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Immune checkpoint blockade has shown significant clinical benefit in multiple cancer indications, but many patients are either refractory or become resistant to the treatment over time. HER2/neu oncogene overexpressed in invasive breast cancer patients associates with more aggressive diseases and poor prognosis. Anti-HER2 mAbs, such as trastuzumab, are currently the standard of care for HER2-overexpressing cancers, but the response rates are below 30% and patients generally suffer relapse within a year. In this study we developed a bispecific antibody (BsAb) simultaneously targeting both PD1 and HER2 in an attempt to combine HER2-targeted therapy with immune checkpoint blockade for treating HER2-positive solid tumors. The BsAb was constructed by fusing scFvs (anti-PD1) with the effector-functional Fc of an IgG (trastuzumab) via a flexible peptide linker. We showed that the BsAb bound to human HER2 and PD1 with high affinities (EC50 values were 0.2 and 0.14 nM, respectively), and exhibited potent antitumor activities in vitro and in vivo. Furthermore, we demonstrated that the BsAb exhibited both HER2 and PD1 blockade activities and was effective in killing HER2-positive tumor cells via antibody-dependent cellular cytotoxicity. In addition, the BsAb could crosslink HER2-positive tumor cells with T cells to form PD1 immunological synapses that directed tumor cell killing without the need of antigen presentation. Thus, the BsAb is a new promising approach for treating late-stage metastatic HER2-positive cancers.

Asthma is characterized by airway hyperresponsiveness and inflammation, as well as underlying structural changes to the airways. Interleukin-4 (IL-4) is a key T-helper type 2 (Th2) cytokine that plays important roles in the pathogenesis of atopic and eosinophilic asthma. We developed a novel humanized anti-IL-4Rα antibody that can potently inhibit IL-4/IL-13-mediated TF-1 cell proliferation. Using monocytes isolated from human peripheral blood mononuclear cells (PBMCs), we revealed a critical role of CD32 in modulating the immune responses of monocytes in response to blockade of IL-4Rα signaling pathway. We, therefore, devised a new strategy to increase the efficacy of the anti-IL-4Rα monoclonal antibody for the treatment of asthma and other atopic diseases by co-engaging CD32 and IL-4Rα on monocytic cells by choosing IgG classes or Fc mutations with higher affinities for CD32. The antibody with selectively enhanced affinity for CD32A displayed superior suppression of IL-4-induced monocytes’ activities, including the down-regulation of CD23 expression. Intriguingly, further analysis demonstrated that both CD32A and CD32B contributed to the enhancement of antibody-mediated suppression of CD23 expression from monocytes in response to blockade of IL-4Rα signaling. Furthermore, inhibition of IgE secretion from human PBMC by the antibody variants further suggests that the complex allergic inflammation mediated by IL-4/IL-4Rα signaling might result from a global network where multiple cell types that express multiple FcγRs are all involved, of which CD32, especially CD32A, is a key mediator. In this respect, our study provides new insights into designing therapeutic antibodies for targeting Th2 cytokine-mediated allergic pathogenesis.

Highlights • The anti-PD1 x anti-EGFR bispecific antibody (BsAb) exhibited all in-vitro bioactivities comparable to that of the parental mAbs and showed anti-tumor efficacies of each of the two arms on par with the mAbs in-vivo. • The anti-PD1 x anti-EGFR bispecific antibody (BsAb) retained full ADCC towards cancer cells but not to T cells. Thus the BsAb is capable of killing tumor cells via ADCC while sparing T cells for T cell-induced anti-tumor immunity. • The anti-PD1 x anti-EGFR bispecific antibody (BsAb) exhibited significantly stronger tumor cell killing effects in the presence of PBMC relative to that of combination of cetuximab with an anti-PD1 mAb, 609A., We developed a strategy to combine conventional targeted therapy with immune checkpoint blockade using a tumor-targeting bispecific antibody (BsAb) to treat solid tumors. The BsAb was designed to simultaneously engage a tumor-associated antigen, epidermal growth factor receptor (EGFR), and programed cell death protein 1 (PD1). In addition to its direct anti-tumor activity via EGFR inhibition, the BsAb mediated efficient antibody-dependent cellular cytotoxicity (ADCC) and activated T cell antitumor im munity through blockade of PD1 from interacting with its counterpart, programed cell death ligand 1 (PDL1). Further, the BsAb exhibited a potent direct tumor cell killing activity in the presence of PBMC, most likely, via activating and, at the same time, physically engaging T cells with tumor cells. Taken together, we here illustrate a new strategy in the design and production of novel BsAbs with enhanced therapeutic efficacy through both direct tumor growth inhibition and T cell activation via tumor-targeted immune checkpoint blockade.

A series of new Zn-porphyrin dyes (LYD-1 similar to 5) with various substituted and anchoring groups have been designed, fabricated and fully characterized by nuclear magnetic resonance (NMR), ultraviolet visible (UV-vis), and High Resolution Mass spectroscopy (HRMS). The solar cells sensitized by new dyes have been systematically investigated by photoelectrochemical experiments of current density-voltage (J - V), incident monochromatic photon-to-electron conversion efficiency (IPCE) and electrochemical impedance spectra (EIS). The results show that the performance of DSC devices is deeply affected by the substituted and anchoring groups of dyes. The best solar cell sensitized by LYD-5 with alkoxyl donors and salicylic acid exhibits excellent photovoltaic conversion efficiency of 8.5% compared with 7.7% of YD2-o-C8-based solar cell under the same conditions. Their structural features and electron distribution of HOMOs and LUMOs are calculated computationally using a DFT method at the level of B3LYP/6-31G(d)/LANL2DZ.

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.

The use of industrial coal char as a fuel source for an anode-supported solid oxide-based carbon fuel cell (SO-CFC) with a yttrium-stabilized zirconia electrolyte and La0.8Sr0.2MnO3 cathode was investigated. Both the Boudouard reactivity and electrochemical performance of the coal char samples are higher than those of activated carbon samples under the same conditions. The inherent catalytic activity of the metal species (FemOn, CaO, etc.) in the coal char mineral matter leads to good cell performance, even in the absence of an external catalyst. For example, the peak power density of a cell fueled with pure coal char is 100 mW cm−2 at 850 °C, and that of a cell fueled with coal char impregnated with an FemOn-alkaline metal oxide catalyst is 204 mW cm−2. These results suggest that using coal char as the fuel in SO-CFCs might be an attractive way to utilize abundant coal resources cleanly and efficiently, providing an alternative for future power generation.

Antibody (Ab) humanization is crucial to generate clinically relevant biologics from hybridoma-derived monoclonal antibodies (mAbs). In this study, we integrated antibody structural information from the Protein Data Bank with known back-to-mouse mutational data to build a universal consensus of framework positions (10 heavy and 7 light) critical for the preservation of the functional conformation of the Complimentarity Determining Region of antibodies. On the basis of FR consensus, we describe here a universal combinatorial library suitable for humanizing exogenous antibodies by CDR-grafting. The six CDRs of the murine anti-human EGFR Fab M225 were grafted onto a distinct (low FR sequence similarity to M225) human FR sequence that incorporates at the 17 FR consensus positions the permutations of the naturally observed amino acid diversities. Ten clones were selected from the combinatorial library expressing phage-displayed humanized M225 Fabs. Surprisingly, 2 of the 10 clones were found to bind EGFR with stronger affinity than M225. Cell-based assays demonstrated that the 10 selected clones retained epitope specificity by blocking EGFR phosphorylation and thus hindering cellular proliferation. Our results suggest that there is a universal and structurally rigid near-CDR set of FR positions that cooperatively support the binding conformation of CDRs.

Acriflavine dye, with a long-lived triplet excited state, has a strong sensitizing ability for photodriven water splitting when [CoIII(dmgH)2(py)Cl] complex is used as a catalyst and triethanolamine as electron donor. This catalytic system is an example of a noble-metal-free homogeneous system for hydrogen generation from water. Moreover, the mechanistic details of the overall reaction pathway, as well as Acriflavine fading, is presented to offer significant guidance to developing more robust photocatalytic systems.

VEGF receptors 1 (FLT-1) and 2 (KDR) are expressed on subsets of acute myeloid leukemia (AML) and acute lymphoid leukemia cells, in which they induce cell survival, proliferation, and migration. However, little is known about possible cofactors that regulate VEGF receptor expression and activation on leukemia cells. Here we show that cholesterol accumulates in leukemia-rich sites within bone marrow of xenotransplanted severe combined immunodeficient (SCID) mice. Therefore, we hypothesized that cholesterol-rich domains might regulate FLT-1 signaling and chemotaxis of acute leukemias. We then showed that FLT-1 accumulates in discrete cholesterol-rich membrane domains where it associates with caveolin-1 and that placenta growth factor (PlGF)/VEGF stimulation promotes FLT-1 localization in such cholesterol-rich domains. Accordingly, FLT-1 localization and its phosphorylation are abrogated by methyl-β-cyclodextrin (MβCD), which removes cellular cholesterol, and by nystatin, an inhibitor of lipid-raft endocytosis. Mechanistically, cholesterol increases FLT-1 expression and promotes PlGF/VEGF-induced leukemia cells viability and also induces VEGF production by the leukemia cells in vitro. Taken together, we conclude that cholesterol regulates VEGF:VEGFR-1 signaling on subsets of acute leukemias, modulating cell migration, and viability, which may be crucial for disease progression. Finally, we provide evidence obtained from human AML samples that primary leukemia cells accumulate significantly more cholesterol than do normal cells and that cholesterol accumulation correlates with disease aggressiveness. Mol Cancer Res; 9(2); 215–24. ©2011 AACR.

The short chain fatty acid (SCFA) butyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive.

Ischemia of the heart, brain, and limbs is a leading cause of morbidity and mortality worldwide. Treatment with tissue type plasminogen activator (tPA) can dissolve blood clots and can ameliorate the clinical outcome in ischemic diseases. But the underlying mechanism by which tPA improves ischemic tissue regeneration is not well understood. Bone marrow (BM)–derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool and mobilizes CD45+CD11b+ proangiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signaling. tPA improves the incorporation of CD11b+ cells into ischemic tissues and increases expression of neoangiogenesis-related genes, including VEGF-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b+ cells and VEGFR-1+ cells, but not carrier-mobilized cells or CD11b− cells, accelerates neovascularization and ischemic tissue regeneration. Inhibition of VEGF signaling suppresses tPA-induced neovascularization in a model of hind limb ischemia. Thus, tPA mobilizes CD11b+ cells from the BM and increases systemic and local (cellular) VEGF-A, which can locally promote angiogenesis during ischemic recovery. tPA might be useful to induce therapeutic revascularization in the growing field of regenerative medicine.

Severe pulmonary arterial hypertension (PAH) is characterized by the formation of plexiform lesions and concentric intimal fibrosis in small pulmonary arteries. The origin of cells contributing to these vascular lesions is uncertain. Endogenous endothelial progenitor cells are potential contributors to this process.To determine whether progenitors are involved in the pathobiology of PAH.We performed immunohistochemistry to determine the expression of progenitor cell markers (CD133 and c-Kit) and the major homing signal pathway stromal cell-derived factor-1 and its chemokine receptor (CXCR4) in lung tissue from patients with idiopathic PAH, familial PAH, and PAH associated with congenital heart disease. Two separate flow cytometric methods were employed to determine peripheral blood circulating numbers of angiogenic progenitors. Late-outgrowth progenitor cells were expanded ex vivo from the peripheral blood of patients with mutations in the gene encoding bone morphogenetic protein receptor type II (BMPRII), and functional assays of migration, proliferation, and angiogenesis were undertaken. measurements and main results: There was a striking up-regulation of progenitor cell markers in remodeled arteries from all patients with PAH, specifically in plexiform lesions. These lesions also displayed increased stromal cell-derived factor-1 expression. Circulating angiogenic progenitor numbers in patients with PAH were increased compared with control subjects and functional studies of late-outgrowth progenitor cells from patients with PAH with BMPRII mutations revealed a hyperproliferative phenotype with impaired ability to form vascular networks.These findings provide evidence of the involvement of progenitor cells in the vascular remodeling associated with PAH. Dysfunction of circulating progenitors in PAH may contribute to this process.

In this article, low-temperature selective catalytic reduction (SCR) reaction of NO on carbon nanotube-supported vanadium (V2O5/CNT) catalysts was carried out. When compared with activated carbon as a support catalyst, carbon nanotubes with low load of V2O5 show excellent catalytic activity than that of activated carbon, and the catalytic activity of V2O5/CNT catalysts increases markedly in the presence of SO2. The transient reaction experiment shows that the reaction of V2O5/CNT catalyst for low-temperature SCR of NO follows the mechanism of Eley–Rideal, namely by the reaction between the adsorbed NH3 and NO in the gaseous or a weakly adsorbed state.

Cancer stem cells (CSC) are predicted to be critical drivers of tumor progression due to their self-renewal capacity and limitless proliferative potential. An emerging area of research suggests that CSC may also support tumor progression by promoting tumor angiogenesis. To investigate how CSC contribute to tumor vascular development, we used an approach comparing tumor xenografts of the C6 glioma cell line containing either a low or a high fraction of CSC. Compared with CSC-low tumors, CSC-high tumors exhibited increased microvessel density and blood perfusion and induced increased mobilization and tumor recruitment of bone marrow–derived endothelial progenitor cells (EPC). CSC-high C6 cell cultures also induced higher levels of endothelial cell proliferation and tubule organization in vitro compared with CSC-low cultures. CSC-high cultures and tumors expressed increased levels of the proangiogenic factors vascular endothelial growth factor and stromal-derived factor1, and when signaling by either factor was blocked, all aspects of angiogenesis observed in CSC-high cultures andtumors, including microvessel density, perfusion, EPC mobilization/recruitment, and stimulation of endothelial cellactivity, were reduced to levels comparable with those observed in CSC-low cultures/tumors. These results suggest that CSC contribute to tumor angiogenesis by promoting both local endothelial cell activity and systemic angiogenic processes involving bone marrow–derived EPC in a vascular endothelial growth factor–dependent and stromal-derived factor 1–dependent manner. [Cancer Res 2009;69(18):7243–51]

SummaryMyelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.

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.

In an attempt to identify novel proteins involved in the emergence of multidrug resistance (MDR) in leukemia cells, we adopted a proteomics approach to analyze protein expression patterns in leukemia cell lines, K562, and its MDR counterpart, K562/A02. Combining high-resolution two-dimensional gel electrophoresis and mass spectrometry, we compared the protein expression profiles between K562 and K562/A02. A total number of 22 protein spots with altered abundances of more than 2-fold were detected and 14 proteins were successfully identified. Consistent with our previous observations by cDNA microarray, sorcin, a 22-kDa calcium-binding protein, was also identified by this proteomic approach with a 10.4-fold up-regulation in K562/A02 cells. Overexpression of sorcin protein in K562 cells by gene transfection led to significantly reduced cytosolic calcium level and increased resistance to cell apoptosis. Further, leukemia cell lines over-expressing sorcin also showed up-regulation of Bcl-2, along with decreased level of Bax. Taken together, our results suggest that sorcin plays an important role in the emergence of MDR in leukemia cells via regulating cell apoptosis pathways, thus may represent both a new MDR marker for prognosis and a good target for anti-MDR drug development.

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG–treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in ∼100 cancer cell lines and ∼300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers. (Cancer Res 2006; 66(18): 9162-70)

Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate glycosaminoglycan side chains of the proteoglycans in extracellular matrix and basement membrane. Heparanase enzymatic activity is important in the promotion of tumor angiogenesis, primary tumor growth, invasion, and metastasis. Expression of heparanase in many tumor types conversely correlates with prognosis. Much progress has been made in studying the regulation of heparanase expression, processing and activation. The interaction between heparanase and its substrate heparan sulfate has been well characterized. The fact that heparanase was identified as the single predominant heparan sulfate-degrading enzyme in human cancer sparked considerable interest in developing heparanase inhibitors for potential therapeutic applications. Recent progress in drug development led to several classes of heparanase inhibitors, including chemically modified natural products, small molecule inhibitors, and antibodies. Some of these inhibitors have demonstrated potent activities to inhibit tumor angiogenesis, tumor progress, or tumor metastasis. A leading compound, PI-88, is currently being evaluated in clinical phase II trials in patients with melanoma, liver, or lung cancers. This review summarizes the recent progress in heparanase biochemical research and the development of heparanase antagonists as novel anti-cancer therapeutics.

The presence of persistent circulating leukemia cells, or engrafted into extramedullary tissues, is a bad prognostic factor for patients with acute leukemia. However, little is known about the mechanisms that regulate the exit of leukemia cells from the bone marrow (BM) microenvironment. We reveal that vascular endothelial growth factor receptor 1 (FLT-1) modulates acute leukemia distribution within the BM, along VEGF and PlGF gradients, regulating leukemia survival and exit into the peripheral circulation. FLT-1 activation on acute lymphoblastic leukemia (ALL) cells results in cell migration and proliferation in vitro, whereas in vivo FLT-1-overexpressing cells accumulate in the BM epiphysis of nonobese diabetic-severe combined immunodeficient (NOD-SCID) recipients and are detected in circulation 2 weeks after inoculation. In turn, FLT-1 neutralization affects leukemia localization (now in the BM diaphysis), increases leukemia apoptosis, and impedes the exit of ALL cells, prolonging the survival of inoculated mice. We demonstrate further that FLT-1-induced cell migration involves actin polymerization and lipid raft formation. Taken together, we show that FLT-1 regulates the BM localization of ALL cells, determining their survival and exit into the circulation and ultimately the survival of inoculated recipients. FLT-1 targeting on subsets of acute leukemias may delay the onset of extramedullary disease, which may be advantageous in combinatorial therapeutic settings.

Monoclonal antibodies have emerged as novel oncology therapeutics. These biologics exert anticancer effects via a variety of mechanisms of action including modulating the function of key regulatory molecules and signaling pathways of tumor cells such as blocking growth factor/receptor interaction and/or down-regulating expression of oncogenic proteins (e.g., growth factor receptors) on the cell surface; recruiting effector mechanisms of the immune system, such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity; as a targeting device in immnuoconjugates; and other mechanisms such as anti-idiotype, catalytic antibodies, or antibodies that modulate patient's immune responses to tumors. In this review, we focus on the research and development experience of four such representative antibody therapeutics, rituximab (Rituxan®), trastuzumab (Herceptin®), cetuximab (Erbitux®), and bevacizumab (Avastin®), approved for treating non-Hodgkin's lymphoma, metastatic breast cancer, and colorectal cancer, respectively. The biology behind each antibody target, their proposed mechanisms of action, as well as preclinical and clinical development of these antibodies are the topics of this article. Experience drawn from the development process of these four antibodies is instrumental for ongoing and future antibody development activities. In addition, perspective views on challenges and opportunities of oncology antibody therapeutics are presented. Drug Dev. Res. 67:699–728, 2006. © 2006 Wiley-Liss, Inc.

Bone marrow (BM)-derived circulating endothelial precursor cells (CEPCs) have been reported to incorporate into newly formed blood vessels under physiologic and pathologic conditions. However, it is unknown if CEPCs contribute to lymphangiogenesis. Here we show that in a corneal lymphangiogenesis model of irradiated mice reconstituted with enhanced green fluorescent protein (EGFP)-positive donor bone marrow cells, CEPCs are present in the newly formed lymphatic vessels. Depletion of bone marrow cells by irradiation remarkably suppressed lymphangiogenesis in corneas implanted with fibroblast growth factor-2 (FGF-2). Further, transplantation of isolated EGFP-positive/vascular endothelial growth factor receptor-3-positive (EGFP+/VEGFR-3+) or EGFP+/VEGFR-2+ cell populations resulted in incorporation of EGFP+ cells into the newly formed lymphatic vessels. EGFP+/CEPCs were also present in peritumoral lymphatic vessels of a fibrosarcoma. These data suggest that BM-derived CEPCs may play a role in “lymphvasculogenesis.”

The molecular and cellular pathways that support the maintenance and stability of tumor neovessels are not well defined. The efficacy of microtubule-disrupting agents, such as combretastatin A4 phosphate (CA4P), in inducing rapid regression of specific subsets of tumor neovessels has opened up new avenues of research to identify factors that support tumor neoangiogenesis. Herein, we show that CA4P selectively targeted endothelial cells, but not smooth muscle cells, and induced regression of unstable nascent tumor neovessels by rapidly disrupting the molecular engagement of the endothelial cell-specific junctional molecule vascular endothelial-cadherin (VE-cadherin) in vitro and in vivo in mice. CA4P increases endothelial cell permeability, while inhibiting endothelial cell migration and capillary tube formation predominantly through disruption of VE-cadherin/beta-catenin/Akt signaling pathway, thereby leading to rapid vascular collapse and tumor necrosis. Remarkably, stabilization of VE-cadherin signaling in endothelial cells with adenovirus E4 gene or ensheathment with smooth muscle cells confers resistance to CA4P. CA4P synergizes with low and nontoxic doses of neutralizing mAbs to VE-cadherin by blocking assembly of neovessels, thereby inhibiting tumor growth. These data suggest that the microtubule-targeting agent CA4P selectively induces regression of unstable tumor neovessels, in part through disruption of VE-cadherin signaling. Combined treatment with anti-VE-cadherin agents in conjunction with microtubule-disrupting agents provides a novel synergistic strategy to selectively disrupt assembly and induce regression of nascent tumor neovessels, with minimal toxicity and without affecting normal stabilized vasculature.

Antibodies represent a unique class of therapeutics because of their high specificity toward a defined target antigen. Recent clinical success with antibody-based cancer therapeutics has led to an upsurge in the development of these agents. Antibodies directed against FLT3 represent a promising approach for the treatment of human leukemia. We discuss some basic aspects of antibody-based cancer therapeutics, including their mechanisms of action, with a focus on recent progress in the generation and development of anti-FLT3 antibodies as well as their therapeutic potentials in the treatment of human hematologic malignancies.

VE-cadherin is an adhesion molecule localized at the adherens junctions of endothelial cells. It is crucial for the proper assembly of vascular structures during angiogenesis and maintaining vascular integrity. We have studied 3 monoclonal antibodies (mAbs) against murine VE-cadherin that inhibit angiogenesis and tumor growth. Two of these, BV13 and 10G4, also disrupted normal vessels, resulting in severe vascular leakage, whereas the third, E4G10, did not. The goal of the current report was to identify the epitope of E4G10 and distinguish it from those of the disruptive mAbs. We mapped the epitope of E4G10 to within the first 10 amino acids of mature VE-cadherin and demonstrated that conserved tryptophan residues in this sequence are required for VE-cadherin–mediated trans-adhesion. The disruptive mAbs target a different epitope within amino acids 45 to 56, which structural homology modeling suggests is not involved in trans-adhesion. From our studies, we hypothesize that E4G10 can only bind the neovasculature, where VE-cadherin has not yet engaged in trans-adhesion and its epitope is fully exposed. Thus, E4G10 can inhibit junction formation and angiogenesis but is unable to target normal vasculature because its epitope is masked. In contrast, BV13 and 10G4 bind an epitope that is accessible regardless of VE-cadherin interactions, leading to the disruption of adherens junctions. Our findings establish the immediate N-terminal region of VE-cadherin as a novel target for inhibiting angiogenesis.

Induction of neoangiogenesis plays an important role in the pathogenesis of multiple myeloma. However, the mechanism by which expression of vascular endothelial growth factor (VEGF)-A and its receptors modulate the interaction of multiple myeloma cells with stromal cells is not known. Here, we describe a novel in vitro coculture system using fetal bone stromal cells as a feeder layer, which facilitates the survival and growth of human primary multiple myeloma cells. We show that stromal-dependent paracrine VEGF-A signaling promotes proliferation of human primary multiple myeloma cells. Primary multiple myeloma cells only expressed functional VEGF receptor (VEGFR)-1, but not VEGFR-2 or VEGFR-3. VEGFR-1 expression was detected in the cytoplasm and the nuclei of proliferating multiple myeloma cells. Inhibition of VEGFR-1 abrogated multiple myeloma cell proliferation and motility, suggesting that the functional interaction of VEGF-A with its cognate receptor is essential for the growth of primary multiple myeloma cells. Collectively, our results suggest that stromal-dependent paracrine and intracrine VEGF-A/VEGFR-1 signaling contributes to human primary multiple myeloma cell growth and therefore, VEGFR-1 blockade is a potential therapeutic strategy for the treatment of multiple myeloma.

Vascular endothelial growth factors (VEGF) and their receptors (VEGFR) have been implicated to play important roles in tumor-associated angiogenesis and lymphangiogenesis, and hence in tumor growth and metastasis. We previously produced a number of fully human antibodies directed against VEGF receptor 2 (VEGFR2) and VEGF receptor 3 (VEGFR3) and showed that these antibodies are capable of inhibiting growth factor (VEGF and VEGF-C)-induced receptor activation, migration, and proliferation of human endothelial cells. In this report, we constructed and produced a bispecific antibody, a diabody, using the variable domain genes of two neutralizing antibodies, IMC-1121 to VEGFR2 and hF4-3C5 to VEGFR3. The diabody binds to both VEGFR2 and VEGFR3 in a dose-dependent manner, and blocks interaction between VEGF/VEGFR2, VEGF-C/VEGFR2, and VEGF-C/VEGFR3. In cell-based assays, the diabody neutralized both VEGF and VEGF-C-stimulated activation of VEGFR2, VEGFR3, and p44/p42 mitogen-activated protein kinase in endothelial cells. Furthermore, the diabody was able to inhibit both VEGF and VEGF-C-induced migration of endothelial cells. Taken together, our results suggest that a dual blockade of both VEGFR2 and VEGFR3 simultaneously may represent a more potent approach to effective cancer therapy.

Aberrant FLT3 expression and/or mutation plays a significant role in leukemogenesis. This has prompted the development of selective small molecule tyrosine kinase inhibitors against FLT3. However, like most tyrosine kinase inhibitors, those against FLT3 are not completely specific and at the doses required to completely inhibit target, significant toxicities may occur. In addition, tyrosine kinase inhibitors for other kinases have been shown to select for cells that become resistant. To overcome some of these limitations we developed two fully human phage display monoclonal antibodies against FLT3 (IMC-EB10 and IMC-NC7). These antibodies inhibited ligand-mediated activation of wild-type FLT3 and constitutively activated mutant FLT3 and in most cell types affected downstream STAT5, AKT, and mitogen-activated protein kinase activation. In addition to interfering with FLT3 signaling, IMC-EB10 and, to a significantly lesser extent, IMC-NC7 initiated antibody-dependent cell-mediated cytotoxicity on FLT3-expressing cells. When IMC-EB10 was used in vivo to treat nonobese diabetic/severe combined immunodeficient mice given injections of primary FLT3/ITD acute myelogenous leukemia samples or myeloid cell lines with FLT3 expression, it significantly decreased engraftment of leukemic cells and increased survival, respectively. In contrast, IMC-EB10 treatment did not reduce engraftment of normal human CD34+ cord blood cells nor did it show any significant inhibition of normal murine hematopoiesis. Thus, these types of antibodies have the potential to be safe and effective new therapeutic agents for acute myelogenous leukemia and possibly other FLT3-expressing malignancies.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Here we show that certain "liquid" tumors such as acute myeloid leukemia not only produce VEGF but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. In addition, the leukemia-derived VEGF can also stimulate the production of growth factors, including interleukin 6 (IL6) and granulocyte-macrophage colony stimulating factor (GM-CSF), by human endothelial cells, which in turn further promotes the growth of leukemia cells (the paracrine loop). A fully human anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-2C6, strongly blocks KDR/VEGF interaction and neutralizes VEGF-stimulated activation of KDR in endothelial cells. In a system where leukemia cells are co-cultured with endothelial cells, IMC-2C6 inhibits both the production of IL6 and GM-CSF by endothelial cells and the growth of leukemia cells. Finally, IMC-2C6 effectively blocks VEGF-induced migration of KDR+ human leukemia cells, and when administered in vivo, significantly prolonged survival of mice inoculated with KDR+ human leukemia cells. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and certain types of leukemia.

One of the major mechanisms of multidrug resistance (MDR) in cancer therapy is the overexpression of P-glycoprotein (Pgp). We previously reported that pyronaridine (PND), a synthetic quinoline derivative in the clinic for the treatment of malaria infections, was capable of reversing MDR phenotype in Pgp-overexpressing tumor cells. Here we further evaluated the reversal activity of PND using two Pgp-overexpressing human tumor cell lines: K562/A02 and MCF-7/ADR. PND significantly enhanced the sensitivity of K562/A02 and MCF-7/ADR cells to doxorubicin (DOX), but had no such effect on the parent K562 and MCF-7 cells. The MDR-modulating effect of PND persisted for longer than 24 h after removal of the agent from the culture. In nude mice bearing K562/A02 xenografts PND significantly enhanced the antitumor activity of DOX when given intraperitoneally or orally without increasing the toxicity of DOX. Our observations suggest that PND represents a promising agent for overcoming MDR in cancer therapy.

The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens. Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH(3) domain of an IgG. The novel BsAb, which we termed "di-diabody", represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH(3) domains. A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography. Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors. The di-diabody retained good antigen-binding activity after incubation at 37 degrees C in mouse serum for 72 h, demonstrating good product stability. Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity. This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.

The variable domain of heavy chain (VH) and light chain (VL) genes of anti-CD20 monoclonal antibody HI47 were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab’ fragment expression vector pYZFcd20. Chimeric anti-CD20 Fab’ fragment was expressed inE. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab’ fragment inhibited binding of HI47 to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab’ fragment inhibited the proliferation of Daudi cells, IC50 = 69 μg/mL. Affinity of chimeric anti-CD20 Fab’ fragment was determined, Ka was about 8.9×108 (mol/L)−1.

Bispecific antibody (BsAb) usually consists of two different antigen-binding arms, by which it is capable of simultaneously binding to target cells and effector cells, and can directly mediate the killing of target cells by retargeting and activating effector cells. The development of BsAb research goes through three main stages: chemical crosslinking of murine-derived monoclonal antibody, hybrid hybridomas and engineered BsAb. Among them, engineered BsAb has more formats than the other two, such as diabody, ScdHLX, ScZip, ScCH3, ScFab and BsIgG, etc. Compared with former murine-derived BsAbs, engineered BsAb has lower immunogenicity and stronger penetrating capacity, and currently, some of them appear suitable for clinical application in yields and qualities. Up to now, several phase I and phase II clinical studies of BsAb, for instance, some (Fab’)2 and Diabodies, have been performed. Among those BsAbs, anti-CD3/anti-tumor BsAbs is most common, they not only can activate T cell and induce CD3AK cytotoxic activity inin vitro experiment, and inhibit the growth of tumor on tumor-bearing mouse by retargeting T cells to lyse tumor cells, but also offer great promise in the therapy of some malignancies in clinic, especially of some advanced cancers as well as elimination of minimal residual tumors, indicated by increasing the tumor/blood ratio of antibody in patients and improving the natural killer cell (NK) anti-tumor activity in tumor sites, and also presenting of an increase level in TNF-α, INF-γ, IL-6, IL-8, IL-10 and soluble CD25, etc. The responses are also shown via improving the quality of life and prolonging the survival of partial patients. The “Knobs into Holes” technology is a new strategy emerging during research on engineered BsAb, it is likely to be useful for heterodimerization and can improve the quantity, purity and stability of BsAb, it is also anticipated to increase the clinical potential of BsAb in the future.

SO2 shows a significant promoting effect on the activity of V2O5/AC catalyst for NO reduction with ammonia at low temperatures (180–250°C). In the present study, the mechanism of the SO2 promotion was studied. It was found that the promoting effect of SO2 on the catalytic activity is due to the formation of a sulfate species on the catalyst surface. The sulfate species is linked to carbon surfaces other than vanadium or mineral surfaces. There is a synergetic role between carbon and V2O5 for the formation of surface sulfate species. A possible mechanism is proposed. SO2 is adsorbed and oxidized by oxygen to SO3 on the vanadium surface, and the formed SO3 shifts to the carbon surface and converts into sulfate species. The formed sulfate species acts as a new acid site, improves significantly the NH3 adsorption, and hence promotes the activity of the catalyst. During the reaction in the presence of SO2 at low temperatures, the sulfate species stays on the catalyst surface, while the ammonium ions react with NO continuously to avoid the formation and deposition of excess ammonium sulfate salts on the catalyst surface.

NO adsorption and reduction with NH3 over activated coke at low temperatures were studied by temperature-programmed desoption (TPD) and step-response experiments. NO adsorption is inhibited by competitive adsorption of NH3 in the absence of oxygen, and is significantly increased in the presence of oxygen. This phenomenon cannot be explained by either the oxidation of NO in the gas phase, nor the creation of oxidized surface on the activated coke. A hypothesis is proposed to interpret the phenomenon, which suggests that on the surface of the activated coke there are at least two types of adsorption sites, one of them adsorbs NO2 and/or the oxidized NO species rather than NO. NO–NH3–O2 reaction proceeds via both the ED mechanism and the LH mechanism. NO conversion decreases with increasing temperature in the range of 30–250°C, which suggests that the adsorption of reactants, especially NH3, is the rate-limiting step.

NO reduction with NH3 over activated carbon-supported copper oxide (CuO/AC) catalyst was studied at low temperatures (30-250 degrees C). The attention was focused on the effects of preparation parameters, reaction temperature and SO2 on the structure and activity of the catalyst. The catalysts show high activities for NO reduction with NH3 in the presence of O-2 at temperatures above 180 degrees C and are gradually deactivated at temperatures below 180 degrees C. Cu2O exists in the catalyst and results in low initial activity but it is easily oxidized into active CuO by O-2 during the NO-NH3-O-2 reaction. Calcination temperature and Cu loading of the catalyst strongly influence the activity and structure of the catalyst. The catalyst with 5 wt% Cu loading and calcined at 250 degrees C shows the highest activity. The activities of the catalysts with higher Cu loadings and/or calcined at higher temperatures are relatively low, mainly derived from aggregation of copper species. The CuO/AC catalyst is greatly deactivated by SO2 due to the formation of CuSO4 which is inactive at low temperatures. (C) 2000 Elsevier Science B.V. All rights reserved.

Emerging data suggest that a subset of circulating human CD34+ cells have phenotypic features of endothelial cells. Whether these cells are sloughed mature endothelial cells or functional circulating endothelial precursors (CEPs) is not known. Using monoclonal antibodies (MoAbs) to the extracellular domain of the human vascular endothelial receptor-2 (VEGFR-2), we have shown that 1.2 ± 0.3% of CD34+ cells isolated from fetal liver (FL), 2 ± 0.5% from mobilized peripheral blood, and 1.4 ± 0.5% from cord blood were VEGFR-2+. In addition, most CD34+VEGFR-2+ cells express hematopoietic stem cell marker AC133. Because mature endothelial cells do not express AC133, coexpression of VEGFR-2 and AC133 on CD34+ cells phenotypically identifies a unique population of CEPs. CD34+VEGFR-2+ cells express endothelial-specific markers, including VE-cadherin and E-selectin. Also, virtually all CD34+VEGFR-2+ cells express the chemokine receptor CXCR4 and migrate in response to stromal-derived factor (SDF)-1 or VEGF. To quantitate the plating efficiency of CD34+ cells that give rise to endothelial colonies, CD34+ cells derived from FL were incubated with VEGF and fibroblast growth factor (FGF)-2. Subsequent isolation and plating of nonadherent FL-derived VEGFR-2+ cells with VEGF and FGF-2 resulted in differentiation of AC133+VEGFR-2+ cells into adherent AC133−VEGFR-2+Ac-LDL+(acetylated low-density lipoprotein) colonies (plating efficiency of 3%). In an in vivo human model, we have found that the neo-intima formed on the surface of left ventricular assist devices is colonized with AC133+VEGFR-2+ cells. These data suggest that circulating CD34+ cells expressing VEGFR-2 and AC133 constitute a phenotypically and functionally distinct population of circulating endothelial cells that may play a role in neo-angiogenesis.

The effect of SO 2 on the V 2 O 5 /AC catalyst for the NO reduction with NH 3 at low temperatures (150–250°C) is studied using a transient method. The results show that the presence of SO 2 in the reaction stream can significantly promote catalytic activity, which is largely conditioned by V 2 O 5 loading and reaction temperature. Lower V 2 O 5 loading and higher reaction temperature are favorable. Higher V 2 O 5 loading causes heavy deactivation of the catalyst. The reason for the behavior of the catalyst is also studied and discussed. The promoting role of SO 2 seems to result from the sulfate species formed and deposited on the carbon surface, which increase the surface acidity and hence the activity of the catalyst.

Objective: To analysis the uptake of free MTX and MTX conjugated to tumor specific monoclonal antibody by target and non-target cells. Methods: The folate antagonist methotrexate (MTX) was conjugated to two monoclonal antibodies (Mab) directed against human chronic lymphocytic leukemia (CLL), Dal B01 and Dal B02, by an active ester method. Both conjugates were more cytotoxic toward the target tumor cell line D10-1 than to the non-target cell line MOLT-3, and Dal B02-MTX conjugate was more inhibitory to D10-1 cells than free MTX in a 6 h pulse exposure assay. Results: Drug uptake studies revealed that D10-1 cells took up much more Dal B01 and Dal B02-conjugated MTX than free MTX. The amounts of drug taken up by D10-1 cells incubated with Dal B01 and Dal B02-conjugated MTX were always 3 to 5-fold higher than that taken up by MOLT-3 cells, although the latter took up more drug when incubated with free MTX. Furthermore, tumor cells incubated with Dal B01 or Dal B02-conjugated MTX retained much larger amounts of drug for a prolonged period of time than those incubated with free MTX. Conclusion: The enhanced specific cytotoxicity of Dal B01 and Dal B02-MTX conjugates toward target tumor cells is therefore likely due to (I) delivery of larger amounts of MTX to target cells when the drug is conjugated to Mab; (ii) longer retention of Mab-conjugated MTX by target cells; and (iii) slow, prolonged release of MTX from the surface-bound or endocytosed conjugates, rendering them into a sustained release dosage form.

Biological evidence suggests that interference with the function of the angiogenic growth factor receptor VEGFR2 (flk1/KDR) is a particularly promising strategy to inhibit tumor-induced angiogenesis. Proof of concept was established by developing a monoclonal rat anti-mouse VEGFR2 antibody (DC101) and showing that it potently blocked the binding of VEGF to its receptor, inhibited VEGF-induced signaling, and strongly blocked tumor growth in mice through an anti-angiogenic mechanism. Since DC101 does not cross-react with the human VEGFR2 KDR, anti-KDR monoclonal antibodies were generated by standard hybridoma technology and by using phage display library. High affinity antibodies (Kd = 4.9 x 10(-10)-1.1 x 10(-9) M) were found with both approaches. The anti-KDR antibodies compete on an equimolar basis with VEGF for binding to KDR and inhibit with similar potency the VEGF-induced signaling and mitogenesis in human endothelial cells. Although these antibodies cannot be tested for in vivo efficacy in standard murine tumor models because of lack of species cross-reactivity, the similarity of their in vitro properties with those of DC101 suggests that they may be effective in blocking KDR function in vivo.

An anti-p185HER2/anti-CD3 humanized bispecific diabody was previously constructed from two cross-over single-chain Fv in which YH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide-stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti-p185HER2.VL/VH interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide-stabilized compared to the parent diabody (> 96% versus 72%), whereas the overall yield was > 60-fold lower. Eleven "knob-into-hole" diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti-p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively. Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer. This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.

The granulocyte colony-stimulating factor (G-CSF) promotes angiogenesis. However, the exact mechanism is not known. We demonstrate that vascular endothelial growth factor (VEGF) was released by Gr-1+CD11b- neutrophils but not Gr-1-CD11b+ monocytes prestimulated with G-CSF in vitro and in vivo. Similarly, in vivo, concomitant with an increase in neutrophil numbers in circulation, G-CSF augmented plasma VEGF level in vivo. Local G-CSF administration into ischemic tissue increased capillary density and provided a functional vasculature and contributed to neovascularization of ischemic tissue. Blockade of the VEGF pathway abrogated G-CSF-induced angiogenesis. On the other hand, as we had shown previously, VEGF can induce endothelial progenitor cell (EPC) mobilization. Here, we show that G-CSF also augmented the number of circulating VEGF receptor-2 (VEGFR2) EPCs as compared with untreated controls. Blocking the VEGF/VEGFR1, but to a much lesser extent, the VEGF/VEGFR2 pathway in G-CSF-treated animals delayed tissue revascularization in a hindlimb model. These data clearly show that G-CSF modulates angiogenesis by increasing myelomonocytic cells (VEGFR1+ neutrophils) and their release of VEGF. Our results indicated that administration of G-CSF into ischemic tissue provides a novel and safe therapeutic strategy to improve neovascularization.

Clinical development of bispecific antibodies (BsAb) has been effectively stymied by the lack of efficient production methods. We therefore attempted to produce a humanized BsAb fragment using an expression system that has proved very successful for secretion of monospecific Ab fragments from E. coli. An anti-p185HER2/anti-CD3 BsF(ab')2 was first recast into the diabody format and then periplasmically secreted from E. coli grown to high cell density in a fermentor. The diabody was recovered in very high yield (up to 935 mg/l) after protein A purification and predominantly (> or = 80%) as a dimer as judged by size exclusion chromatography. Diabody dimers were found to be mainly functional heterodimers (approximately 75%) by titration with p185HER2 extracellular domain. The diabody binds p185HER2 extracellular domain and human T lymphocytes with affinities close to those of the parent BsF(ab')2. Furthermore, the diabody is capable of simultaneous binding to tumor cells overexpressing p185HER2 and CD3 on T cells as shown by cellular rosetting. The diabody is equally potent as the parent BsF(ab')2 in retargeting IL-2 activated T-enriched peripheral blood lymphocytes to lyse tumor cells overexpressing p185HER2.

The recent clinical successes of immune checkpoint inhibitors have fueled the intense interest in novel immuno-oncology (I/O) therapeutics. The lack of relevant animal models remains a major hurdle in understanding the mechanism of action and evaluating the efficacy of such therapeutics. Patient derived xenograft (PDX), considered to most closely mimic patient tumors in both histo- and molecular pathology1,2, is however rarely used in I/O studies because it grow only in immune-compromised hosts. In reality, many PDXs grow well in nude mice where certain immune functions remain intact, excluding T-cells/T-cell functions. Therefore, PDX could still potentially be of practical use for studying T-cell independent I/O therapy. This study set out to evaluate a biologics for the treatment of a patient derived xenograft disease, by activating mouse natural killer (NK). NK and CD8 T cells are two major immune effector cell types that mediate cytotoxicity to tumor cells in vivo. One of the immunomodulatory agents, an anti-PD-L1 antibody-based IL-15 immunocytokine, was recently tested as a novel I/O treatment of cancer3. This molecule was engineered to be cross-species for both human and mouse PD-L1 and IL-15 that antagonize PD1/PD-L1 checkpoint-mediated immune suppression and also target the PD-L1-expressing tumor with IL-15 stimulated NK and CD8 T effector cells into local tumor sites, thus synergizing tumor-located anti-tumor immunity. In fact, our previous studies have demonstrated a greatly enhanced anti-tumor activity in the PDL-1-expressing syngeneic mouse tumor models via the mobilization of tumor infiltrating lymphocyte (TILs), over the PD-L1 antibody alone3. Since IL-15 also stimulates NKs in addition to T-cells, we reasoned that this bifunctional agent could also have potential activity against a PD-L1-expressing PDX in nude mice where NK remains functional. LU1901 is previously described NSCLC-PDX1, and was confirmed to express high level of PD-L1 by both RNAseq and flow analysis, after screening of a large panel of PDXs. We implanted LU1901 in Balb/c nude mice, and started to treat the mice by the bifunctional agent when the tumor reached to 150 mm3. Our result clearly demonstrated a significant inhibition of LU1901 growth by the bifunctional agent in nude mice, in the apparent absence of T-cells. When the treated tumors were examined at the termination, significantly infiltrate NK cells were found inside the treated tumors, as measured by both flow cytometry and immunohistochemistry (IHC). The number of infiltrating NK also statistically correlates to the amplitude of the tumor responses. Together, our data suggest that one of important mechanisms of action of this bifunctional agent relies on the tumor-targeting NK activation, and also that PDX could be a useful model suitable for in vivo efficacy analysis of T-cell independent immunotherapy. References 1. Yang, M., et al. Overcoming erlotinib resistance with tailored treatment regimen in patient-derived xenografts from naive Asian NSCLC patients. International journal of cancer. Journal international du cancer 132, E74-84 (2013). 2. Guo, S., Wubin Qian, Jie Cai, Likun Zhang, Jean-Pierre Wery, Qi-Xiang Li. Molecular pathology of patient tumors, patient derived xenografts and cancer cell lines EORTC-NCI-AACR. (2015). 3. Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Zhanna Polonskaya, Xenia Luna, Haifan Zhang, Zhikai Zhang, Zhun Wang*, Leo Liu*, Jeegar Patel, James Tonra, Henry Li*, Larry Witte, Sam Waksal, Zhenping Zhu. Anti-PD-L1 antibody-based IL-15 immunocytokine has enhanced antitumor immunity. EORTC-NCI-AACR Abstract (2015). Citation Format: Henry Q. Li, Zhun Wang, Xiaoyu An, Jinping Liu, Likun Zhang, Jean-Pierre Wery, Yan Wu, James Tonra, Sam Waksal, Zhenping Zhu. Modeling an immunotherapy of NK mechanism on a NSCLC patient derived xenograft. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A30.

Target-based immunocytokine approaches have been reported to be efficacious in the control of tumor growth in preclinical and clinical studies. By targeting cytokines-activated immune effectors to local tumor sites, an antibody-based immunocytokine is able to achieve antitumor immunity in the tumor microenvironment while reducing cytokines-mediated systemic side effects. Recently, immune checkpoint antagonists to PD-1/PD-L1 have shown success in certain clinical settings across multiple cancer types. However, the full potential of the checkpoint inhibitor is limited due to impaired overall antitumor immunity. It is therefore desirable to develop immunotherapeutics with the capacity of simultaneously inhibiting immunosuppressive pathways and stimulating immune effector cells to potentiate innate and adaptive immune responses against tumor growth. To this end, we generated a bifunctional fusion protein, KD033, composed of an antibody specific for PD-L1 and IL-15 as a novel immunocytokine for achieving better immunotherapeutic efficacy against tumors. Previously, we demonstrated that KD033 has an enhanced immunological activity and stronger antitumor efficacy in some syngeneic mouse tumor models in comparison to single agents. In the present report, we show that the mechanisms of actions of the bifunctional protein in the enhancement of antitumor immune responses results from an increase in Th1 cytokine secretion, the expansion and cytotoxicity of CD8 T-cells and NK cells and a decrease in immunosuppressive cells, i.e. regulatory T cells and myeloid derived suppressive cells in a number of preclinical experimental models. In the preclinical studies, KD033 regimens, with the unique immunological properties, led to stronger anti-tumor efficacy in controlling primary tumor growth and prolonging the survival of tumor bearing mice in a number of mouse tumor models including PDX and GEMM tumor models. Importantly, the PD-L1-targeted IL-15 bifunctional protein had significantly less cytokine-related toxicity when compared to non-targeted full IgG antibody-IL-15 fusion protein in vivo. These results further elucidate the capacity of targeting IL-15-stimulated innate and adaptive immune effector cells into tumor microenvironment, thereby effectively controlling tumor progression while having minimized adverse effect in vivo. These encouraging preclinical results of the novel immunotherapeutics suggest further advancement of this innovative therapeutic candidate towards clinical development for cancer treatment. Citation Format: Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Haifan Zhang, Zhanna Polonskaya, Xenia Luna, Zhikai Zhang, Zhun Wang, Leo Liu, Jeegar Patel, James Tonra, Henry Li, Larry Witte, Sam Waksal, Zhenping Zhu. Novel anti-PD-L1/IL-15 bifunctional immunotherapeutics potentiates antitumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4997.

The importance of VEGF/VEGFR2 signaling in cancer growth and metastasis has been clearly highlighted by the therapeutic benefits of bevacizumab (Avastin), a humanized antibody to VEGF, and Cyramza, a fully human antibody to VEGFR2, in multiple cancer treatment modalities. Recently, immune checkpoint antagonistic antibodies to PD1 and PDL1 have shown some success in a variety of clinical settings across multiple cancer types. The overall clinical efficacy of these individual antibody therapies has been, however, rather limited and, only evident in a fraction of patients. To this end, combinations of antibodies against both VEGF/VEGFR2 and PD1/PDL1 may represent promising approaches to further enhance the antitumor efficacy of individual antibody therapies. In this study, we engineered a bispecific anti-PDL1 x anti-VEGFR2 antibody using two fully human antibodies derived from antibody phage display libraries. In this bispecific format, a high affinity single chain antibody to PDL1 was genetically fused to the C-terminus of the heavy chain of a conventional IgG antibody against VEGFR2. The bispecific antibody was efficiently expressed in mammalian cells and could be purified to homogeneity via single step affinity chromatography. The bispecific antibody retained the binding activity of its parental antibodies to PDL1 and VEGFR2, and strongly blocked both VEGF/VEGFR2 and PDL1/PD1 interaction. Further, the bispecific antibody inhibited VEGF-stimulated VEGFR2 phosphorylation and proliferation of endothelial cells, and promoted proliferation of human T cells and secretion of cytokine such as IL2 and INFγ. The bispecific antibody is currently being evaluated in vivo in relevant animal models. Citation Format: Dan Lu, Zhanna Polonskaya, Haifan Zhang, Xenia Luna, Stella Martomo, Zhikai Zhang, Zhaojing Zhong, Yan Wu, Jeegar Patel, James Tonra, Larry Witte, Sam Waksal, Zhenping Zhu. A novel anti-PDL1 x anti-VEGFR2 bispecific antibody for enhanced antitumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 572.

The anthracyclin antineoplastic agent doxorubicin (Adriamycin) was linked by four different methods of linkage to DalB02, an IgG1 kappa murine monoclonal antibody (mAb) against surface-associated antigens on human chronic lymphocytic leukaemia (CLL) B cells. All the four conjugates fully retained the immunoreactivity of the parent DalB02. When the inhibitory effect of these conjugates was evaluated in vitro against the target D10-1 cells (a clone derived from an Epstein-Barr-virus-transformed human CLL B cell line that binds DalB02) it was observed that one conjugate was more potent than the free drug but the others were not. When 131I-labelled unmodified DalB02 and the 131I-labelled DalB02-containing conjugate that was found to be potent were injected i.v. into nude mice bearing a subcutaneous D10-1 xenograft, the percentages of the injected dose (%ID) of both 131I-DalB02 and the 131I-DalB02-containing conjugate that localized in the tumour were much higher than the %ID of the respective preparations that localized in normal tissues of D10-1-xenografted mice. The systemic toxicity of the conjugate was less than that of the free drug. At an equitoxic dose level, this conjugate was a more effective inhibitor of established D10-1 xenografts than the free drug.

Immune checkpoint antagonists to PD-1/PD-L1 and immunostimulating cytokines such as IL-15 have shown success to some extent in certain clinical settings across multiple cancer types. However, the full potential of the checkpoint inhibitor is limited due to impaired overall antitumor immunity, and cytokine as single agent has insufficient half-life and systemic toxicities due to the lack of target specificity. To overcome these challenging hurdles, we developed a bifunctional fusion protein, KD-033, composed of an antibody specific for PD-L1 and complex of IL-15Rα sushi domain/IL-15 as a novel immunotherapeutic agent for achieving better antitumor efficacy. Previously, we presented the generation and characteristics of a prototype of bifunctional fusion protein and its potential of in vivo antitumor activity. Here, we report a genetically modified fusion protein that has enhanced immunological activity and capability to achieve stronger antitumor efficacy in tumor models in comparison with either single agent. Our data indicate that the improved bifunctional fusion protein has favorable thermal stability and can be efficiently expressed in mammalian cells. The bifunctional fusion protein has higher affinity to PD-L1, silenced binding activity to Fc receptors and better ability to increase the secretion of Th1 cytokine, i.e. gamma IFN and the cytotoxicity of CD8 T-cells and NK cells to tumor cells as assessed in immunological assays. In preclinical study, KD033 had stronger anti-tumor efficacy in controlling primary tumor growth and prolonging the survival of tumor bearing mice in a number of mouse tumor models including those aggressive tumor models. Furthermore, the PD-L1 targeted bifunctional protein had significantly less cytokine-related toxicity when compared to non-targeted full IgG/IL-15Rα sushi domain/IL-15 fusion protein in vivo. These results demonstrate that KD033 has the capacity of targeting IL-15-stimulated innate and adaptive immune effectors into local tumor sites, thereby effectively controlling tumor progression while having minimized potential adverse effect in vivo. The preclinical studies of the novel immunotherapeutics warrant further investigation towards the clinical development of the bifunctional immunotherapeutic agent for cancer treatment. Citation Format: Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Zhanna Polonskaya, Xenia Luna, Haifan Zhang, Zhikai Zhang, Zhun Wang, Leo Liu, Jeegar Patel, James Tonra, Henry Li, Larry Witte, Sam Waksal, Zhenping Zhu. Anti-PD-L1 antibody-based IL-15 immunocytokine has enhanced antitumor immunity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C173.