective in inducing cell growth arrest and apoptosis, the functions of their tetravalent versions, 2F2(ScFvHL)(4)-Fc and C2B8(ScFvHL)(4)-Fc, were significantly different. 2F2(ScFvHL)(4)-Fc exhibited exceptionally more potent antiproliferative and apoptosis-inducing activity than that of C2B8(ScFvHL)(4)-Fc. Immunotherapeutic studies further showed that 2F2(ScFvHL)(4)-Fc was far more effective in prolonging the survival of severe combined immunodeficient mice bearing systemic Daudi or Raji tumors than C2B8, 2F2, and C2B8(ScFvHL)(4)-Fc, suggesting that it might be a promising therapeutic agent for B-cell lymphoma.

Treatment of collagen-induced arthritis with an anti-osteopontin monoclonal antibody through promotion of apoptosis of both murine and human activated T cells

OBJECTIVE To test the effects of a novel monoclonal antibody (mAb) against human osteopontin (OPN) in the prevention and treatment of collagen-induced arthritis (CIA) and to elucidate the underlying mechanisms of these effects. METHODS DBA/1J mice immunized with type II collagen to induce CIA were monitored to assess the effects of anti-OPN mAb on the clinical severity of the disease, and pathologic changes in the joints were examined histologically. The effects of anti-OPN mAb on survival of activated T cells from arthritic mice and from the synovial fluid of patients with rheumatoid arthritis (RA) were determined by TUNEL assay or annexin V assay. The levels of apoptosis-related proteins (Bim, Bax, and Bcl-2) and NF-kappaB were detected by immunoblot analysis. RESULTS One anti-OPN mAb, 23C3, was effective in inhibiting the development of CIA and even reversing established disease in DBA/1J mice. Monoclonal antibody 23C3 reduced the levels of serum type II collagen-specific autoantibodies and proinflammatory cytokines, and suppressed T cell recall responses to type II collagen. Mechanistic studies demonstrated that OPN prevented the death of type II collagen-activated murine T cells and synovial T cells from RA patients. Monoclonal antibody 23C3 promoted apoptosis of the activated T cells, particularly CD4+ T cells, by inhibiting activation of NF-kappaB and by altering the balance among the proapoptotic proteins Bim and Bax and the antiapoptotic protein Bcl-2. Screening of a phage display peptide library led to identification of the epitope ATWLNPDPSQKQ as being recognized by this novel antibody. CONCLUSION Because of its ability to effectively promote apoptosis of activated T cells, mAb 23C3 may be a novel therapeutic agent for the treatment of RA.

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.

Construction and characterization of a humanized anti‐human CD3 monoclonal antibody 12F6 with effective immunoregulation functions

12F6 is a murine anti‐human CD3 monoclonal antibody, which competes with OKT3 for binding to human T cells and possesses more effective T‐cell suppression and activation properties compared to OKT3. It thus exhibits the potential to be developed as an immunoregulation agent for manipulating T‐cell functions and preventing acute allograft rejection. In an attempt to minimize the immunogenicity of murine 12F6 (m12F6) for potential clinical application, a humanized version of 12F6, denoted as hu12F6, was successfully constructed by complementary determining region (CDR) grafting and shown to maintain both T‐cell activation and suppression activities similar to m12F6. Furthermore, in order to reduce the first dose reaction syndrome caused by T‐cell activation following the first administration of anti‐CD3 antibodies, two amino acid mutations were introduced into the Fc region of hu12F6, resulting in the Fc‐mutated 12F6 humanized antibody (hu12F6mu). This Fc‐mutated version displayed a similar antigen‐binding affinity and specificity compared with hu12F6 and m12F6 but with much weaker FcR binding activity. hu12F6mu was shown to be much less potent in the induction of T‐cell proliferation, cytokine release (tumour necrosis factor‐α, interferon‐γ and interleukin‐10) and early activation marker expression on the cell surface (CD69 and CD25) than parental 12F6 and OKT3 did. In contrast, hu12F6mu was effective in modulating T‐cell receptor/CD3 and inhibiting mixed lymphocyte reaction with a similarity as compared to m12F6 and OKT3. In conclusion, the resultant hu12F6mu was much less mitogenic to T cells but retained potent immunosuppression, suggesting it might be an alternative to OKT3 as an immunosuppressive drug with less immunogenicity and toxicity for clinical application.

Bispecific Antibody to ErbB2 Overcomes Trastuzumab Resistance through Comprehensive Blockade of ErbB2 Heterodimerization

The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. However, resistance to trastuzumab is common. Heterodimerization between ErbB2 and other ErbBs may redundantly trigger cell proliferation signals and confer trastuzumab resistance. Here, we developed a bispecific anti-ErbB2 antibody using trastuzumab and pertuzumab, another ErbB2-specific humanized antibody that binds to a distinct epitope from trastuzumab. This bispecific antibody, denoted as TPL, retained the full binding activities of both parental antibodies and exhibited pharmacokinetic properties similar to those of a conventional immunoglobulin G molecule. Unexpectedly, TPL showed superior ErbB2 heterodimerization-blocking activity over the combination of both parental monoclonal antibodies, possibly through steric hindrance and/or inducing ErbB2 conformational change. Further data indicated that TPL potently abrogated ErbB2 signaling in trastuzumab-resistant breast cancer cell lines. In addition, we showed that TPL was far more effective than trastuzumab plus pertuzumab in inhibiting the growth of trastuzumab-resistant breast cancer cell lines, both in vitro and in vivo. Importantly, TPL treatment eradicated established trastuzumab-resistant tumors in tumor-bearing nude mice. Our results suggest that trastuzumab-resistant breast tumors remain dependent on ErbB2 signaling and that comprehensive blockade of ErbB2 heterodimerization may be an effective therapeutic avenue. The unique potential of TPL to overcome trastuzumab resistance warrants its consideration as a promising treatment in the clinic.

A humanized anti-osteopontin antibody inhibits breast cancer growth and metastasis in vivo

Osteopontin (OPN) has been implicated as an important mediator of breast cancer progression and metastasis and has been investigated for use as a potential therapeutic target in the treatment of breast cancer. However, the in vivo antitumor effect of anti-OPN antibodies on breast cancer has not been reported. In this study, a mouse anti-human OPN antibody (1A12) was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. A humanized version of 1A12, denoted as hu1A12, was shown to possess affinity comparable to that of its parental antibody. The ability of hu1A12 to inhibit cell migration, adhesion, invasion and colony formation was assessed in a highly metastatic human breast cancer cell line MDA-MB-435S. The results indicated that hu1A12 was effective in inhibiting the cell adhesion, migration, invasion and colony formation of MDA-MB-435S cells in vitro. hu1A12 also showed significant efficacy in suppressing primary tumor growth and spontaneous metastasis in a mouse lung metastasis model of human breast cancer. The specific epitope recognized by hu1A12 was identified to be 212NAPSD216, adjacent to the calcium binding domain of OPN. Our data strongly support that OPN is a potential target for the antibody-based therapies of breast cancer. The humanized anti-OPN antibody hu1A12 may be a promising therapeutic agent for the treatment of human breast cancer.