Regression In Mice Research Articles

A phase 1 trial of PT-100 in patients receiving myelosuppressive chemotherapy

2572 Background: PT-100 is a small molecule which competitively inhibits dipeptidyl peptidase activity of fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). It rapidly increases cytokine (G-CSF, IL-8) production, accelerates neutrophil and erythrocyte regeneration, and causes tumor regression in mice via inhibition of FAP and DPP-IV. This dose-escalation study was conducted to evaluate the safety of PT-100 in patients receiving myelosuppressive chemotherapy (a doxorubicin or taxane based regimen) and to assess its effects on neutrophil recovery. Methods: Patients received 2 cycles of chemotherapy: the first cycle (C1) served as each patient's individual control. PT-100 was administered orally for 7 days in cycle 2 (C2) as a 200, 400, 800, and 1200mcg total daily dose (divided twice daily) to 6, 6, 13, and 4 patients, respectively. Most patients received PT-100 on Days 2–8 of chemotherapy in C2, except at 800mcg where one cohort was treated on a Day 5–11 schedule. Patients had to have Grade 3/4 neutropenia in C1 to receive PT-100 in C2. Results: Five of 13 patients receiving PT-100 800mcg experienced a ≥ 2-day improvement in ≥ Grade 3 neutropenia, and a 62% improvement in median AUC in C2 vs. C1 was observed in patients treated on the Day 2–8 schedule. A corresponding upregulation in G-CSF, IL-6, and IL-8 was observed in most patients. Overall, PT-100 was well-tolerated. Edema/peripheral swelling, hypotension, and hypovolemia were the most common non-hematologic AEs considered related to PT-100. Two Grade 3 AEs were considered related to PT-100: syncope (1200mcg) and orthostatic hypotension (800mcg). An MTD was not reached. Conclusions: Given the accelerated neutrophil recovery, strong preclinical evidence of antitumor activity, and tolerable toxicities of PT-100, studies using a longer PT-100 dosing schedule are warranted to investigate its antitumor and neutrostatic effects. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Point Therapeutics Point Therapeutics Point Therapeutics Cell Genesys; ONYX Pharmaceuticals Corixa

In vitro and in vivo cytotoxic activities of recombinant immunotoxin 8H9(Fv)-PE38 against breast cancer, osteosarcoma, and neuroblastoma.

The 8H9 monoclonal antibody (MAb) is highly reactive with a cell surface glycoprotein expressed on human breast cancers, childhood sarcomas, and neuroblastomas but is not reactive with the cell surface of normal human tissues. This specific reactivity suggests that MAb 8H9 may be useful for targeted cancer therapy. To explore this possibility, we generated two recombinant immunotoxins (ITs) using the single-chain Fv (scFv) of MAb 8H9. Initially the 8H9(scFv) cDNA was fused to a DNA encoding a 38-kDa truncated form of Pseudomonas exotoxin (PE38) to generate the IT 8H9(scFv)-PE38. The fusion gene was expressed in Escherichia coli, and the IT was purified to near homogeneity from inclusion bodies. The purified IT showed specific cytotoxicity on nine different cancer cell lines derived from breast cancer, osteosarcoma, and neuroblastomas, known to react with MAb 8H9. The cytotoxic activity was inhibited by MAb 8H9, showing the cytotoxic activity is specific. The antitumor activity of 8H9(scFv)-PE38 was evaluated in severe combined immunodeficient mice bearing MCF-7 breast cancers or OHS-M1 osteosarcomas. The IT showed a specific dose-dependent antitumor activity at 0.075 and 0.15 mg/kg. Next, a more stable disulfide-linked IT, 8H9(dsFv)-PE38, was constructed. It was produced in high yield (16%) and showed cytotoxic and antitumor activities similar to those of 8H9(scFv)-PE38. 8H9(dsFv)-PE38 was given to two cynomolgus monkeys at doses of 0.1 and 0.2 mg/kg i.v. QOD x 3 and was well tolerated. This shows that a dose that causes significant tumor regressions in mice is well tolerated by monkeys. These results make 8H9(dsFv)-PE38 a candidate for further development as a therapeutic agent for breast cancers, osteosarcomas, and neuroblastomas.

RPE cells modulate subretinal neovascularization, but do not cause regression in mice with sustained expression of VEGF.

Previous studies using models of choroidal neovascularization (CNV) in which the angiogenic stimulus is not sustained, have concluded that the retinal pigmented epithelium (RPE) causes regression of neovascularization (NV). However, the withdrawal of angiogenic stimuli may actually be the major modulator of NV, and RPE cells may simply be responding to withdrawal of the angiogenic stimuli or something released by NV because of the withdrawal. In this study, the long-term course of NV and the behavior of the RPE in rhodopsin/VEGF transgenic mice, in which there is a sustained angiogenic stimulus, was investigated. Hemizygous mice from the V-6 line were killed at 0.75, 1, 3, 6, and 12 months after birth, and at each time point mRNA for VEGF, VEGF-R1, and VEGF-R2 was measured by RT-PCR. Some mice were perfused with fluorescein-labeled dextran and retinal flatmounts were examined by fluorescence microscopy. Light and electron microscopy was performed on Epon-embedded eyes. The mRNA levels for VEGF, VEGF-R1, and VEGF-R2 remained constant from the earliest to the latest time point. Retinal flatmounts showed numerous small areas of subretinal NV at 3 weeks and at 1 month, and there were a similar number of larger lesions. By 6 months, many of the individual NV lesions had grown together to form large networks of new vessels. At 12 months, NV networks were similar to those at 6 months, but some of the vessels were not perfused. Light microscopy showed serous retinal detachments overlying NV lesions in mice up to 3 months of age, but at 6 and 12 months, the RPE completely surrounded new vessels and formed tight junctions to reestablish the outer blood-retinal barrier, and there were no serous detachments. Electron microscopy showed that compared with more acute NV lesions, chronic lesions contained thinner endothelial cells, similar to those of the choriocapillaris in that they had scant cytoplasm and numerous fenestrations, or pinocytotic vesicles with thick basement membrane surrounded by extracellular matrix (ECM). Bruch's membrane remained intact. Despite persistent high expression of VEGF and its receptors, NV stopped growing and reached a plateau in older V-6 mice. RPE cells modulated the NV by surrounding it and reestablishing the blood-retinal barrier, but did not cause regression, although some vessels in chronic lesions were not perfused. These data do not support the conclusion of several previously reported studies, that RPE cells cause regression of CNV.

Signalling pathways of the TNF superfamily: a double-edged sword.

Two different tumour-necrosis factors (TNFs), first isolated in 1984, were found to be cytotoxic to tumour cells and to induce tumour regression in mice. Research during the past two decades has shown the existence of a superfamily of TNF proteins consisting of 19 members that signal through 29 receptors. These ligands, while regulating normal functions such as immune responses, haematopoiesis and morphogenesis, have also been implicated in tumorigenesis, transplant rejection, septic shock, viral replication, bone resorption, rheumatoid arthritis and diabetes; so indicating their role as 'double-edged swords'. These cytokines either induce cellular proliferation, survival, differentiation or apoptosis. Blockers of TNF have been approved for human use in treating TNF-linked autoimmune diseases in the United States and other countries.

Potential involvement of monocyte chemoattractant protein (MCP)-1/CCL2 in IL-4-mediated tumor immunity through inducing dendritic cell migration into the draining lymph nodes

We previously observed that IL-4 gene transduction into a mouse colon 26 adenocarcinoma cell line abrogated its tumorigenicity due to the generation of anti-tumor CTL. DEC-205- and CD11c-double positive cells were increased in the lymph nodes of mice injected with IL-4-transfected cells between 2 and 3 days after the tumor injection, compared with those injected with parental cells. Most of these double positive cells expressed CD86 antigen. Among the chemokines with chemotactic activities against dendritic cells, monocyte chemoattractant protein (MCP)-1/CCL2, ABCD-1/CCL22, and liver and activation-regulated chemokine (LARC)/CCL20 gene expression was enhanced no later than 3 days after the tumor injection, in the draining lymph nodes of IL-4-transfected cell bearing mice. Moreover, gene expression of the receptor for MCP-1/CCL2, CCR2, was enhanced in the draining lymph nodes of the mice injected with IL-4-transfected cells, and most DEC-205-positive cells in the lymph nodes expressed CCR2. Finally, the administration of anti-MCP-1/CCL2 antibodies retarded the rate of tumor regression in mice injected with IL-4-tranfected cells, concomitantly with a decrease in DEC-205- and CD11c-double positive cell number in the draining lymph nodes. Thus, locally produced MCP-1/CCL2 may be responsible for IL-4-mediated tumor rejection presumably based on the induction of dendritic cell migration into the draining lymph nodes.

Protoporphyrin-IX accumulation and cutaneous tumor regression in mice using a ferrochelatase inhibitor

The use of endogenously created porphyrins as an alternative to photosensitizer injection for photodynamic therapy (PDT) of cancer is a rapidly evolving area of study. In-situ accumulation of protoporphyrin-IX (PpIX) by hemebiosynthesis inhibition represents a novel method for PDT of cancer cells. The kinetics of PpIX accumulation and cutaneous tumor regression in mice was studied using lead (a known and effective inhibitor of ferrochelatase). Cutaneous tumors were exposed to various doses of ferrochelatase enzyme inhibitor (lead) and to different durations and doses of visible light. The maximum increase in PpIX levels (blood, skin and tumor) was observed 48 h after the parenteral administration of second injection of lead within a period of 1 month. The maximum tumor regression was observed in mice that were exposed to visible light at a light dose of 648 J/cm 2 (1 h exposure in four sessions of 15 min, with a gap of 10 min between each exposure). Continuous treatment for 6 consecutive days resulted in almost complete regression of the tumors in most of the animals. Histopathological sections of tumors after light exposure showed necrotic tissue with degenerated lymphocytes, monocytes, and neutrophils. Since the ferrochelatase inhibitor (lead) used in the present study is toxic, the search must continue for a safe, non-toxic inhibitor to enhance sensitizer-mediated PDT.

Vaccination with recoverin, a cancer-associated retinopathy antigen, induces autoimmune retinal dysfunction and tumor cell regression in mice.

Recoverin (Rec)-specific CTL present in peripheral blood recognize Rec-expressing tumor cells of patients with cancer-associated retinopathy (CAR), a paraneoplastic retinopathy syndrome. To evaluate the effects of Rec on retina and cancer cells, we generated an experimental mouse model, tested the induction of Rec-specific anti-tumor CTL, and analyzed retinal function using electroretinogram (ERG) in these animals. We observed a Rec-specific CTL response in BALB/c mice and significant growth inhibition of Rec-expressing syngeneic MethA fibrosarcoma cells in vivo. R64 (AYAQHVFRSF) peptide, derived from Rec that induces anti-tumor CTL in humans, produced anti-tumor effects in BALB/c mice. Furthermore, elevated anti-Rec antibodies correlated with decreased ERG amplitudes in Rec, Rec-expressing tumor and R64-treated mice. These data suggest that Rec contains amino acid sequences which cause retinal dysfunction, but they also induce anti-tumor CTL and tumor regression. These observations describe initial characterization of the CAR mouse model, a necessary step in developing new insight into immunological mechanisms of paraneoplastic syndromes and tumor immunity for potential immunotherapeutic approaches to cancer.

SU6668 inhibits Flk-1/KDR and PDGFRbeta in vivo, resulting in rapid apoptosis of tumor vasculature and tumor regression in mice.

SU6668 is a small molecule inhibitor of the angiogenic receptor tyrosine kinases Flk-1/KDR, PDGFRbeta, and FGFR1. In mice, SU6668 treatment resulted in regression or growth arrest of all large established human tumor xenografts examined associated with loss of tumor cellularity. The events underlying loss of tumor cellularity were elucidated in detail in several tumor models. SU6668 treatment induced apoptosis in tumor microvessels within 6 h of the initiation of treatment. Dose-dependent decreases in tumor microvessel density were observed within 3 days of the first treatment. These changes were accompanied by decreased tumor cell proliferation and increased tumor cell apoptosis. Rapid increases in VEGF transcript levels were seen, consistent with the induction of tumor hypoxia. Using Western blot analyses, we determined that these in vivo antiangiogenic and proapoptotic effects of SU6668 occur at doses comparable to those required to inhibit Flk-1/KDR and PDGFRbeta phosphorylation in tumors. Potent, dose-dependent inhibition of Flk-1/KDR activity in vivo was independently demonstrated using vascular permeability as a readout. These data demonstrate that SU6668-induced inhibition of angiogenic receptor tyrosine kinase activity in vivo is associated with rapid vessel killing in tumors, leading to broad and potent antitumor effects.

Mouse α-fetoprotein-specific DNA-based immunotherapy of hepatocellular carcinoma leads to tumor regression in mice

alpha-Fetoprotein (AFP) is a tumor-associated protein that is frequently expressed at high levels in hepatocellular carcinoma (HCC). The aim of the study was to characterize self-reactive cytotoxic T lymphocytes (CTLs) directed against murine AFP (mAFP) after DNA-based immunization in mice. To study CTL responses, mAFP-expressing recombinant vaccinia viruses were generated. An HCC tumor model was established in C57L/J mice by injection of syngeneic endogenously mAFP-expressing Hepa1-6 cells. Gene gun and intramuscular coimmunizations of DNA expression vectors encoding mAFP with plasmids encoding murine interleukin (IL)-12, granulocyte-macrophage colony-stimulating factor, or IL-18 induced weak CTL activity against mAFP in different mouse strains. Some mice developed anti-mAFP antibody responses, suggesting breaking of immunologic ignorance. No hepatocyte damage was detectable despite low-level endogenous hepatic mAFP expression. Therapeutic immunizations of mice bearing mAFP-expressing murine HCCs induced partial regression of tumors. A significant survival benefit was observed in mice immunized with mAFP expression vector DNA but not in untreated mice or in mice immunized with mock/cytokine plasmid DNA. The data show that AFP may be used as a potential self tumor antigen to induce CTL and CD4(+) T cell-mediated regression of AFP-expressing HCC by DNA-based immunization.

Mouse α-fetoprotein specific DNA-based immunotherapy of hepatocellular carcinoma to tumor regression in mice

Mouse α-fetoprotein specific DNA-based immunotherapy of hepatocellular carcinoma to tumor regression in mice

Upregulated Expression of Fibronectin Receptors Underlines the Adhesive Capability of Thymocytes to Thymic Epithelial Cells During the Early Stages of Differentiation: Lessons From Sublethally Irradiated Mice

AbstractA 250-cGy whole-body γ-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3−CD25hiCD44+ and CD3−CD25in/hiCD44−cells occurred. Enhanced expression of L-selectin, α4, and α5 integrin chains (L-selhi α4hiα5hi) was also exhibited by these cells. This pattern of expression was maintained until the CD4+CD8+ (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4+ or CD8+ single positive (SP) thymocytes (L-selin α4inα5in). In some recently generated SP cells, α4 expression was downregulated before the α5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the α6 integrin chain was downregulated only in maturing CD4+cells. Importantly, the increased expression of L-selectin and α4 and α5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the α4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for α5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the β1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25+ precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.

Upregulated Expression of Fibronectin Receptors Underlines the Adhesive Capability of Thymocytes to Thymic Epithelial Cells During the Early Stages of Differentiation: Lessons From Sublethally Irradiated Mice

A 250-cGy whole-body γ-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3−CD25hiCD44+ and CD3−CD25in/hiCD44−cells occurred. Enhanced expression of L-selectin, 4, and 5 integrin chains (L-selhi 4hi5hi) was also exhibited by these cells. This pattern of expression was maintained until the CD4+CD8+ (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4+ or CD8+ single positive (SP) thymocytes (L-selin 4in5in). In some recently generated SP cells, 4 expression was downregulated before the 5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the 6 integrin chain was downregulated only in maturing CD4+cells. Importantly, the increased expression of L-selectin and 4 and 5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the 4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for 5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the β1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25+ precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.

Tumor-specific killer cells in paraneoplastic cerebellar degeneration.

Models for immune-mediated tumor regression in mice have defined an essential role for cytotoxic T lymphocytes (CTLs); however, naturally occurring tumor immunity in humans is poorly understood. Patients with paraneoplastic cerebellar degeneration (PCD) provide an opportunity to explore the mechanisms underlying tumor immunity to breast and ovarian cancer. Although tumor immunity and autoimmune neuronal degeneration in PCD correlates with a specific antibody response to the tumor and brain antigen cdr2, this humoral response has not been shown to be pathogenic. Here we present evidence for a specific cellular immune response in PCD patients. We have detected expanded populations of MHC class I-restricted cdr2-specific CTLs in the blood of 3/3 HLA-A2.1+ PCD patients, providing the first description, to our knowledge, of tumor-specific CTLs using primary human cells in a simple recall assay. Cross-presentation of apoptotic cells by dendritic cells also led to a potent CTL response. These results indicate a model whereby immature dendritic cells that engulf apoptotic tumor cells can mature and migrate to draining lymph organs where they could induce a CTL response to tissue-restricted antigens. In PCD, peripheral activation of cdr2-specific CTLs is likely to contribute to the subsequent development of the autoimmune neuronal degeneration.

Transfectant Influenza A Viruses Are Effective Recombinant Immunogens in the Treatment of Experimental Cancer

Using reverse genetics methods, we constructed three different transfectant influenza A viruses encoding an Ld-restricted, nine amino-acid-long fragment, corresponding to amino-acid residues 876–884, of β-galactosidase (β-gal). Sequences encoding this epitope were nested within the hemagglutinin (HA) or neuraminidase (NA) open reading frames. Alternatively, an independent β-gal mini-gene, preceded by an endoplasmic reticulum insertion signal sequence, was placed in a bicistronic arrangement in the NA RNA segment of the virus. All three transfectants mediated the presentation of the epitope to a β-gal-specific CTL clone. Furthermore, each of the three transfectant viruses expressing the β-gal fragment elicited specific cytolytic responsesin vivo.Most importantly, these H1N1 transfectants mediated the regression of established murine pulmonary metastases. Tumor regression in mice was also achieved in the presence of preexisting immunity against an H3N2 influenza A virus serotype. Nononcogenic and nonintegrating, transfectant influenza A viruses are attractive candidates for development as antitumor vaccines.

Tumor regression in mice following vaccination with human papillomavirus E7 recombinant protein in PROVAX.

Induction of CD8+ cytotoxic T lymphocytes (CTLs) specific for human papillomavirus (HPV) antigens provides an attractive strategy for immunotherapy of HPV-related cancers in humans. In this study, we investigated the potential of utilizing soluble E7 protein of HPV 16 in an adjuvant formulation, PROVAX as a vaccine against a progressively growing E7 transfected K1735-X21 (H-2k) metastatic melanoma cells (HOPE2) in a mouse model. Vaccination of HOPE2 tumor bearing mice (C3H) with E7 protein in PROVAX resulted in significant inhibition of tumor growth, compared to mice vaccinated with E7 in Alum or saline. In vivo depletion of CD8+ or CD4+ cells indicated that CD8+ cells are the major effector cells in mediating the anti-tumor activity in this model. Furthermore, E7-specific CTL activity in vitro was detected in tumor bearing mice vaccinated with E7-PROVAX. Our studies suggest that recombinant HPV antigens in combination with PROVAX could serve as an effective subunit vaccine to stimulate tumor specific CD8+ T cell mediated immunity against HPV-related cancers.

Strategies to Enhance the Localization of Anticancer lmmunoconjugates

Abstract: Monoclonal antibodies (mAb) that bind to tumor-associated antigens have been used to selectively deliver toxic agents such as drugs, radionuclides, or protein toxins to tumors. A number of mAb conjugates have been effective in killing tumor cells in vitro and in causing tumor regressions in mice; but as yet have not been curative against solid tumors in humans. One of the problems that has been identified for mAb tumor targeting is that systemic clearance tends to be slow, resulting in low tumor-to-blood ratios even many days after administration. Many strategies have been explored to accelerate mAb conjugate clearance without affecting intratumoral uptake. This can be accomplished by extracorporeal adsorption or plasmapheresis to remove the conjugate from the blood, or by complexing the conjugate with an agent that is cleared very quickly from the blood. Such agents include carbohydrates, avidin, and mAbs directed against the immunoconjugate. Each of these strategies have been employed to enhance the tumor-to- blood ratio of immunoconjugates.

Efficacy of Liposomal Muramyl Tripeptide (CGP 19835A) in the Treatment of Relapsed Osteosarcoma

Muramyl tripeptide phosphatidylethanolamine (MTP-PE) is a synthesized lipophilic analogue of muramyl dipeptide. MTP-PE encapsulated in liposomes (L-MTP-PE) allows selective delivery to pulmonary macrophages and circulating monocytes. In vivo administration has resulted in tumor regression in mice with B16 melanoma lung and lymph node metastases and a 40% long-term disease-free survival in dogs with osteosarcoma. Phase I studies have demonstrated that the drug is well tolerated. A Phase II trial using L-MTP-PE was undertaken in relapsed osteosarcoma patients to determine whether L-MTP-PE therapy could improve the progression-free interval in this high-risk group of patients. Patients had histologically proven osteosarcoma and pulmonary metastases that had developed during adjuvant chemotherapy or that were present at diagnosis and had persisted despite chemotherapy. Patients were rendered disease free by surgery. L-MTP-PE, 2 mg/m2, was infused over a 1-hour period twice a week for 12 weeks in 12 patients (Group 1). Sixteen patients (Group 2) received 2 mg/m2 L-MTP-PE twice a week for 12 weeks, then once a week for 12 weeks, for a total of 24 weeks of therapy. Progression-free intervals in each group were calculated from the day of surgery to the day of relapse and compared with the progression-free interval of a historical control group (Group 3) treated postoperatively with chemotherapy at M. D. Anderson Cancer Center between 1980 and 1990. Patients who received 24 weeks of L-MTP-PE therapy had a significant prolongation in time to relapse but those who received 12 weeks of therapy did not. The median time to relapse for group 2 patients was 9.0 months compared with 4.5 months for the control group (Group 3). These data suggest that L-MTP-PE deserves further investigation in a more appropriate adjuvant setting. A nationwide randomized Phase III trial is now underway in newly diagnosed osteosarcoma patients in conjunction with the Children's Cancer Study Group and the Pediatric Oncology Group.

Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interleukin-2 receptor-expressing human carcinoma

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin composed of the variable domains of the monoclonal antibody anti-Tac, which binds to the p55 subunit of the interleukin-2 receptor (IL-2R), and a truncated form of Pseudomonas exotoxin (PE), which does not bind to the PE receptor (Chaudhary et al, Nature 339:394, 1989). Whereas its cytotoxic activity toward autoimmune and malignant target cells has been established, its efficacy in vivo remains unknown. To establish an animal model, we produced ATAC-4 cells by transfecting the gene encoding the low-affinity IL-2R (p55) into A431 epidermoid carcinoma cells. ATAC-4 cells contained low-affinity IL-2Rs (2 x 10(5)/cell) and formed tumors in nude mice. In tissue culture, protein synthesis in ATAC-4 cells was inhibited 50% (IC50) at 0.06 ng/mL (0.9 pmol/L) of anti-Tac(Fv)-PE40. IC50s for the derivatives anti-Tac(Fv)-PE38, which is missing PE amino acids 365–380, and anti-Tac(Fv)-PE38KDEL, which contains the same deletion plus the KDEL carboxyl terminus, were 0.04 and 0.025 ng/mL, respectively. All the agents produced complete tumor regressions in ATAC-4 tumor-bearing mice and anti-Tac(Fv)-PE38KDEL had significant antitumor activity at 1% of the LD50. The dose limiting toxicity of anti-Tac(Fv)-PE38KDEL was from hemorrhagic liver necrosis, which was observed at approximately 55% of the LD50.

Recombinant Immunotoxins Containing Anti-Tac(Fv) and Derivatives of Pseudomonas Exotoxin Produce Complete Regression in Mice of an Interleukin-2 Receptor-Expressing Human Carcinoma

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin composed of the variable domains of the monoclonal antibody anti-Tac, which binds to the p55 subunit of the interleukin-2 receptor (IL-2R), and a truncated form of Pseudomonas exotoxin (PE), which does not bind to the PE receptor (Chaudhary et al, Nature 339:394, 1989). Whereas its cytotoxic activity toward autoimmune and malignant target cells has been established, its efficacy in vivo remains unknown. To establish an animal model, we produced ATAC-4 cells by transfecting the gene encoding the low-affinity IL-2R (p55) into A431 epidermoid carcinoma cells. ATAC-4 cells contained low-affinity IL-2Rs (2 x 10(5)/cell) and formed tumors in nude mice. In tissue culture, protein synthesis in ATAC-4 cells was inhibited 50% (IC50) at 0.06 ng/mL (0.9 pmol/L) of anti-Tac(Fv)-PE40. IC50s for the derivatives anti-Tac(Fv)-PE38, which is missing PE amino acids 365-380, and anti-Tac(Fv)-PE38KDEL, which contains the same deletion plus the KDEL carboxyl terminus, were 0.04 and 0.025 ng/mL, respectively. All the agents produced complete tumor regressions in ATAC-4 tumor-bearing mice and anti-Tac(Fv)-PE38KDEL had significant antitumor activity at 1% of the LD50. The dose limiting toxicity of anti-Tac(Fv)-PE38KDEL was from hemorrhagic liver necrosis, which was observed at approximately 55% of the LD50.

Combined effects of interferon alpha and interleukin 2 on the induction of a vascular leak syndrome in mice.

Immunotherapy with interleukin 2 (IL-2) alone or in combination with lymphokine-activated killer cells can mediate tumor regression in mice and in man. Further dose escalation of IL-2 along with lymphokine-activated killer cells has been prevented by the development of a vascular leak syndrome produced by IL-2. Because we have found that interferon alpha (IFN-alpha) or tumor necrosis factor (TNF-alpha) has synergistic antitumor effects when administered together with IL-2, we have tested the vascular leakage induced by these lymphokine combinations. We used a murine model to quantify vascular leakage by measuring the extravasation of 125I-albumin from the intravascular space as well as the wet and dry lung weights after treatment with different cytokines. Cytokines (or Hanks balanced salt solution) were administered to C57BL/6 mice and 4 h after the last injection the vascular leak was quantified. IFN-alpha alone did not cause extravasation of radiolabel or increase in wet lung weights, though when given in combination with IL-2, significantly greater extravasation (P less than 0.01) as well as increase in lung water weights (P less than 0.05) was observed compared to the response in mice treated with IL-2 alone. IFN-alpha in combination with IL-2 induced significant vascular leakage earlier than the response induced by IL-2 alone. For example treatment with IFN-alpha and IL-2 induced accumulation of 14,674 +/- 605 cpm in the lungs at day 1 while IL-2 alone induced 12,340 +/- 251 cpm. The degree of vascular leakage was highly related to the dose of IFN-alpha administered along with IL-2 and increased vascular leak syndrome was evident even at low doses (5000 units) of IFN-alpha. Immunosuppression of mice by pretreatment irradiation (500 rad) markedly decreased the development of vascular leak syndrome induced by IL-2 and IFN-alpha. Interestingly IFN-gamma and TNF-alpha did not induce vascular leakage in the lungs when given alone, and did not add or synergize with IL-2 in causing the syndrome. Thus the administration of IFN-alpha in combination with IL-2 produces a dose-limiting vascular leakage that is more severe than that caused by IL-2 alone, and may be mediated, directly or indirectly by host radiosensitive cells.