Brain Disposition of Antibody-Based Therapeutics: Dogma, Approaches and Perspectives.

Due to their high specificity, monoclonal antibodies have been widely investigated for their application in drug delivery to the central nervous system (CNS) for the treatment of neurological diseases such as stroke, Alzheimer's, and Parkinson's disease. Research in the past few decades has revealed that one of the biggest challenges in the development of antibodies for drug delivery to the CNS is the presence of blood-brain barrier (BBB), which acts to restrict drug delivery and contributes to the limited uptake (0.1-0.2% of injected dose) of circulating antibodies into the brain. This article reviews the various methods currently used for antibody delivery to the CNS at the preclinical stage of development and the underlying mechanisms of BBB penetration. It also describes efforts to improve or modulate the physicochemical and biochemical properties of antibodies (e.g., charge, Fc receptor binding affinity, and target affinity), to adapt their pharmacokinetics (PK), and to influence their distribution and disposition into the brain. Finally, a distinction is made between approaches that seek to modify BBB permeability and those that use a physiological approach or antibody engineering to increase uptake in the CNS. Although there are currently inherent difficulties in developing safe and efficacious antibodies that will cross the BBB, the future prospects of brain-targeted delivery of antibody-based agents are believed to be excellent.

Spatio-temporal biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells by PET imaging in a preclinical tumor model.

Quantitative in vivo monitoring of cell biodistribution offers assessment of treatment efficacy in real-time and can provide guidance for further optimization of chimeric antigen receptor (CAR) modified cell therapy. We evaluated the utility of a non-invasive, serial 89Zr-oxine PET imaging to assess optimal dosing for huLym-1-A-BB3z-CAR T-cell directed to Lym-1-positive Raji lymphoma xenograft in NOD Scid-IL2Rgammanull (NSG) mice. In vitro experiments showed no detrimental effects in cell health and function following 89Zr-oxine labeling. In vivo experiments employed simultaneous PET/MRI of Raji-bearing NSG mice on day 0 (3 h), 1, 2, and 5 after intravenous administration of low (1.87 ± 0.04 × 106 cells), middle (7.14 ± 0.45 × 106 cells), or high (16.83 ± 0.41 × 106 cells) cell dose. Biodistribution (%ID/g) in regions of interests defined over T1-weighted MRI, such as blood, bone, brain, liver, lungs, spleen, and tumor, were analyzed from PET images. Escalating doses of CAR T-cells resulted in dose-dependent %ID/g biodistributions in all regions. Middle and High dose groups showed significantly higher tumor %ID/g compared to Low dose group on day 2. Tumor-to-blood ratios showed the enhanced extravascular tumor uptake by day 2 in the Low dose group, while the Middle dose showed significant tumor accumulation starting on day 1 up to day 5. From these data obtained over time, it is apparent that intravenously administered CAR T-cells become trapped in the lung for 3-5 h and then migrate to the liver and spleen for up to 2-3 days. This surprising biodistribution data may be responsible for the inactivation of these cells before targeting solid tumors. Ex vivo biodistributions confirmed in vivo PET-derived biodistributions. According to these studies, we conclude that in vivo serial PET imaging with 89Zr-oxine labeled CAR T-cells provides real-time monitoring of biodistributions crucial for interpreting efficacy and guiding treatment in patient care.

Principles of N-Linked Glycosylation Variations of IgG-Based Therapeutics: Pharmacokinetic and Functional Considerations.

The development of recombinant therapeutic proteins has been a major revolution in modern medicine. Therapeutic-based monoclonal antibodies (mAbs) are growing rapidly, providing a potential class of human pharmaceuticals that can improve the management of cancer, autoimmune diseases, and other conditions. Most mAbs are typically of the immunoglobulin G (IgG) subclass, and they are glycosylated at the conserved asparagine position 297 (Asn-297) in the CH2 domain of the Fc region. Post-translational modifications here account for the observed high heterogeneity of glycoforms that may or not impact the stability, pharmacokinetics (PK), efficacy, and immunogenicity of mAbs. These modifications are also critical for the Fc receptor binding, and consequently, key antibody effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Moreover, mAbs produced in non-human cells express oligosaccharides that are not normally found in serum IgGs might lead to immunogenicity issues when administered to patients. This review summarizes our understanding of the terminal sugar residues, such as mannose, sialic acids, fucose, or galactose, which influence therapeutic mAbs either positively or negatively in this regard. This review also discusses mannosylation, which has significant undesirable effects on the PK of glycoproteins, causing a decreased mAbs' half-life. Moreover, terminal galactose residues can enhance CDC activities and Fc-C1q interactions, and core fucose can decrease ADCC and Fc-FcγRs binding. To optimize the therapeutic use of mAbs, glycoengineering strategies are used to reduce glyco-heterogeneity of mAbs, increase their safety profile, and improve the therapeutic efficacy of these important reagents.

A Humanized Lym-1 CAR with Novel DAP10/DAP12 Signaling Domains Demonstrates Reduced Tonic Signaling and Increased Antitumor Activity in B-Cell Lymphoma Models.

PURPOSE: The murine Lym-1 mAb targets a discontinuous epitope (Lym-1 epitope) on several subtypes of HLA-DR, which is upregulated in a majority of human B-cell lymphomas and leukemias. Unlike CD19, the Lym-1 epitope does not downregulate upon crosslinking, which may provide an advantage as a target for CAR T-cell therapy. Lym-1 CAR T cells with a conventional 4-1BB and CD3ζ (BB3z) signaling domain exhibited impaired ex vivo expansion. This study aimed to identify the underlying mechanisms and develop strategies to overcome this effect. EXPERIMENTAL DESIGN: A functional humanized Lym-1 antibody (huLym-1-B) was identified and its scFv form was used for CAR design. To overcome observed impaired expansion in vitro, a huLym-1-B CAR using DAP10 and DAP12 (DAP) signaling domains was evaluated for ex vivo expansion and in vivo function. RESULTS: Impaired expansion in huLym-1-B-BB3z CAR T cells was shown to be due to ligand-dependent suboptimal CAR signaling caused by interaction of the CAR binding domain and the surface of human T cells. Using the novel DAP signaling domain construct, the effects of suboptimal CAR signaling were overcome to produce huLym-1-B CAR T cells with improved expansion ex vivo and function in vivo. In addition, the Lym-1 epitope does not significantly downregulate in response to huLym-1-B-DAP CAR T cells both ex vivo and in vivo. CONCLUSIONS: DAP intracellular domains can serve as signaling motifs for CAR, and this new construct enables nonimpaired production of huLym-1-B CAR T cells with potent in vivo antitumor efficacy.

Generation of a Monoclonal Antibody to Detect Elastin-like Polypeptides.

The identification and use of antibodies dominate the biologic, clinical diagnostic, and therapeutic landscapes. In particular, antibodies have become essential tools in a variety of protein analytical experiments and to study the disposition of biologic therapeutics. One emerging class of peptide biologics is known as the elastin-like polypeptides (ELPs), which are repetitive protein polymers inspired by human tropoelastin. A major limitation in the clinical translation of ELP biologics has been a lack of a monoclonal antibody (mAb) to characterize their identity during expression. To facilitate these studies, we successfully generated a new mAb that is specific toward ELPs and ELP fusion proteins. A purified antibody was evaluated in an ELISA, western blotting, and immunofluorescence assay. The optimal anti-ELP mAb proved to be highly reactive and specific toward ELPs. Moreover, they were able to detect ELPs with a variety of aliphatic guest residues. ELPs phase-separate in response to heating; furthermore, when incubated at a great excess of ELPs, the anti-ELP mAb partially blocks phase separation. These findings are direct evidence that novel murine mAbs can be raised against purified ELPs. This new reagent will enable purification, experimental detection, and characterization of these biopolymers.

Costimulation of type-2 innate lymphoid cells by GITR promotes effector function and ameliorates type 2 diabetes.

Metabolic syndrome is characterized by disturbances in glucose homeostasis and the development of low-grade systemic inflammation, which increase the risk to develop type 2 diabetes mellitus (T2DM). Type-2 innate lymphoid cells (ILC2s) are a recently discovered immune population secreting Th2 cytokines. While previous studies show how ILC2s can play a critical role in the regulation of metabolic homeostasis in the adipose tissue, a therapeutic target capable of modulating ILC2 activation has yet to be identified. Here, we show that GITR, a member of the TNF superfamily, is expressed on both murine and human ILC2s. Strikingly, we demonstrate that GITR engagement of activated, but not naïve, ILC2s improves glucose homeostasis, resulting in both protection against insulin resistance onset and amelioration of established insulin- resistance. Together, these results highlight the critical role of GITR as a novel therapeutic molecule against T2DM and its fundamental role as an immune checkpoint for activated ILC2s.

Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma.

T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

Systemic delivery of chTNT-3/CpG immunoconjugates for immunotherapy in murine solid tumor models.

CpG oligodeoxynucleotides (CpG) potently activate the immune system by mimicking microbial DNA. Conjugation of CpG to chTNT-3, an antibody targeting the necrotic centers of tumors, enabled CpG to accumulate in tumors after systemic delivery, where it can activate the immune system in the presence of tumor antigens. CpG chemically conjugated to chTNT-3 (chTNT-3/CpG) were compared to free CpG in their ability to stimulate the immune system in vitro and reduce tumor burden in vivo. In subcutaneous Colon 26 adenocarcinoma and B16-F10 melanoma models in BALB/c and C57BL/6 mice, respectively, chTNT-3/CpG, free CpG, or several different control constructs were administered systemically. Intraperitoneal injections of chTNT-3/CpG delayed tumor growth and improved survival and were comparable to intratumorally administered CpG. Compared to saline-treated mice, chTNT-3/CpG-treated mice had smaller average tumor volumes by as much as 72% in Colon 26-bearing mice and 79% in B16-bearing mice. Systemically delivered free CpG and CpG conjugated to an isotype control antibody did not reduce tumor burden or improve survival. In this study, chTNT-3/CpG retained immunostimulatory activity of the CpG moiety and enabled delivery to tumors. Because systemically administered CpG rapidly clear the body and do not accumulate into tumors, chTNT-3/CpG provide a solution to the limitations observed in preclinical and clinical trials.

Phase 1 Dose-Escalation Study with LEC/chTNT-3 and Toceranib Phosphate (Palladia®) in Dogs with Spontaneous Malignancies.

OBJECTIVES: LEC chemokine promotes TH1 responses and recruits immune cells to inflammatory sites. By linking LEC to an antibody targeting tumor necrosis, LEC/chTNT-3 can be used for the immunotherapeutic treatment of tumors. The primary objective of this study was to determine the safety profile of LEC/chTNT-3 and toceranib phosphate (Palladia®) combination therapy in dogs with spontaneous malignancies. Secondary purpose was to determine objective responses to treatment. METHODS: Twenty-three dogs with cancer were enrolled, covering nine different malignancies. In this dose escalation study, dogs received LEC/chTNT-3 for five days, and toceranib every 48 hours for the remainder of the study. Dogs received physical exams, chemistry panel, urinalysis, and complete blood counts on days 0, 10, 28 of the study, and every 6-8 weeks thereafter. RESULTS: Lethargy was noted in 13% dogs. There were no statistical differences in the prevalence of anorexia, diarrhea, thrombocytopenia, renal toxicity, or hepatic toxicity before or during the study. There were trends in increases in the prevalence of vomiting, lymphopenia, and neutropenia (all grade 2 or lower, p=0.07) over the initial 28 days of the study. By day 28, 10% of dogs had partial responses, 58% had stable disease, and 32% had progressive disease. CONCLUSIONS: LEC/chTNT-3 and toceranib were well tolerated. This combination therapy showed some biological activity against a variety of cancers at a low dose and short duration of LEC/chTNT-3 administration.

A hybrid protein-polymer nanoworm potentiates apoptosis better than a monoclonal antibody.

B-cell lymphomas continue to occur with a high incidence. The chimeric antibody known as Rituximab (Rituxan) has become a vital therapy for these patients. Rituximab induces cell death via binding and clustering of the CD20 receptor by Fcγ expressing effector cells. Because of the limited mobility of effector cells, it may be advantageous to cluster CD20 directly using multivalent nanostructures. To explore this strategy, this manuscript introduces a nanoparticle that assembles from a fusion between a single chain antibody and a soluble protein polymer. These hybrid proteins express in Escherichia coli and do not require bioconjugation between the antibody and a substrate. Surprisingly a fusion between an anti-CD20 single chain antibody and a soluble protein polymer assemble worm-like nanostructures, which were characterized using light scattering and cryogenic transmission electron microscopy. These nanoworms competitively bind CD20 on two B-cell lymphoma cell lines, exhibit concentration-dependent induction of apoptosis, and induce apoptosis better than Rituximab alone. Similar activity was observed in vivo using a non-Hodgkin lymphoma xenograft model. In comparison to Rituximab, systemic nanoworms significantly slowed tumor growth. These findings suggest that hybrid nanoworms targeted at CD20 may be useful treatments for B-cell related malignancies. Because of the ubiquity of antibody therapeutics, related nanoworms may have uses against other molecular targets.

CD8+ T cell-independent tumor regression induced by Fc-OX40L and therapeutic vaccination in a mouse model of glioma.

Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Although some promising advances have been made, the immune response stimulated as a result of immunotherapeutic protocols has been inefficient at complete tumor elimination, primarily due to our lack of understanding of the necessary effector functions of the immune system. We previously demonstrated that a tumor lysate vaccine/Fc-OX40L therapy is capable of inducing enhanced survival and tumor elimination in the GL261 mouse glioma model. The following experiments were performed to determine the mechanism(s) of action of this therapy that elicits a potent antitumor immune response. The evidence subsequently outlined indicates a CD8(+) T cell-independent and CD4(+) T cell-, NK cell-, and B cell-dependent means of prolonged survival. CD8(+) T cell-independent tumor clearance is surprising considering the current focus of many cancer immunotherapy protocols. These results provide evidence for CD8(+) T cell-independent means of antitumor response and should lead to additional examination of the potential manipulation of this mechanism for future treatment strategies.

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.

Generation of tumor-targeted antibody-CpG conjugates.

A number of monoclonal antibodies against tumor-associated antigens have been developed for the treatment of cancer. The anti-tumor effects of such antibodies can be enhanced by conjugation to immune stimulatory ligands, such as the toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG). The present study describes methods for the conjugation of CpG to two clinically approved monoclonal antibodies (rituximab and trastuzumab) via a Sulfo-EMCS maleimide linker. This conjugation method yielded stable joining of CpG and antibody (molar range 2.2-4.3:1). Immunofluorescence studies showed intact antigen-specific antibody binding of the immunoconjugates, that were comparable to unmodified antibody. Furthermore, antibody-CpG conjugates demonstrated improved (rituximab) or equivalent (trastuzumab) immune stimulatory activity compared to free CpG in vitro. These studies demonstrate the feasibility of antibody-CpG immunoconjugates and provide the foundation for future in vivo immunotherapy evaluation.

An in vivo immunotherapy screen of costimulatory molecules identifies Fc-OX40L as a potent reagent for the treatment of established murine gliomas.

PURPOSE: We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecules to identify an immunotherapeutic approach capable of curing established murine gliomas. EXPERIMENTAL DESIGN: Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide. RESULTS: Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100% depending on the timing, regimen, and combination with temozolomide. Cured mice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine/Fc-OX40L-treated mice had superior tumoricidal function relative to all other groups. Vaccine/Fc-OX40L-treated mice exhibited a significant increase in proliferation of brain-infiltrating CD4 and CD8 T cells, as indicated by Ki67 staining. Fc-OX40L had single-agent activity in transplanted and spontaneous glioma models, and the pattern of inflammatory gene expression in the tumor predicted the degree of therapeutic response. CONCLUSIONS: These data show that Fc-OX40L has unique and potent activity against experimental gliomas and warrants further testing.

Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys.

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K(d) values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5-32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1-8.8% and (3.0-9.0)%, respectively with the inter-assay precision and accuracy were 5.7-11.5% and (10.7-9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32-320 µg/kg).

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.

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 microg 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 microg) 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.

Immunogenicity of Iodine 131 chimeric tumor necrosis therapy monoclonal antibody in advanced lung cancer patients.

PURPOSE: Iodine-131 radiolabeled chimeric tumor necrosis therapy monoclonal antibody ((131)I-TNT) has been approved for the treatment of advanced lung cancer in China. In the present study, the immunogenicity of TNT was studied in advanced lung cancer patients using BIACORE and enzyme linked immunosorbent assay (ELISA) methods. EXPERIMENTAL DESIGN: Serum samples from 78 advanced lung cancer patients were analyzed for antibody development to TNT after systemic or intratumoral administration of two doses of (131)I-TNT. Patients' sera were obtained before, and 2 weeks and 2 months after (131)I-TNT radioimmunotherapy. RESULTS: Four of 78 lung cancer patients (4/78 or 5.13%) developed antibodies to TNT as measured by ELISA method, and 7 of 78 patients (8.97%) development anti-TNT antibody as measured by BIACORE biosensor after 2 doses of (131)I-TNT administration (P > 0.05). All the 4 ELISA-positive patients were also BIACORE-positive. Among the 7 BIACORE-positive patients, 5 (of 42, 11.9%) patients receiving intravenous TNT injection developed antibodies to TNT, and 2 (of 36, 5.56%) patients, receiving intratumoral therapy developed antibodies to TNT. The route of administration of the radiolabeled TNT antibody was not a statistically significant factor in the incidence of anti-TNT antibody. Detailed BIACORE serological analysis showed that the induced antibodies were mostly of the IgG1 subclass. CONCLUSIONS: (131)I-TNT was immunogenic in only a small minority of advanced lung cancer patients (8.97%). The route of administration did not statistically influence the incidence of anti-TNT antibody after TNT radioimmunotherapy in lung cancer patients.

Lym-1-induced apoptosis of non-Hodgkin's lymphomas produces regression of transplanted tumors.

Lym-1 was one of the first antibodies to be used successfully for the radioimmunotherapy of the human malignant lymphomas. This antibody, which recognizes the HLA-DR10 antigen preferentially expressed in B-cell lymphomas, was recently shown to induce apoptosis upon binding to lymphoma cells. In this study, Lym-1-induced apoptosis was studied to identify the potential molecular pathways of programmed cell death and to demonstrate the clinical potential of this antibody in the treatment of the human malignant lymphomas. Immunofluorescence microscopy revealed that Lym-1 stained focal areas of the cell surface, consistent with the fact that the HLA-DR10 antigen is associated with lipid rafts, a known prerequisite for apoptosis signaling. Likewise, Annexin V/propidium iodide staining and TUNEL assays demonstrated that both murine Lym-1 and chimeric Lym-1 induced both early and late apoptosis, respectively, unlike anti-CD20 rituximab. Furthermore, Lym-1 was found to produce a rapid loss of mitochondrial membrane potential and mitochondrial release of cytochrome C 14 hours post-Lym-1 treatment. Although it was found to activate caspase-3, inhibitors of caspase pathways showed that the Lym-1-induced apoptosis in lymphoma cell lines is independent of caspase induction. Finally, treatment studies in vivo demonstrated that, compared with murine anti-CD20 (2B8), Lym-1 was more effective in inducing the regression of human lymphoma xenografts. Based upon these results, chimeric Lym-1 should be especially effective in treating lymphoma patients, as, in addition to being able to elicit immune effector functions such as chimeric anti-CD20, it can also induce apoptosis directly upon cell binding.