Remodeling of the Tumor Microenvironment by a Chemokine/anti-PD-L1 Nanobody Fusion Protein (original) (raw)
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Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors
Immunotherapy, 2011
In this article, the role of chemokines and costimulatory molecules in the immunotherapy of experimental murine solid tumors and immunotherapy used in ongoing clinical trials are presented. Chemokine networks regulate physiologic cell migration that may be disrupted to inhibit antitumor immune responses or co-opted to promote tumor growth and metastasis in cancer. Recent studies highlight the potential use of chemokines in cancer immunotherapy to improve innate and adaptive cell interactions and to recruit immune effector cells into the tumor microenvironment. Another critical component of antitumor immune responses is antigen priming and activation of effector cells. Reciprocal expression and binding of costimulatory molecules and their ligands by antigen-presenting cells and naive lymphocytes ensures robust expansion, activity and survival of tumor-specific effector cells in vivo. Immunotherapy approaches using agonist antibodies or fusion proteins of immunomodulatory molecules si...
Nature Biotechnology, 1999
Chemokines are a group of small (7-15 kDa) secreted proteins that induce inflammatory response by orchestrating the selective migration, diapedesis, and activation of blood-borne leukocytes 1-3. Chemokines act by binding to a specific cell-surface heptahelical Gprotein-coupled receptor, which is internalized after binding with the ligand 4-6. Moreover, chemokines may have potent effects in promoting tumor immunity 7-9. For example, interferon inducible protein 10 (IP-10) can generate a T-lymphocyte-dependent antitumor response by recruiting other lymphocytes, neutrophils, and monocytes 1-3,7-9. In addition, monocyte chemotactic protein 3 (MCP-3) is a potent chemoattractant for monocytes and dendritic cells, T lymphocytes, basophils, and eosinophils 1-3,10. Importantly, most antigen presenting cells (APCs), particularly dendritic cells, express CCR1, CCR2, and CCR3 chemokine receptors, which bind MCP-3 (refs. 1, 11, and 12). Clonal B-cell malignancies express variable region sequences (idiotypes) on the Ig receptor, which can serve as potential tumor-specific antigens 13. However, a major obstacle is that syngeneic lymphoma Ig protein is nonimmunogenic and therefore requires conjugation to immunogenic, exogenous carriers (e.g., keyhole-limpet hemocyanin [KLH]) 14,15. Even then, Ig-KLH conjugates elicit primarily antibody responses, with little evidence of T-cell responses against the idiotype. Recent advances in antibody engineering make it possible to produce a refined lymphoma-specific single-chain Ig (sFv), consisting solely of linked V H and V L domains that retain the conformation of the native Ig 16,17. However, KLH crosslinked to sFv failed to induce the anti-idiotypic antibody response usually observed with native Ig-KLH conjugates, due to disruption of the fragile conformation of sFv (data not shown). We explore a novel strategy for induction of antitumor immunity, in which APCs are specifically targeted in vivo by a fusion protein consisting of chemokine and tumor antigen. We demonstrate that genetic fusion of chemokine moieties to lymphoma-derived sFv, a model nonimmunogenic antigen, converted this "self " tumor antigen into a potent immunogen. In both murine lymphoma models tested, 38C-13 (ref. 18) and A20 (ref. 19), immunization with either chemokine-sFv protein or naked DNA vaccines encoding the fusion elicited a T-cell dependent, protective antitumor immunity. Results Chemokine-fused sFv proteins retain conformation of native lymphoma-derived Ig and functional properties of chemokine. Ig variable region fragments were cloned by reverse transcription PCR from two different B-cell lymphomas, 38C-13 (ref. 19) and A20 (ref. 20). The fragments were arranged as sFv and produced as recombinant fusion proteins with either IP-10, MCP-3, control viral epitopes DomA (a 40-amino acid fragment containing major Band T-cell epitopes of S antigen), or PreS2 of hepatitis B surface antigen (HBsAg) (Fig. 1). Recombinant fusion proteins were purified from Escherichia coli 20 and fully characterized. The proper folding of sFv proteins was demonstrated by their ability to inhibit polyclonal or monoclonal anti-idiotypic antibodies binding to 38C-13-derived native Ig protein (Ig38) (Fig. 2). In contrast, control fusions lacking a spacer (IP10dsFv38), fusions with sFv derived from the irrelevant lymphoma, or fusions with viral epitopes DomA and PreS2 failed to inhibit binding to native 38C-13, suggesting that they did not retain proper folding of the antigen. Folding
Optimized Procedures for Producing Biologically Active Chemokines
Protein expression and …, 2009
We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6ÂHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6ÂHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L. Ó 2009 Elsevier Inc. All rights reserved.
Cancer Gene Therapy, 2006
Our goal in the present study was to evaluate antitumor effects and frequency of tumor-infiltrating immune cells upon intratumoral injection of RGD fiber-mutant adenoviral vector (AdRGD) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1, and CX3CL1. Among eight kinds of chemokine-expressing AdRGDs, AdRGD-CCL19 injection most efficiently induced infiltration of T cells into established B16BL6 tumor parenchyma, whereas most of these T cells were perforin-negative in immunohistochemical analysis. Additionally, the growth of AdRGD-CCL19-injected tumors decreased only slightly as well as that of other tumors treated with each chemokine-expressing AdRGD, which indicated that accumulation of naive T cells in tumor tissue does not effectively damage the tumor cells. Tumor-bearing mice, in which B16BL6-specific T cells were elicited by dendritic cellbased immunization, demonstrated that intratumoral injection of AdRGD-CCL17, -CCL22, or -CCL27 could considerably suppress tumor growth and attract activated T cells. On the other hand, AdRGD-CCL19-injection in the immunized mice showed slight increase of tumor-infiltrating T cells compared to treatment using control vector. Collectively, although AdRGD-mediated chemokine gene transduction into established tumors would be very useful for augmentation of tumor-infiltrating immune cells, a combinational treatment that can systemically induce tumor-specific effector T cells is necessary for satisfactory antitumor efficacy.
Frontiers in immunology, 2017
Following the clinical success achieved with the first generation of adoptive cell therapy (ACT) utilizing in vitro expanded tumor-infiltrating lymphocytes (TILs), the second and third generations of TIL ACT are evolving toward the use of genetically modified TIL. TIL therapy generally involves the transfer of a high number of TIL, ranging from 10(9) to 10(11) cells. One of the technical difficulties in genetically modifying TIL, using a retroviral vector, is the ability to achieve large expansion of transduced TIL, while keeping the technique suitable to a Good Manufacturing Practices (GMP) environment. Consequently, we developed and optimized a novel method for the efficient production of large numbers of GMP-grade, gene-modified TIL for the treatment of patients with ACT. The chemokine receptor CXCR2 was used as the gene of interest for methodology development. The optimized procedure is currently used in the production of gene-modified TIL for two clinical trials for the treatme...
In Vivo Expression of the Novel CXC Chemokine BRAK in Normal and Cancerous Human Tissue
American Journal of Pathology, 2000
Using differential display, we cloned a gene with reduced expression in short-term explants of head and neck squamous cell carcinoma (HNSCC) tumors compared to cultured normal oral epithelial cells. The differentially expressed gene was identical to the recently cloned CXC chemokine BRAK, which is ubiquitously expressed in normal tissue extracts but is absent from many tumor cell lines in vitro. To define the cell populations expressing BRAK in vivo, in situ mRNA hybridization was performed on normal and cancerous tissues from six different histological sites.
Journal of Leukocyte Biology, 2004
The ideal vaccine carrier should be able to target antigen delivery and possibly recruit antigen-presenting cells (APC) and deliver an activation signal to promote adaptive immune responses. Ligands for chemokine receptors expressed on APC may be attractive candidates, as they can both target and attract APC. To investigate the requirement for APC recruitment, we used a pair of viral chemokines, agonist virus 8-derived chemokine macrophage inflammatory protein-I (vMIP-I) and antagonist MC148, which induce and suppress chemotaxis, respectively. Chemokine-antigen fusions efficiently delivered a model nonimmunogenic tumor antigen to APC for processing and presentation to antigen-specific T cells in vitro. Physical linkage of chemokine and antigen and specific binding of chemokine receptor by the fusion protein were required. Mice immunized with vMIP-I or MC148 fusion DNA vaccines elicited protection against tumor challenge. Therefore, vaccine efficacy depends primarily on the ability of the carrier to target antigen delivery to APC for subsequent processing and presentation, and chemotaxis directly induced by the chemokine moiety in the fusion may not be necessary.
Expression, Purification and Biological Activity Study of Viral Chemokine VM IP2
2003
Background: Romiplostim is a peptibody analogue of thrombopoietin (TPO) which regulates platelet production. This molecule consists of two main parts: Peptide sequences which like wild type TPO, mimics stimulation of TPO receptor and IgG1Fc, (Peptide + Antibody = Peptibody). This drug is used in treatment of chronic Immune Thrombocytopenic Purpura (ITP). Methods: In this project E. coli bacteria were transformed by a construct harboring peptibody fusion gene. This construct consisted of two repeated peptide sequences which have fused to Carboxyl group of IgG1Fc. Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body. The inclusion bodies were separated, washed and after denaturation and solubilization, in the last stage the desired peptibodies were refolded and purified. The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting. The bioactivity were assessed in vivo using subcutaneous injection in mice. Results: Results showed accurate molecules were produced and purified. Also, in vivo experiment showed significant increment (more than two fold) of platelets compared to control group. Conclusion: In this study laboratory scale production of recombinant romiplostim showed proper in-vivo bioactivity. This new approach in expression and purification of this recently introduced thrombopoietin receptor agonist drug may be followed by scale up of its production to response the chronic ITP patient's demand.
Journal of Investigative Dermatology, 2010
Skin inflammation can induce local expression of CCL21, which is subsequently drained to lymph nodes (LNs) influencing their cellular composition. To determine whether the same can be achieved by dermal administration of a plasmid DNA (pDNA) encoding CCL21, we generated a pDNA-based gene construct allowing high-level expression of CCL21. Expression and secretion of biologically active CCL21 were confirmed in vitro by immunohistochemistry, western blot analysis, ELISA, and transwell chemotactic assays. In vivo experiments showed cellular expression of transgenic CCL21 after particle-mediated gene gun delivery of pDNA into skin. CCL21 was expressed in the epidermis, consequently secreted into the upper dermis, and transported into the draining LNs, which resulted in increased CCL21 concentration, total cell number, and frequencies of CD11c þ DCs and CD4 þ /CD62L þ naïve, CD4 þ /CD62L À , and CD8 þ /CD62L À effector memory Tcells (expressing CCL21 receptors CCR7 or CXCR3), as well as retention of adoptively transferred T-lymphocytes, in the draining LNs of plt/plt mice (lacking endogenous expression of CCL21). Our studies show that biologically active CCL21 can be overexpressed by genetic means in vitro and in vivo. This strategy allows reconstitution of a genetic defect and colocalization of different cell types in the secondary lymphoid organs, an important prerequisite for targeted cell migration.