Aizhi Zhao

Novel Recombinant Human B7-H4 Antibodies Overcome Tumoral Immune Escape to Potentiate T-Cell Antitumor Responses

B7-H4 (VTCN1, B7x, B7s) is a ligand for inhibitory coreceptors on T cells implicated in antigenic tolerization. B7-H4 is expressed by tumor cells and tumor-associated macrophages (TAM), but its potential contributions to tumoral immune escape and therapeutic targeting have been less studied. To interrogate B7-H4 expression on tumor cells, we analyzed fresh primary ovarian cancer cells collected from patient ascites and solid tumors, and established cell lines before and after in vivo passaging. B7-H4 expression was detected on the surface of all fresh primary human tumors and tumor xenotransplants, but not on most established cell lines, and B7-H4 was lost rapidly by tumor xenograft cells after short-term in vitro culture. These results indicated an in vivo requirement for B7-H4 induction and defined conditions for targeting studies. To generate anti-B7-H4-targeting reagents, we isolated antibodies by differential cell screening of a yeast-display single-chain fragments variable (scFv) library derived from patients with ovarian cancer. We identified anti-B7-H4 scFv that reversed in vitro inhibition of CD3-stimulated T cells by B7-H4 protein. Notably, these reagents rescued tumor antigen-specific T-cell activation, which was otherwise inhibited by coculture with antigen-loaded B7-H4+ APCs, B7-H4+ tumor cells, or B7-H4- tumor cells mixed with B7-H4+ TAMs; peritoneal administration of anti-B7-H4 scFv delayed the growth of established tumors. Together, our findings showed that cell surface expression of B7-H4 occurs only in tumors in vivo and that antibody binding of B7-H4 could restore antitumor T-cell responses. We suggest that blocking of B7-H4/B7-H4 ligand interactions may represent a feasible therapeutic strategy for ovarian cancer.

Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy

Abstract Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.

Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform.

Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purpura (TTP), as autoimmune disorders may produce self-reactive specificities. The yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage in the novel vector pAGA2 for yeast-display expression. The TTP yeast-display library (10⁹ members) was screened by magnetic and flow sorting with human TEM1 recombinant protein. A pool of yeast-display scFv able to detect 2nM of TEM1 was obtained and transformed into yeast-secreted scFv by homologous recombination using the novel p416 BCCP vector for yeast secretion of biotinylated scFv. Anti-TEM1 yeast-secreted scFv were independently validated in vitro by flow cytometry analysis and ELISA assays, then in vivo biotinylated in N-termini to produce biobodies. Biobody-78 bound specifically to Endosialin/TEM1-expressing ovarian tumor in vivo, with functional stability over 48 h. Our results suggest that our novel paired display-secretory yeast libraries can serve as an ideal platform for the rapid isolation of high-affinity reagents, and that anti-TEM1 biobody-78 can be used for in vitro assays including flow cytometry analysis, as well as in vivo for targeted imaging and therapy of cancer.

Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody.

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206low macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.

A novel anti-PSMA human scFv has the potential to be used as a diagnostic tool in prostate cancer

Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of cancer related death in men. The early diagnosis and treatment of PCa are still challenging due to the lack of efficient tumor targeting agents in traditional managements. Prostate specific membrane antigen (PSMA) is highly expressed in PCa, while only has limited expression in other organs, providing an ideal target for the diagnosis and therapy of PCa. The antibody library technique has opened the avenue for the discovery of novel antibodies to be used in the diagnosis and therapy of cancer. In this paper, by screening a large yeast display naive human single chain antibody fragment (scFv) library, we obtained a high affinity scFv targeting PSMA, called gy1. The gy1 scFv was expressed in E.coli and purified via a C terminal 6His tag. The binding affinity of gy1 was shown to be at the nanomolar level and gy1 can specifically bind with PSMA positive cancer cells, and binding triggers its rapid internalization through the endosome-lysosome pathway. The specific targeting of gy1 to PSMA positive tumor tissues was also evaluated in vivo. We showed that the IRDye800CW labeled gy1 can efficiently target and specifically distribute in PSMA positive tumor tissues after being injected into xenograft nude mice. This study indicated that the novel antibody gy1 could be used as a great tool for the development of PSMA targeted imaging and therapy agents for PCa.

A fusion-protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development: Tumor Targeting Protein Domains for Therapeutic Development

A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (VH) and light (VL) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

Correction to: Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy

Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional (3D) structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a mouse tumor model that has highly endogenous mTEM1 expression in the vasculature. Our data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.AbstractTumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional (3D) structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a mouse tumor model that has highly endogenous mTEM1 expression in the vasculature. Our data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

Immunotargeting of tumor vasculature: preclinical development of novel antibody-based imaging and therapy against TEM1/CD248

The success of antibody-based theranostics depends on the identification of tumor specific biomarkers and the development of corresponding antibodies with high-affinity and specificity. Tumor endothelial marker-1 (TEM1) is highly expressed in tumor vasculature of multiple cancers but not in normal organs. The expression of TEM1 was first evaluated and confirmed by immunohistochemistry from 53 cases of metastatic serous ovarian cancer at HUP. TEM1 positive tumor stroma was observed in >95% of the cases studied. Hence, developing sensitive and effective theranostic agents against TEM1 are of utmost significance in improving diagnosis and treatment of ovarian cancer. Our goals are: 1) engineer TEM1-specific antibodies; 2) evaluate these engineered antibodies in imaging and immunotherapies in preclinical models. To generate TEM1-targeting agents, we designed a panel of multivalent fusion proteins from scFv78, a previously isolated single chain variable fragment specifically recognizing the extracellular domain of TEM1. scFv78 was fused with different huIgG1 Fc region (CH2-, CH3-, or hinge). Proteins were expressed in 293F cells and purified by affinity chromatography. All scFv78 variants exhibited comparable thermo and serum stability in vitro. Among them, the scFv78-Fc fusion (78Fc) has the highest affinity to TEM1 (Kd = 0.15nM, 15X higher than scFv78). Pharmacokinetics (PK) and biodistribution of the protein panel were evaluated in naive and TEM1+ tumor bearing animals. 78Fc has a t1/2 of 5.1hr, which is suitable for in vivo therapeutic and imaging applications. Therefore, 78Fc was further developed as imaging tool and antibody-drug conjugate (ADC) based on its favorable affinity, stability, half-life and PK profile. In pilot studies with preclinical animal models of tumor vasculature, fluorophore- and [124-I]-labeled 78Fc demonstrated specific enrichment in TEM1+ grafts, but not in control tumor or other organs, by both optical and immunoPET imaging. In addition, 78Fc-MMAE conjugate exerted specific killing of TEM1+ cells. In summary, we have developed a panel of innovative theranostics agents targeting TEM1 on the vasculature of ovarian cancer and several other solid tumors. Our long term goal is to translate such combined approach into the clinic: Using TEM1-antibody as imaging tools to select, and monitor patients for TEM1-antibody based targeted therapies.

Abstract B68: Targeted theranostics of tumor vasculature by multivalent scFv constructs against TEM1

Background: It is direly needed to develop new therapy and diagnosis for ovarian cancer. There are molecular markers on tumor vessels but not normal; thus provide strong rationale to develop early detection and targeting agents for such “signature”. One such protein, Tumor endothelial marker-1 (TEM1), is overexpressed in tumor endothelial cells as a suitable marker for targeting tumor vasculature. Objective: To better assist TEM1-specific theranostics, we sought to 1)develop TEM1-specific affinity agents from scFv-display library by protein engineering, 2)develop immunoPET/optical imaging; and 3)develop TEM1-targeting immunotherapies such as radioimmunotherapy, antibody-drug conjugate and bispecific antibodies. Methods: To determine the expression of TEM1 in tumor vasculature, 53 cases of metastatic serous ovarian cancer (stages IIIC and IV) resected at HUP were studied by immunohistochemistry. We previously isolated TEM1-specific scFv,”scFv78”, binding to extracellular domain (aa.324-390) of TEM1 with Kd ~2 nM. To improve thermal/serum stability and affinity, a series of multivalent variants were engineered, including scFv78 fusion with huIgG1 Fc region, or partial Fc (CH2-, CH3-, or hinge). Protein production was carried out with 293F cells and purified with affinity chromatography. These variants are characterized in vitro for Kd, serum and thermo stability. In vivo PK/PD and biodistribution studies were carried out in naive and TEM1+ tumor bearing animals. Protein with the most favorable affinity, PK/PD and stability was developed into agent for in vivo imaging, such as NIR optical imaging and immunoPET by 124-Iodine labeling. Antibody-drug conjugate (ADC) was designed and tested by conjugation to MMAE. Results: Over 99% of patients have positive staining on either fibroblast or vessel. To develop TEM1 antibody for theranostics, we characterized a panel of multivalent scFv78 variants. All variants exhibit comparable thermo and serum stability. ScFv78-Fc exhibited higher affinity (KD=0.15nM, 15 fold of parental scFv78) to TEM1. In vivo pharmacokinetics study of these variants suggested that scFv78-Fc is suitable for further development as targeted therapeutics and imaging. In pilot optical and PET imaging study, florouphore-labeled and radiolabeled scFv78-Fc showed enrichment in TEM1+ tumor, but not control tumor or normal organs. Pilot experiment showed specific killing of TEM1+ cells by 78Fc-MMAE conjugate. Conclusions: We developed an innovative approach of tumor therapy by targeting tumor vasculature marker such as TEM1, which is common on ovarian cancer and some other solid tumors. Citation Format: Chunsheng Li, Jia Hu, Ann Marie Chacko, Junying Wang, Aizhi Zhao, Xingmei Duan, Vladimir Muzykantov, George Coukos. Targeted theranostics of tumor vasculature by multivalent scFv constructs against TEM1. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B68.

Abstract 3809: Tumor-released GPI-anchored proteins bind to mannose receptor and polarize tumor-infiltrating macrophages

Tumors polarize their microenvironment to escape immune-mediated destruction and to promote their growth. Macrophages respond to microenvironmental signals and can be activated toward a classical (M1) or an alternative (M2) phenotype. Tumor-associated macrophages (TAM) resemble to M2 macrophages with a phenotype characterized by the overexpression of IL10, scavenger receptor SR-A, CD68 and MR phenotype but hampered levels of IL-12. M2, in contrast to M1 macrophages, promote TH2 immune responses as well as tissue remodelling and M2 infiltration in solid tumors has been shown to significantly correlate with poor prognosis. Recent evidences suggest that macrophage differentiation towards TAM involves a “chemical conversation” via exchange of unknown soluble extracellular mediators between tumor cells and macrophages. In addition, previous work showed that the cross-linking mannose receptor (MR) with an anti-MR mAb could cause dendritic cell differentiation into APCs promoting T-cell anergy. We hypothesized that MR expressed by macrophages could be bound by tumor-released GPI-anchored glycoproteins through the GPI-anchor mannose residues freed by cleavage. We also hypothesized that MR binding by GPI-anchor could trigger macrophage polarization toward TAMs. To test our hypotheses, we established an in vitro model system of cell co-culture in transwell allowing chemical exchanges between human monocyte-derived macrophages and human ovarian cancer cell line Ovcar3 that sheds mesothelin, a cancer biomarker and a GPI-anchored glycoprotein. Macrophage phenotype changes were monitored by flow cytometry and qRT-PCR. We also isolated three novel recombinant antibodies (scFv) against MR by magnetic and flow sortings from a novel yeast-display human scFv library. We describe here for the first time that tumor-released mesothelin bound to macrophage cell surface in presence of calcium, and that mesothelin binding to macrophages could be blocked by the anti-MR scFv. Furthermore, macrophage phenotype switch toward M2 during coculture with tumor cells could be prevented by one of the anti-MR scFv. Finally, we demonstrated that M1 phenotype could be rescued during coculture with tumor cells with one of the anti-MR scFv. To further characterize the role of MR in vivo, we have implanted mannose receptor knock-out mice intraperitoneally (IP) with luminescent-labeled ovarian cancer cells that overexpress mesothelin. We are now monitoring the tumor growth by in vivo imaging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3809.