Peisheng Hu

Pivotal Study of Iodine-131-Labeled Chimeric Tumor Necrosis Treatment Radioimmunotherapy in Patients With Advanced Lung Cancer

PURPOSE Tumor necrosis treatment (TNT) uses degenerating tumor cells and necrotic regions of tumors as targets for radioimmunotherapy. Previous studies in animal tumor models and clinical trials have demonstrated that when linked to the therapeutic radionuclide iodine-131, recombinant chimeric TNT antibody ((131)I-chTNT) can deliver therapeutic doses to tumors regardless of the location or type of malignancy. Therapeutic efficacy and toxicity of (131)I-chTNT in advanced lung cancer patients were studied in this pivotal registration trial. PATIENTS AND METHODS Patients with advanced lung cancer were treated with systemic or intratumoral injection of (131)I-chTNT in eight oncology centers in China. The objective response rate (ORR) was assessed as the primary end point. RESULTS All 107 patients who were entered onto the study and completed therapy had experienced treatment failure after prior radiotherapy or chemotherapy a mean of three times. The results showed an ORR of 34.6% (complete response, 3.7%; partial response, 30.8%; no change, 55.1%; and progressive disease, 10.3%) in all patients and 33% in 97 non-small-cell lung cancer patients. A biodistribution study demonstrated excellent localization of the radioactivity in tumors in both systemically and intratumorally injected patients. The most obvious adverse side effect was mild and reversible bone marrow suppression. CONCLUSION Radioimmunotherapy with (131)I-chTNT was well tolerated and can be used systemically or locally to treat refractory tumors of the lung.

Generation of Rituximab Polymer May Cause Hyper-Cross-linking–Induced Apoptosis in Non-Hodgkin's Lymphomas

Purpose: Although Rituximab has produced significant tumor regressions in lymphoma patients, only 50% respond. Clinically, it has been shown that the major mechanism of action of Rituximab is antibody-dependent cytotoxicity requiring presentation by Fc-bearing cells. To improve the clinical efficacy of Rituximab for the treatment of CD20+ lymphomas, we now describe a new formulation of Rituximab, which, on direct binding to target, can induce apoptosis. Methods: In this report, enhanced apoptosis was observed by treating CD20+ lymphoma cells with a new polymer formulation of Rituximab. The polymer was produced by formation of a peptide bond using the sugar moiety of dextran (MW 6,000) to generate a clinically relevant reagent for use in vivo. Results: Comparison of Rituximab with a previously described dimer and the newly generated polymer shows that the polymer induced apoptosis more effectively in CD20+ cells as shown by the terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay (Rituximab, 3%; dimer, 3%; polymer, 58%). Consistent with these results, the polymer produced marked regression in CD20+ lymphoma xenografts, whereas the dimer and monomer reagents showed little effect. In addition, we were able to show that the level of apoptosis induced in human lymphoma cell lines was in accordance with the extent of both surface CD20 clustering and caspase-3 activation. Conclusions: These data suggest that hyper-cross-linking–induced apoptosis can be simulated by the use of a dextran polymer of Rituximab, which, when used in vivo, can directly kill CD20+ lymphoma cells and improve the clinical efficacy of this important therapeutic for human B-cell lymphomas.

Immunogenicity of murine solid tumor models as a defining feature of in vivo behavior and response to immunotherapy

Immune profiling has been widely used to probe mechanisms of immune escape in cancer and identify novel targets for therapy. Two emerging uses of immune signatures are to identify likely responders to immunotherapy regimens among individuals with cancer and to understand the variable responses seen among subjects with cancer in immunotherapy trials. Here, the immune profiles of 6 murine solid tumor models (CT26, 4T1, MAD109, RENCA, LLC, and B16) were correlated to tumor regression and survival in response to 2 immunotherapy regimens. Comprehensive profiles for each model were generated using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and flow cytometry techniques, as well as functional studies of suppressor cell populations (regulatory T cells and myeloid-derived suppressor cells), to analyze intratumoral and draining lymphoid tissues. Tumors were stratified as highly or poorly immunogenic, with highly immunogenic tumors showing a significantly greater presence of T-cell costimulatory molecules and immune suppression in the tumor microenvironment. An absence of tumor-infiltrating cytotoxic T lymphocytes and mature dendritic cells was seen across all models. Delayed tumor growth and increased survival with suppressor cell inhibition and tumor-targeted chemokine+/−dendritic cells vaccine immunotherapy were associated with high tumor immunogenicity in these models. Tumor MHC class I expression correlated with the overall tumor immunogenicity level and was a singular marker to predict immunotherapy response with these regimens. By using experimental tumor models as surrogates for human cancers, these studies demonstrate how select features of an immune profile may be utilized to identify patients most likely to respond to immunotherapy regimens.

Cytokine, Chemokine, and Co-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors

This chapter describes the generation of novel reagents for the treatment of cancer using fusion proteins constructed with natural ligands of the immune system. Immunotherapy is a powerful therapeutic modality that has not been fully harnessed for the treatment of cancer. We and others have hypothesized that if the proper immunoregulatory ligands can be targeted to the tumor, an effective immune response can be mounted to treat both established primary tumors and distant metastatic lesions. Though it is generally believed that immunotherapy has the potential to treat only residual disease, we offer evidence that this approach can, by itself, destroy large tumor masses and produce lasting remissions of experimental solid tumors. From these studies, three major classes of immune activators, namely, cytokines, chemokines, and costimulatory molecules, have been shown to generate antitumor responses in animal models. In addition, the reversal of immune tolerance by the deletion of T regulatory (Treg) cells has been shown to be equally important for effective immunotherapy. In an attempt to identify reagents that can provide an enhanced immune stimulation and treatment of cancer, our laboratory has developed a novel monoclonal antibody targeting approach, designated Tumor Necrosis Therapy (TNT), which utilizes stable intracellular antigens present in all cell types but which are only accessible in dead and/or dying cells. Since tumors contain necrotic and degenerating regions that account for 30-80% of the tumor mass, this targeting approach can be used to deliver therapeutic reagents to the core of tumors, a site abundant in tumor antigens. In our first set of reagents, a panel of cytokine fusion proteins was genetically engineered using monoclonal antibody chimeric TNT-3 (chTNT-3) directed against necrotic regions of tumors (single-stranded DNA) fused with IL-2, or GM-CSF, or TNFalphaa, or IFNgamma. Tested against different solid tumors, these reagents were found to mount an effective although transient immune response to tumor especially when used in combination. To improve upon these results, additional chTNT-3 fusion proteins using the liver-expression chemokine (LEC) and the costimulatory molecule B7.1 were constructed. Both of these reagents were found to work significantly better than the above cytokine fusion proteins due to their ability to stimulate multiple arms of the immune system deemed useful for cancer immunotherapy. Finally, the Tumor Necrosis Factor Superfamily (TNFSF) gene DC137L was used to generate chTNT-3 antibody (targeted) and soluble Fc (untargeted) fusion proteins. When used alone, both forms of costimulatory fusion proteins were found to produce in a s dose-dependent manner, complete regression of murine solid tumors. Evidence is presented to show that Treg cells play an important role in suppressing antitumor immunity since the deletion of these cells, when used in combination with LEC or costimulatory fusion proteins, produced profound and effective treatment with sustained memory. It is hoped that these data will further the preclinical development of soluble Fc and antibody based fusion proteins fro the immunotherapy of cancer.

Targeted and untargeted CD137L fusion proteins for the immunotherapy of experimental solid tumors.

Introduction: CD137L is a member of the tumor necrosis factor superfamily that provides a costimulatory signal to T cells. In this study, two novel CD137L fusion proteins were produced and compared with the CD137 agonist antibody 2A. Materials and Methods: Murine CD137L was linked to the COOH terminus of either the Fc fragment of immunoglobulin (untargeted version) or TNT-3 (targeted version), an antibody that binds to necrotic regions of tumors. Groups of mice bearing established Colon 26 tumors were then treated daily ×5 with each fusion protein or 2A to determine their immunotherapeutic potential. Results: Both fusion proteins retained CD137L activity in vitro and TNT-3/CD137L showed tumor-binding activity by biodistribution analysis in tumor-bearing mice. The fusion proteins also produced similar responses in vivo at the 1 nmol per dose range and showed a 60% (TNT-3/CD137L) or 40% (Fc/CD137L) survival of treated mice at 150 days after tumor implantation, similar to the effects of 2A. Morphologic and immunohistochemical analyses showed massive central necrosis and infiltration of granzyme B–positive cells in necrotic areas and viable peripheral regions of treated tumors. Finally, cell depletion studies showed that CD137L-mediated tumor regression was CD8+ T cell dependent. Conclusions: From these studies, it was determined that both targeted and untargeted CD137L fusion proteins showed effective antitumor activity, but that the targeted version was more potent. Therefore, the use of the natural CD137 ligand is a promising approach to the treatment of solid tumors by virtue of its ability to produce physiologic costimulation within the tumor, limiting side effects often seen with agonist antibody therapies.

Pharmacokinetic characteristics and biodistribution of radioiodinated chimeric TNT-1, -2, and -3 monoclonal antibodies after chemical modification with biotin

To improve the clinical potential of monoclonal antibodies (MAbs), new methods are required to augment antibody uptake in the tumor while minimizing binding in normal tissues. Our laboratory has pioneered the use of chemical modification to accomplish this goal. Using three chimeric MAbs, chTNT-1, chTNT-2, and chTNT-3, which target solid tumors by binding to common antigens found in the central necrotic core, we now demonstrate the potential of chemical modification to improve the pharmacokinetic characteristics of these unique MAbs. To identify optimal modification conditions, TNT MAbs were reacted with biotin at various ratios and tested by clearance and biodistribution analyses. The biodistribution results revealed that the numbers of biotin molecules per MAb yielding optimal tumor uptake were 3:1 for chTNT-1, 5:1 for chTNT-2, and 8:1 for chTNT-3. Biotinylated MAbs were found to have faster whole body clearance times and better biodistribution profiles compared to unmodified antibodies. Although chTNT-2 showed only a modest improvement after biotinylation, biodistribution results indicated that this MAb had the highest uptake in tumor. By reducing the charge of the antibody molecule, chemical modification appears to be a useful method for improving the pharmacokinetics and biodistribution of TNT antibodies directed to the necrotic region of solid tumors.

Construction and Preclinical Characterization of Fc-mGITRL for the Immunotherapy of Cancer

Purpose: To provide proper costimulation required for effective cancer T-cell immunity, Fc-GITRL fusion proteins were generated for use in immunotherapy protocols. Experimental Design: Soluble fusion proteins consisting of the Fc fragment of immunoglobulin and the murine glucocorticoid-induced tumor necrosis factor–related receptor ligand (mGITRL) connected with different linkers were genetically engineered and tested for their potency in two BALB/c solid tumor models. Results:In vivo, construct #178-14 (−5aa, −linker) showed the best activity (>90% tumor reduction) at doses ranging from 5 to 25 μg and was found to be intact by gel electrophoresis. Similar doses used with construct #175-2 (-linker) produced good but not as high tumor regression. Construct #5-1 (+linker), which was found to be relatively unstable by SDS gel electrophoresis, produced <60% tumor regression and required a higher dose (100 μg) to produce optimal results. Survival curves showed that Fc-mGITRL treatment extended the life of 80% of tumor-bearing mice to >3 months compared with controls that died by day 40. T-cell depletion studies showed that CD8+ T cells play a major role in Fc-mGITRL immunotherapy, and tumors removed from Fc-mGITRL– and DTA-1–treated mice showed a significant influx of granzyme B+ lymphocytes compared with controls. Finally, T regulatory (Treg) cell assays showed that, unlike other Fc fusion proteins, all three Fc-mGITRL constructs profoundly suppressed Treg activity. Conclusions: These studies suggest that a stable, intact Fc-mGITRL fusion protein can provide missing costimulation for the immunotherapy of solid tumors. In addition, Fc-mGITRL may alter Treg activity to enhance its effectiveness for tumor immunotherapy.

A Therapeutic OX40 Agonist Dynamically Alters Dendritic, Endothelial, and T Cell Subsets within the Established Tumor Microenvironment

Little preclinical modeling currently exists to support the use of OX40 agonists as therapeutic agents in the setting of advanced cancers, as well as the mechanisms through which therapeutic efficacy is achieved. We show that treatment of mice bearing well-established day 17 sarcomas with a novel OX40 ligand-Fc fusion protein (OX40L-Fc) resulted in tumor regression or dormancy in the majority of treated animals. Unexpectedly, dendritic cells (DC) in the progressive tumor microenvironment (TME) acquire OX40 expression and bind fluorescently labeled OX40L-Fc. Furthermore, longitudinal analyses revealed that DCs become enriched in the tumor-draining lymph node (TDLN) of both wild-type and Rag-/- mice within 3 days after OX40L-Fc treatment. By day 7 after treatment, a significant expansion of CXCR3+ T effector cells was noted in the TDLN, and by day 10 after treatment, type 1 polarized T cells exhibiting a reactivated memory phenotype had accumulated in the tumors. High levels of CXCL9 (a CXCR3 ligand) and enhanced expression of VCAM-1 by vascular endothelial cells (VEC) were observed in the TME early after treatment with OX40L-Fc. Notably, these vascular alterations were maintained in Rag-/- mice, indicating that the OX40L-Fc-mediated activation of both DC and VEC occurs in a T-cell-independent manner. Collectively, these findings support a paradigm in which the stimulation of DC, T cells, and the tumor vasculature by an OX40 agonist dynamically orchestrates the activation, expansion, and recruitment of therapeutic T cells into established tumors.

Fc-mOX40L fusion protein produces complete remission and enhanced survival in 2 murine tumor models.

OX40L is a member of the tumor necrosis factor superfamily that provides a costimulatory signal to CD4+ and CD8+ T cells while inhibiting the effects of suppressive CD4+CD25+ regulatory T cells. Because of this dual activity, OX40L may provide significant antitumor immunity in tumor-bearing mice. To study its clinical potential, a fusion protein consisting of mOX40L linked to the C-terminus of the Fc fragment of immunoglobulin was genetically engineered. After demonstrating its potency in vitro, several assays were performed to evaluate its antitumor effect in comparison to the OX40 agonist antibody OX86. Dosing studies in Colon 26-bearing and renal cell carcinoma (RENCA)-bearing mice showed that although OX86 produced modest tumor regression, Fc-mOX40L produced complete remission in both tumor models. Survival studies confirmed these results and showed that Fc-mOX40L treatment produced lasting responses throughout the 5-month observation period. Flow cytometric analysis of treated and untreated tumors and tumor-draining lymph nodes identified a qualitative difference in the activity of Fc-mOX40L compared with OX86 treatment as evidenced by differences in lymphoid and macrophage populations. These studies reflect the profound therapeutic potential of Fc-mOX40L, which substantially exceeds the agonist antibody OX86 in ability to produce complete tumor remissions and promote long-term survival in solid tumor models.

LEC/chTNT-3 fusion protein for the immunotherapy of experimental solid tumors.

The human chemokine liver-expression chemokine (LEC) was originally found in an expressed sequence tag library, and later the LEC gene was located to chromosome 17q in the ML chemokine gene cluster. LEC has been shown to chemoattract monocytes, lymphocytes, and polymorphonuclear leukocytes (PMNs) by its binding to CCR1 and CCR8 chemokine receptors. Because of its potency as a chemoattractant for immune cells, LEC was used to genetically engineer a fusion protein with chTNT-3, a monoclonal antibody previously shown to target tumors by binding to DNA exposed in necrotic zones. Because the N-terminus of chemokines is important for their activity, the C-terminus of LEC was genetically linked to the chTNT-3 heavy chain variable region and, along with the light chain gene, cotransfected into NSO murine myeloma cells using the glutamine synthetase gene amplification system. The expressed LEC/chTNT-3 fusion protein was purified by tandem protein-A affinity and ion-exchange chromatography and chemotaxis and binding assays confirmed the bioactivity of the purified fusion protein. Pharmacokinetic and biodistribution studies in vivo showed that LEC/chTNT-3 had a biologic half-life of 3 hours and had good uptake in tumor (2.4% injected dose/g), which remained stable at 12 and 24 hours postinjection. Immunotherapy studies performed in three solid tumor models of the BALB/c mouse showed between 37% and 55% tumor reduction at 19 days post-implantation. Immunohistochemical studies using tumor sections obtained at different time points after the administration of control chTNT-3 and LEC/chTNT-3 showed heavy infiltration of CD4+ and CD8+ T cells, PMNs, B cells, and CD11c+CD11b+ dendritic cells in the LEC/chTNT-3 treated groups. The results of these studies demonstrate that this novel fusion protein has potent antitumor activity that is associated with the infiltration of different subpopulations of immune cells. The targeting of LEC to necrotic areas of tumors where the release of tumor antigens is prevalent may be a new approach for the immunotherapy of solid tumors.