Richard P. Junghans

FINALLY ! THE BRAMBELL RECEPTOR (FCRB) MEDIATOR OF TRANSMISSION OF IMMUNITY AND PROTECTION FROM CATABOLISM FOR IGG

F. W. Rogers Brambell was the father of the field of transmission of immunity, which he entered 50 years before the present era. As part of his quantitative and temporal studies on transmission, he defined the first Fc receptor system for IgG, and furthermore recognized the link between transmission of passive immunity from mother to young and protection from catabolism for IgG. This article provides a historical overview of the efforts of Professor Brambell and summarizes the subsequent elaboration of the details of the physiology and molecular biology of this remarkable receptor system.

Anti-Tac-H, a humanized antibody to the interleukin 2 receptor, prolongs primate cardiac allograft survival.

High-affinity interleukin 2 receptors (IL-2Rs) are expressed by T cells activated in response to foreign histocompatibility antigens but not by normal resting T cells. To exploit this difference in IL-2R expression, anti-Tac-M, a murine monoclonal antibody specific for the IL-2R alpha chain, was used to inhibit organ allograft rejection. However, the use of murine anti-Tac as an immunosuppressive agent was limited by neutralization by human anti-murine antibodies and by weak recruitment of effector functions. To circumvent these difficulties, a humanized antibody to the IL-2R, anti-Tac-H, was prepared. This molecule is human with the exception of the hypervariable segments, which are retained from the mouse. In vivo survival of anti-Tac-H is 2.5-fold longer than simultaneously administered anti-Tac-M (terminal t1/2, 103 hr vs. 38 hr). In addition, anti-Tac-H is less immunogenic than anti-Tac-M when administered to cynomolgus monkeys undergoing heterotopic cardiac allografting. Specifically, all monkeys treated with anti-Tac-M developed measurable anti-anti-Tac-M levels by day 15 (mean onset, 11 days). In contrast, none of the animals receiving anti-Tac-H produced measurable antibodies to this monoclonal antibody before day 33. Finally, there was a prolongation of graft survival in the cynomolgus heterotopic cardiac allograft model in animals receiving anti-Tac. In animals that received anti-Tac-M, the allograft survival was prolonged compared to that of the control group (mean survival, 14 +/- 1.98 days compared to 9.2 +/- 0.48 days; P less than 0.025). Graft survival was further prolonged by anti-Tac-H with a mean survival of 20.0 +/- 0.55 days (compared to controls, P less than 0.001; compared to anti-Tac-M, P less than 0.02). There was no toxicity attributable to the administration of either form of anti-Tac. Thus, anti-Tac-H significantly prolonged allograft survival in primates, without toxic side effects, and may be of value as an adjunct to standard immunosuppressive therapy in humans.

Suppression of human glioma xenografts with second-generation IL13R-specific chimeric antigen receptor-modified T cells.

Purpose: Glioblastoma multiforme (GBM) remains highly incurable, with frequent recurrences after standard therapies of maximal surgical resection, radiation, and chemotherapy. To address the need for new treatments, we have undertaken a chimeric antigen receptor (CAR) “designer T cell” (dTc) immunotherapeutic strategy by exploiting interleukin (IL)13 receptor α-2 (IL13Rα2) as a GBM-selective target. Experimental Design: We tested a second-generation IL13 “zetakine” CAR composed of a mutated IL13 extracellular domain linked to intracellular signaling elements of the CD28 costimulatory molecule and CD3ζ. The aim of the mutation (IL13.E13K.R109K) was to enhance selectivity of the CAR for recognition and killing of IL13Rα2+ GBMs while sparing normal cells bearing the composite IL13Rα1/IL4Rα receptor. Results: Our aim was partially realized with improved recognition of tumor and reduced but persisting activity against normal tissue IL13Rα1+ cells by the IL13.E13K.R109K CAR. We show that these IL13 dTcs were efficient in killing IL13Rα2+ glioma cell targets with abundant secretion of cytokines IL2 and IFNγ, and they displayed enhanced tumor-induced expansion versus control unmodified T cells in vitro. In an in vivo test with a human glioma xenograft model, single intracranial injections of IL13 dTc into tumor sites resulted in marked increases in animal survivals. Conclusions: These data raise the possibility of immune targeting of diffusely invasive GBM cells either via dTc infusion into resection cavities to prevent GBM recurrence or via direct stereotactic injection of dTcs to suppress inoperable or recurrent tumors. Systemic administration of these IL13 dTc could be complicated by reaction against normal tissues expressing IL13Ra1. Clin Cancer Res; 18(21); 5949–60. ©2012 AACR.

Autoimmunity associated with immunotherapy of cancer

In this age of promise of new therapies for cancer, immunotherapy is emerging as an exciting treatment option for patients. Vaccines and cytokines are being tested extensively in clinical trials, and strategies using monoclonal antibodies and cell transfer are mediating dramatic regression of tumors in patients with certain malignancies. However, although initially advocated as being more specific for cancer and having fewer side effects than conventional therapies, it is becoming increasingly clear that many immunotherapies can lead to immune reactions against normal tissues. Immunotoxicities resulting from treatment can range from relatively minor conditions, such as skin depigmentation, to severe toxicities against crucial organ systems, such as liver, bowel, and lung. Treatment-related toxicity has correlated with better responses in some cases, and it is probable that serious adverse events from immune-mediated reactions will increase in frequency and severity as immunotherapeutic approaches become more effective. This review introduces immunotherapeutic approaches to cancer treatment, provides details of toxicities arising from therapy, and discusses future potential ways to avoid or circumvent these side effects.

The Multichain Interleukin-2 Receptor: A Target for Immunotherapy

Activation of resting T-lymphocytes induces synthesis of interleukin-2 (IL-2) and expression of cell surface receptors for this lymphokine. In contrast to resting normal T-cells that do not express high-affinity IL-2 receptors (IL-2R), abnormal T-cells of patients with leukemia-lymphoma, certain autoimmune disorders, and individuals rejecting allografts express this receptor. Exploiting this difference in receptor expression, antibodies to the IL-2 receptor have been used effectively to treat patients with leukemia and lymphoma. One approach is to use monoclonal antibodies produced in mice; the disadvantage is that they are highly immunogenic. In an effort to reduce the immunogenicity of the mouse monoclonal antibodies, monoclonal-antibody-mediated therapy has been revolutionized by generating humanized antibodies produced by genetic engineering in which the molecule is human except for the antigen-combining regions, which are retained from the mouse. Further, to increase its cytotoxic effectiveness, the monoclonal antibody has been armed with toxins or radionuclides. Alternatively, IL-2 itself has been linked to a toxin to kill IL-2 receptor-bearing cells. Thus, IL-2 receptor-directed therapy provides a new method for treating certain neoplastic diseases and autoimmune disorders and for preventing allograft rejection.

Phase I Hepatic Immunotherapy for Metastases study of intra-arterial chimeric antigen receptor modified T cell therapy for CEA+ liver metastases

Purpose: Chimeric antigen receptor–modified T cells (CAR-T) have demonstrated encouraging results in early-phase clinical trials. Successful adaptation of CAR-T technology for CEA-expressing adenocarcinoma liver metastases, a major cause of death in patients with gastrointestinal cancers, has yet to be achieved. We sought to test intrahepatic delivery of anti-CEA CAR-T through percutaneous hepatic artery infusions (HAIs). Experimental Design: We conducted a phase I trial to test HAI of CAR-T in patients with CEA+ liver metastases. Six patients completed the protocol, and 3 received anti-CEA CAR-T HAIs alone in dose-escalation fashion (108, 109, and 1010 cells). We treated an additional 3 patients with the maximum planned CAR-T HAI dose (1010 cells × 3) along with systemic IL2 support. Results: Four patients had more than 10 liver metastases, and patients received a mean of 2.5 lines of conventional systemic therapy before enrollment. No patient suffered a grade 3 or 4 adverse event related to the CAR-T HAIs. One patient remains alive with stable disease at 23 months following CAR-T HAI, and 5 patients died of progressive disease. Among the patients in the cohort that received systemic IL2 support, CEA levels decreased 37% (range, 19%–48%) from baseline. Biopsies demonstrated an increase in liver metastasis necrosis or fibrosis in 4 of 6 patients. Elevated serum IFNγ levels correlated with IL2 administration and CEA decreases. Conclusions: We have demonstrated the safety of anti-CEA CAR-T HAIs with encouraging signals of clinical activity in a heavily pretreated population with large tumor burdens. Further clinical testing of CAR-T HAIs for liver metastases is warranted. Clin Cancer Res; 21(14); 3149–59. ©2015 AACR.

The role of the Brambell receptor (FcRB) in liver: protection of endocytosed immunoglobulin G (IgG) from catabolism in hepatocytes rather than transport of IgG to bile

The Brambell receptor (FcRB) mediates functions of both immunoglobulin G (IgG) transport, transmitting immunity from mother to young, and IgG protection, making IgG the longest surviving of all plasma proteins. Reflecting its role as transport receptor (termed FcRn, for neonatal rat intestine, the tissue from which it was first cloned), FcRB is expressed antenatally in the rabbit, mouse and rat fetal yolk sac and in human placental syncytiotrophoblasts, and neonatally in the intestinal epithelium of mice and rats. Reflecting its role as protection receptor (FcRp), FcRB is expressed in the vascular endothelium throughout life, where it protects IgG from the on-going catabolic activities of this tissue. FcRB detected in hepatocytes was hypothesized to mediate transport of IgG from serum to bile, thus potentially extending the transport expression (FcRn) of this receptor beyond the perinatal period. Our results show serum-to-bile transport of IgG to be unaffected in mice functionally deleted for FcRB. Accordingly, the hypothesis is rejected that FcRB functions as transport receptor (FcRn) in liver. The default conclusion is that FcRB in hepatocytes functions as FcRp, serving to protect IgG from catabolism in hepatocytes that accompanies the endocytic activity of these cells. We conclude that there remains to date no evidence of an FcRn-like transport function of the Brambell receptor beyond the perinatal period, after which the FcRp function of the receptor predominates, paralleling the endocytic activities of the associated tissues.

Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal Tumors

Purpose: The aims of this study are to compare antitumor activities of two generations of GD3-specific chimeric antigen receptors (CAR) in human primary T lymphocytes in vitro and to evaluate the antitumor efficacy of using a combination of systemic infusion of interleukin-2 (IL2) and designer T cells to eradicate subcutaneous established GD3+ melanoma in nude mice. Experimental Design: Antitumor activities were compared for two generations of designer T cells, the progenitor first-generation with immunoglobulin T-cell receptor (TCR) with Signal 1 and the second-generation designer T cells with Signal 1+2. Osmotic IL2 pumps were used to deliver the maximum tolerated dose of IL2 to enhance the antitumor effects of designer T cells on subcutaneous established melanoma in nude mice. Results: Melanoma is associated with high expression of ganglioside GD3, which has been targeted with modest effect in antibody therapies. We previously showed that an anti-GD3 CAR (sFv-TCRζ) will recruit T cells to target this non–T-dependent antigen, with potent killing of melanoma cells. Here, we report the addition of a CD28 costimulation domain to create a second-generation CAR, called Tandem for two signals. We show that this Tandem sFv-CD28/TCRζ receptor on T cells confers advantages of improved cytokine secretion, cytotoxicity, proliferation, and clonal expansion on tumor contact versus the same CAR without costimulation. In an adoptive transfer model using established melanoma tumors, designer T cells with CD28 showed a 50% rate of complete remissions but only where IL2 was supplemented. Conclusions: As a reagent for clinical development, the second-generation product is shown to have superior properties to warrant its preference for clinical designer T-cell immunotherapy for melanoma and other tumors. Systemic IL2 was required for optimal activity in an established tumor model. Clin Cancer Res; 16(10); 2769–80. ©2010 AACR.

An optimized method for cell-based phage display panning

BACKGROUND Traditional methods of phage display panning, bind purified antigen to plates or other solid phases to which libraries are then applied, followed by vigorous washings in detergent-supplemented buffers to select for specific phage Fab. These methods are not directly applicable to antigens in their native environment on cell surfaces or in settings where the target antigen is unknown. OBJECTIVES To develop a model antigen system employing whole cells rather than purified protein immobilized on a substrate; to optimize methods for phage display panning using a cell-based system. RESULTS Specificity of binding of phage Fab to antigen on cells was demonstrated by output titer and by flow cytometry. Output titers showed a plateau and binding advantage, after four washes, corresponding to removal of most non-specifically bound phage. Enrichment advantage was independent of input phage number. Longer incubation times, to cell tolerance, improved specific binding. Temperature had modes impact as a variable in the panning and washing. An increase in output titers paralleled enrichment for specific phage Fab. CONCLUSION An optimized method applied to whole cells can productively enrich specific phage Fab in mixtures with large excesses of non-specific phage Fab over several rounds of panning.

Retroviral DNA H structures: Displacement-assimilation model of recombination

The avian retroviruses are unique among known RNA and DNA viruses in their extremely high frequencies of genetic recombination. We propose that these high frequencies can be explained by the facts that the closely associated RNA genomes of this diploid virus can be reverse-transcribed concurrently and that strand displacement is a fundamental property of the reverse transcription reaction. We have elaborated a specific model to describe this process that was suggested by the properties of novel structures observed with high frequency in the electron microscope: DNA duplexes in dimer arrangement that are linked at homologous regions by single-stranded DNA bridges. These structures are presumed to be intermediates of recombination, trapped because their generation in vitro prevents the subsequent resolution steps that would normally take place via the cellular apparatus during infection. The model generates several hypotheses whose exploration should help to test its accuracy.