Immunotoxin Therapy Research Articles

CD3 immunotoxin therapy of cutaneous T cell lymphoma

e19511 Background: T cell lymphomas represent 12% of lymphoma cases in the U.S. Cytotoxic chemotherapies, radiation therapies, monoclonal antibodies, transcription modulators and topical therapies yield remissions, but over half of patients relapse and die with progressive disease. Patients ineligible for allogeneic transplant need additional therapeutic options. One class of T cell directed agents are immunotoxins composed of protein synthesis inactivating peptide toxins covalently linked to antibodies or hormone ligands. We prepared a new immunotoxin, A-dmDT390-bisFv(UCHT1) composed of the catalytic and translocation domains of diphtheria toxin fused to two single chain antibody fragments reactive with an acidic loop on the extracellular domain of CD3epsilon. We prepared a clinical batch of drug and obtained FDA approval for a phase I trial (IND#100712). Methods: Cohort of three-six CTCL patients were treated with immunotoxin via 15min IV infusion twice daily for four days at 2.5–5ug/kg. CTCAEv3.0 toxicity grading, blood samples counts, chemistries, CMV/EBV PCR, PK, immune response and flow cytometry, and disease assessments by CT scans, skin mapping and bone marrow biopsies were done. Results: Six patients received all eight 2.5ug/kg doses. Toxicities were mild- moderate with fever, chills, nausea, transaminasemia, hypoalbuminemia, lymphopenia and reactivation of EBV and CMV. Side effects responded to antipyretics, anti-emetics, albumin infusions, rituximab and valgancyclovir. Lymphopenia was marked at the end of treatment (99.9% reduction) and was followed by partial recovery at two weeks. Circulating T regulatory cells doubled. Cmax occured 5 min post-infusion and was 18ng/mL. Half-life was 49 min. All patient had anti-immunotoxin antibodies at a median of 1.3ug/mL which increased after 30 days to 9–1700ug/mL. Two of five evaluable patients had PRs lasting one and 6+ months. Conclusions: This novel immunotoxin has dramatic clinical activity even at the lowest dose in CTCL patients and merits applications at higher doses in CTCL and other CD3+ T cell leukemia/lymphoma patients. The lymphodepletion with recovery of T regulatory cells suggests the drug may be beneficial for immunosuppression of T cell autoimmune disorders. No significant financial relationships to disclose.

Intravesical Mycobacterial Cell Wall-DNA Complex in the Treatment of Carcinoma In Situ of the Bladder After Standard Intravesical Therapy Has Failed

We assessed the clinical efficacy and safety of mycobacterial cell wall-DNA complex after intravesical administration in patients with carcinoma in situ in whom prior therapy with bacillus Calmette-Guerin failed or in those who were treatment naïve. Patients received 6 weekly instillations of 4 or 8 mg mycobacterial cell wall-DNA complex (formulated as an emulsion) followed by 3 weekly instillations at weeks 12 and 24. Efficacy and safety were evaluated throughout the treatment phase and at months 12 and 18. A total of 55 patients (mean age 74 years, 74.6% male) received 4 mg (25) or 8 mg (30) mycobacterial cell wall-DNA complex emulsion. All patients were previously treated with bacillus Calmette-Guerin except for 8 who were treatment naïve and 2 who received chemotherapy. In the intent to treat population the complete response rate was 27.3% at weeks 12 and 26 in the 4 mg group while 46.4% of patients receiving 8 mg had a complete response at both points. Mycobacterial cell wall-DNA complex was well tolerated by both dose groups. Overall 90% of all adverse events were mild to moderate in severity. Mycobacterial cell wall-DNA complex has shown antineoplastic activity in patients with bladder cancer with less toxicity than that associated with bacillus Calmette-Guerin administration. The tolerance and efficacy of mycobacterial cell wall-DNA complex might hold promise for the treatment of carcinoma in situ of the bladder.

Site-directed mutagenesis at amino terminus of recombinant ricin A chain

Successful immunotoxin therapy may depend upon reduction of the size of the components in order to decrease antigenicity and rate of clearance. In initial attempts to modify the A chain of ricin by deletion analyses within a prokaryotic expression system, coding sequences were modified by the insertion of unique restriction endonuclease sites and by the removal of 22 codons near the 5' terminus. The work presented here examines the expression, solubility, and activity of these mutant proteins and demonstrates that while amino acid residues may be altered in this region, the deletion of residues 19 through 40 yields an insoluble and inactive toxin molecule.

Back-burning to cure HIV: Temporary depletion of all CD4 + cells and elimination of the extracellular reservoir with HIV Immunotoxin Therapy

Temporary elimination of all host cells for the human immunodeficiency virus (HIV) combined with dislodging HIV from its extracellular reservoir could cure acquired immunodeficiency syndrome (AIDS). This combination would be effective because the virus is dependent on host cell integration or on the membrane protection of B cells or of follicular dendritic cells (FDCs) for its survival and because the CD4(+) host cells are leukocytes that are naturally renewable through hematopoiesis. By treating HIV patients with a combination of humanized antibodies it should be possible to achieve both goals. To deplete HIV host cells, a humanized antibody against CD4 should be fused to an apoptosis-inducing toxin; and to void the extracellular reservoir, a fragment of a humanized antibody against CD21 should be used. Because only CD4(+) cells would be destroyed, hematopoietic stem cells would be spared, and would spontaneously replace the depleted cells. We call this hypothetical new HIV treatment "HIV Immunotoxin Therapy (HIT)". Once the HIV viral load reaches zero, the HIT would be withdrawn and IL-2 or luteinizing hormone releasing hormone analogues (LHRH-A) might be administered to accelerate the natural replacement of the CD4(+) T(H) cells and macrophages. Killing all HIV host cells may seem counterintuitive at first, because it requires the purposeful destruction of the very cells that we ultimately hope to preserve for AIDS patients, but just as controlled back-burning purposefully creates a trap to stop a wildfire from burning out of control, this method could provide a mechanism to extinguish HIV.

Non-Clinical Pharmacology of Anti-CD22 Antibody Drug Conjugates.

CD22 is a B-cell specific antigen expressed on both normal and malignant cells. It is internalized rapidly upon binding to its antibody, making it an ideal target for immunotoxin therapy. Anti-CD22 is a humanized monoclonal antibody that recognizes human and cynomolgus monkey CD22 but not the rodent CD22. When conjugated with mitotic inhibitors such as DM1 (a maytansinoid) or monomethylauristatin F (MMAF) as anti-CD22-MCC-DM1 or anti-CD22-MC-MMAF, the conjugates have been shown to regress tumors completely after a single IV bolus in a variety of xenograft mouse models. Pharmacokinetics (PK), pharmacodynamics (PD), efficacy and safety studies were conducted to explore the non-clinical pharmacology of the two anti-CD22 conjugates (ADC). Dose response of anti-tumor activity, characterized in the Bjab-Luc xenograft model, yielded an ED50 of ∼1 mg/kg for MC-MMAF. MCC-DM1 was about 2-fold less potent compared to MC-MMAF in this model. Anti-CD22 antibody demonstrated very limited antibody dependent cell mediated cytotoxicity and no complement dependent cytotoxicity in vitro. Antigen-independent pharmacokinetics were assessed in mouse (single IV bolus, 0.5 or 5 mg/kg) and rat (single IV bolus, 10 mg/kg), while the effect of target mediated disposition was studied in cynomolgus monkey (two IV bolus with three weeks apart, vehicle, 10, 20 or 30 mg/kg). Conjugation with DM1 or MMAF appeared to have minimal impact on the PK of anti-CD22 when the PK of naked anti-CD22 was compared with the total antibody PK profile in all three species. Pharmacokinetic parameters of both total antibody and ADCs were generally dose proportional at the dose range studied. Free DM1 in plasma increased with dose, while free MMAF was not detected. Safety and PD were also characterized in the cynomolgus monkey. Both ADCs were well tolerated up to 30 mg/kg through out the entire study period (42 days). For PD, B-cell depletion was analyzed via FACS. Substantial decreases of CD20+ B-cells were observed in peripheral blood at all dose levels. In tissues, reduction of CD20+ B-cells was observed in spleen, bone marrow and in the lymph nodes, although to a lesser extent. No anti-drug antibodies were detected in the cynomolgus monkey at the doses tested. Based on the preclinical data, human efficacious dose and therapeutic window were estimated to be ∼2–5 mg/kg and 5–10, respectively. In summary, both ADCs exhibit an encouraging nonclinical efficacy, pharmacokinetic, pharmacodynamic, and safety profile that supports clinical development for the potential treatment of NHL.

Expression, purification, and characterization of an immunotoxin containing a humanized anti-CD25 single-chain fragment variable antibody fused to a modified truncated Pseudomonas exotoxin A

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In the present study, we have constructed and expressed a recombinant immunotoxin CD25-PE38KDELKQK containing humanized anti-CD25 single-chain antibody (scFv) genetically fused to PE38KDELKQK in Escherichia coli. After washing with buffer containing 2 M urea, the purity of inclusion body was about 82%. The denatured inclusion bodies were refolded on-column in Tris buffer (pH 8.0) containing 4 mM of GSH and 1 mM of GSSG using a gradient of decreasing urea. We found that the presence of GSH/GSSG (4:1) in the on-column refolding buffer was important for efficient refolding. In addition, slow flow rate was another important factor could increase refolding. Under these conditions, the activity of the refolded protein could reach about 90% of that of the native protein. The refolded proteins were purified to homogeneity (∼95% purity) by a combination of His-Ni 2+ metal affinity chromatography and gel filtration chromatography. The in vitro cytotoxicity assay indicated the purified immunotoxin CD25-PE38KDELKQK had specific cytotoxicity to CD25-positive leukemic cells comparable to wild-type CD25-PE38KDEL (wt). In contrast, CD25-PE38KDELKQK was shown to be much weaker in inducing VLS in mice than wt. The protein expression, purification, and refolding system established in this paper is important for further study on immunotoxin CD25-PE38KDELKQK.

Treatment of hepatocellular carcinoma in a mouse xenograft model with an immunotoxin which is engineered to eliminate vascular leak syndrome.

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin and interleukin-2 therapy. It has been evidenced that VLS-inducing molecules share a three-amino acid consensus motif, (x)D(y), which may be responsible for initiating VLS. Here we have constructed a recombinant immunotoxin (SMFv-PE38KDEL) by genetically fusing PE38KDEL to a single-chain antibody derived from SM5-1 monoclonal antibody, which has a high specificity for melanoma, hepatocellular carcinoma and breast cancer. In order to eliminate VLS induced by this PE38KDEL-based immunotoxin, a panel of mutants were generated by changing amino acid residues adjacent to its three (x)D(y) motifs in the three-dimensional structure. One of the SMFv-PE38KDEL mutants, denoted as mut1, displayed a similar protein synthesis inhibitory in a reticulocyte lysate translation assay compared to the wild-type SMFv-PE38KDEL (wt). The in vitro cytotoxicity assay indicated that mut1 specifically killed SM5-1 binding protein-positive tumor cells, although its cytotoxicity was slightly less than wt. In contrast, mut1 was shown to be much weaker in inducing VLS in mice than wt. The LD(50) values of wt and mut1 in mice were investigated with the result that the LD(50) of mut1 was about tenfold higher than that of wt. The in vivo antitumor activity of wt and mut1 were also compared in tumor-bearing nude mice. Both wt and mut1 were effective in inhibiting the tumor growth but mut1 showed improved therapeutic efficacy. These studies suggest mut1 may be a novel PE-based immunotoxin with much less toxicity for clinical use.

Expression and structure of interleukin 4 receptor (IL-4R) complex in human invasive pituitary adenomas

Pituitary adenomas are frequently invasive of surrounding tissues, which adversely affects the surgical outcome and the disease-free survival of patients. In the present study, Interleukin 4 receptor (IL-4R) complex has been investigated to figure out whether the three subunits are overexpressed in human invasive pituitary adenomas. Reverse transcription-polymerase chain reaction (RT-PCR) analysis for interleukin 4 receptor α (IL-4Rα), interleukin 13 receptor α1 (IL-13Rα1), interleukin 2 receptor γc (IL-2Rγc) were performed on total RNA extracted from 10 non-invasive pituitary adenomas, 30 invasive pituitary adenomas, one glioblastoma multiforme, one normal human pituitary tissue sample and one normal human brain tissue sample. Quantitative real-time PCR and in situ immunofluorescence assay were performed in five invasive functioning pituitary adenoma samples and five invasive nonfunctioning pituitary adenoma samples. RT-PCR analysis for IL-4Rα, IL-13Rα1 and IL-2Rγc chains were overexpressed in invasive pituitary adenomas. The transcripts for three subunits were not/weakly expressed in normal pituitary tissue and normal brain tissue. The quantitative real-time PCR and in situ immunofluorescence assay confirmed the results of the RT-PCR analysis. Our results indicate that human invasive pituitary adenomas express type III IL-4R complex. These receptors may serve as a novel target for immunotoxin therapy in patients with invasive pituitary adenomas who are not amenable to total surgical resection or for recurrent cases.

Characterization of the B Cell Epitopes Associated with a Truncated Form of Pseudomonas Exotoxin (PE38) Used to Make Immunotoxins for the Treatment of Cancer Patients

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

Eradication of Tumor Colonization and Invasion by a B Cell–Specific Immunotoxin in a Murine Model for Human Primary Intraocular Lymphoma

Human primary intraocular lymphoma (PIOL) is predominantly a B cell-originated malignant disease with no appropriate animal models and effective therapies available. This study aimed to establish a mouse model to closely mimic human B-cell PIOL and to test the therapeutic potential of a recently developed immunotoxin targeting human B-cell lymphomas. Human B-cell lymphoma cells were intravitreally injected into severe combined immunodeficient mice. The resemblance of this tumor model to human PIOL was examined by fundoscopy, histopathology, immunohistochemistry, and evaluated for molecular markers. The therapeutic effectiveness of immunotoxin HA22 was tested by injecting the drug intravitreally. Results showed that the murine model resembles human PIOL closely. Pathologic examination revealed that the tumor cells initially colonized on the retinal surface, followed by infiltrating through the retinal layers, expanding preferentially in the subretinal space, and eventually penetrating through the retinal pigment epithelium into the choroid. Several putative molecular markers for human PIOL were expressed in vivo in this model. Tumor metastasis into the central nervous system was also observed. A single intravitreal injection of immunotoxin HA22 after the establishment of the PIOL resulted in complete regression of the tumor. This is the first report of a murine model that closely mimics human B-cell PIOL. This model may be a valuable tool in understanding the molecular pathogenesis of human PIOL and for the evaluation of new therapeutic approaches. The results of B cell-specific immunotoxin therapy may have clinical implications in treating human PIOL.

Immunotoxin therapy of cancer

Rationally designed anticancer agents that target cell-surface antigens or receptors represent a promising approach for treating cancer patients. However, antibodies that bind these targets are often, by themselves, non-cytotoxic. By attaching potent toxins we can dramatically improve the clinical utility of some anti-tumour antibodies. Here we describe the construction and clinical utility of several recombinant immunotoxins; each of which is composed of antibody Fv fragments fused to powerful bacterial toxins. Results from clinical trials indicate that recombinant immunotoxins and similar agents that are designed to combine antibody selectivity with toxin cell-killing potency will be useful additions to cancer therapy.

IL-2 Immunotoxin Therapy Modulates Tumor-Associated Regulatory T Cells and Leads to Lasting Immune-Mediated Rejection of Breast Cancers in neu-Transgenic Mice

Studies in cancer patients have suggested that breast tumors recruit regulatory T cells (Tregs) into the tumor microenvironment. The extent to which local Tregs suppress antitumor immunity in breast cancer is unknown. We questioned whether inhibiting systemic Tregs with an IL-2 immunotoxin in a model of neu-mediated breast cancer, the neu-transgenic mouse, could impact disease progression and survival. As in human breast cancer, cancers that develop in these mice attract Tregs into the tumor microenvironment to levels of approximately 10-25% of the total CD4+ T cells. To examine the role of Tregs in blocking immune-mediated rejection of tumor, we depleted CD4+CD25+ T cells with an IL-2 immunotoxin. The treatment depleted Tregs without concomitant lymphopenia and markedly inhibited tumor growth. Depletion of Tregs resulted in a persistent antitumor response that was maintained over a month after the last treatment. The clinical response was immune-mediated because adoptive transfer of Tregs led to a complete abrogation of the therapeutic effects of immunotoxin treatment. Further, Treg down-modulation was accompanied by increased Ag-specific immunity against the neu protein, a self Ag. These results suggest that Tregs play a major role in preventing an effective endogenous immune response against breast cancer and that depletion of Tregs, without any additional immunotherapy, may mediate a significant antitumor response.

Multiepitope HER2 Targeting Enhances Photoimmunotherapy of HER2-Overexpressing Cancer Cells with Pyropheophorbide-a Immunoconjugates

Multi-targeting strategies improve the efficacy of antibody and immunotoxin therapies but have not yet been thoroughly explored for HER2-based cancer treatments. We investigated multi-epitope HER2 targeting to boost photosensitizer immunoconjugate uptake as a way of enhancing photoimmunotherapy. Photoimmunotherapy may allow targeted photodynamic destruction of malignancies and may also potentiate anticancer antibodies. However, one obstacle preventing its clinical use is the delivery of enough photosensitizer immunoconjugates to target cells. Anti-HER2 photosensitizer immunoconjugates were constructed from two monoclonal antibodies (mAb), HER50 and HER66, using a novel method originally developed to label photosensitizer immunoconjugates with the photosensitizer, benzoporphyrin derivative verteporfin. Photosensitizer immunoconjugates were labeled instead with a promising alternative photosensitizer, pyropheophorbide-a (PPa), which required only minor changes to the conjugation procedure. Uptake and phototoxicity experiments using human cancer cells were conducted with the photosensitizer immunoconjugates and, for comparison, with free PPa. SK-BR-3 and SK-OV-3 cells served as HER2-overexpressing target cells. MDA-MB-468 cells served as HER2-nonexpressing control cells. Photosensitizer immunoconjugates with PPa/mAb molar ratios up to approximately 10 specifically targeted and photodynamically killed HER2-overexpressing cells. On a per mole basis, photosensitizer immunoconjugates were less phototoxic than free PPa, but photosensitizer immunoconjugates were selective for target cells whereas free PPa was not. Multiepitope targeted photoimmunotherapy with a HER50 and HER66 photosensitizer immunoconjugate mixture was significantly more effective than single-epitope targeted photoimmunotherapy with a single anti-HER2 photosensitizer immunoconjugate, provided photosensitizer immunoconjugate binding was saturated. This study shows that multiepitope targeting enhances HER2-targeted photoimmunotherapy and maintains a high degree of specificity. Consequently, it seems that multitargeted photoimmunotherapy should also be useful against cancers that overexpress other receptors.

Clinical Evaluation of BR96 sFv-PE40 Immunotoxin Therapy in Canine Models of Spontaneously Occurring Invasive Carcinoma

The immunotoxin BR96 sFv-PE40 is an effective antitumor agent against human breast and lung carcinoma xenografts in rodents. This study was designed to (a) determine the frequency with which canine carcinoma cells express Lewis(y) (Le(y)) antigen, thereby identifying canine carcinoma types suitable for the clinical evaluation of BR96 sFv-PE40, and (b) determine the safety and efficacy of BR96 sFv-PE40 in a canine model of spontaneously occurring cancers for investigation of targeted therapy. Carcinoma tissue samples were obtained from client-owned dogs presented for medical care. The tissues were assessed for Le(y) antigen expression using immunohistochemical methods. Dogs with tumors expressing Le(y) antigen were offered enrollment in a clinical trial to receive twice-weekly infusions of 4 to 12 mg/m(2) BR96 sFv-PE40. Clinical toxicity and response data were assessed at each treatment. Twenty-two of 61 carcinomas evaluated were positive for Le(y) expression, including mammary, prostate, lung, and rectal carcinomas, and 12 dogs were enrolled in the clinical trial. The primary side effect was transient emesis. Partial responses or disease stabilization were noted in dogs with inflammatory mammary, bronchogenic, rectal, and tonsillar carcinoma. At least nine of the dogs developed antibodies to the immunotoxin after two to five infusions. Although development of anti-BR96 sFv-PE40 antibodies limited the long-term effectiveness of this immunotoxin in dogs, rapid clinical responses in several aggressive canine carcinomas suggest the immunotoxin has utility for treatment of certain naturally occurring tumors and that its clinical evaluation for treatment of similar human carcinomas is warranted.

Immunotoxin therapy of hematologic malignancies

Patients with chemotherapy relapsed or refractory hematologic malignancies may be effectively treated with allogeneic or autologous stem cell transplants. However, many patients cannot be transplanted due to age, comorbidities, or lack of suitable donors. Further, a fraction of patients relapse post-transplant. Novel therapeutic agents that can kill multidrug-resistant malignant stem cells and are not myelosuppressive are needed. One class of such agents is immunotoxins. Immunotoxins consist of cell-selective ligands covalently linked to peptide toxins. The ligand delivers the molecule to specific cell surface receptors on malignant cells. The toxin triggers cell death either by reaching the cytosol and catalytically inactivating vital cell processes or by modifying the tumor cell surface membrane. We have synthesized immunotoxins for therapy of chemoresistant hematologic diseases. In this review, we will detail the synthesis of a number of these drugs and describe their preclinical and clinical activity. Several of these agents have shown dramatic antitumor effects in patients with hematologic neoplasms, and one immunotoxin has been approved for use by the US Food and Drug Administration (FDA). Over the next several decades, a growing number of these agents should reach the clinic.

Immunotoxin therapy for CNS tumor.

Surgery, chemotherapy, and radiation therapy have become standard of practice in treating malignant brain tumors. Unfortunately, the prognosis of these malignant tumors still remains poor. Immunotoxins are a relatively new adjuvant treatment for brain tumors. Within the last few years an increased amount of clinically-oriented research involving immunotoxins has been published. This has led to numerous clinical trials which although encouraging have not yet born out the "magic bullet" concept envisioned for immunotoxins. In this review article the history, design, toxicity, and pharmokinetics of immunotoxins will be discussed in detail.

Immunotoxin therapy for primary malignant brain tumors

The prognosis of malignant brain tumors is poor in spite of aggressive treatment. Immunotoxin therapy is a novel approach for the treatment of tumors. Targeted fusion toxins, chimeric protein consisting of the cytotoxic domain of the natural toxin and carrier ligands such as a growth factor or an antibody, have been introduced in the treatment of malignant central nervous system (CNS) tumors, with promising results. The toxin is internalized into the cytosol of the target cell via cell-surface receptors and it is essential for these receptors on the tumor cell to be highly expressed in order for the immunotoxin to have specific anti-tumor activity. Studies on expression of cell-surface receptor for immunotoxin targeting was performed on transferring (TR), insulin-like growth factor-1 (IGF-1R), and interleukin-4 (IL-4R) receptors of human glioblastoma (U373-MG and T98-G) and medulloblastoma (Daoy) cell lines, and the effect of irradiation on expressibility of these receptors was studied. Recombinant diphtheria toxin–murine interleukin-4 conjugate (DT 390–mIL4) was developed and its cytotoxic efficacy against murine glioblastoma (SMA-560) and neuroblastoma (Neuro-2a and NB41-A3) cell lines was examined, and the combined effect with irradiation was tested. Receptors were expressed in all cases except IGF-1R on T98-G. After irradiation, TR expression was increased for Daoy, IGF-1R expression was increased on Daoy and U373-MG, and IL-4R expression was decreased on Daoy and U373-MG. DT 390–mIL4 exhibited dose-dependent, specific cytotoxic effects on all cell lines tested with IC 50 (concentration of DT 390–mIL4 that inhibit 50% of protein synthesis) value of 0.56×10 −9 M in SMA-560, 1.28×10 −9 M in Neuro-2a and 0.95×10 −10 M in NB41-A3. Cytotoxicity was additive when DT 390–mIL4 at 10 −9 M was administered with irradiation. Targeted fusion toxins are characterized by great potency and high specificity with minimal damage to normal neuronal tissue, and interleukin-4 receptor is one of the proper targets for fusion toxin. The cytotoxicity of the immunotoxin is additive when it is administrated with irradiation. Targeted toxin therapy is a promising adjuvant treatment modality for the malignant brain tumors.

Augmentation of the Activity of an Immunotoxin, Anti-Tac(Fv)-PE40KDEL, in T Cell Lines Infected with Human T Cell Leukemia Virus Type-I

Therapy with an immunotoxin, anti-Tac(Fv)-PE38, which is a conjugate of the variable domains of an anti-Tac monoclonal antibody and Pseudomonas exotoxin, was reported to be useful for adult T cell leukemia (ATL) patients but a considerable amount of the immunotoxin is needed for the therapy and some side effects were also observed. We have previously demonstrated that an immunotoxin, anti-Tac(Fv)-PE40KDEL, showed strong cytotoxic effects on malignant cells from ATL patients. Therefore, we searched for agents that enhance the effects of the immunotoxin. PAK-200, FK-506, quinidine, cepharanthine and cyclosporine A (CsA) augmented the ability of the immunotoxin to inhibit protein synthesis in two human T cell leukemia virus type-I infected T cell lines, KUT-1 and KUT-2. CsA was the most potent agent in both the cell lines. Augmentation of the cytotoxic effect of the immunotoxin by these agents, especially CsA, may be useful in the immunotoxin therapy of ATL.

Synthesis and characterization of membrane-active GALA-OKT9 conjugates.

Cellular processing of immunotoxins is inefficient, limiting the overall effectiveness of current immunotoxin therapies. Specifically, translocation of ribosome-inactivating toxins across intracellular membranes is agonizingly slow. In one strategy to improve immunotoxin efficacy, membrane-active peptides are attached to immunotoxins to facilitate transfer of the toxic moiety across a cellular membrane to the cytosol. pH-sensitive peptides are of particular interest, as the membrane activity can be localized to the endosomal/lysosomal pathway, reducing nonspecific interactions at the cell surface. In this study, GALA, a pH-sensitive peptide that forms multimeric pores in membranes, was chemically attached to OKT9, an anti-transferrin receptor mAb. Conjugates were tested by measuring release of encapsulated dyes from liposomes to determine the extent to which the membrane-lytic properties of GALA were retained. The most significant feature affecting the lytic properties of GALA-OKT9 conjugates was the number of attached GALA per OKT9. Conjugates with a single GALA per OKT9 caused almost no leakage while conjugates with two or three GALA per OKT9 caused significant leakage in a concentration-dependent manner. Invariably, GALA-OKT9 conjugates were significantly less active than unconjugated GALA, attributable to a decrease both in partitioning and in surface aggregation. No improvement in membrane-lytic activity was achieved by using a longer, more flexible poly(ethylene glycol) cross-linker. Attachment of GALA via C- versus N-terminal linkage had no effect on membrane-lytic properties. Size-selective release of high molecular weight dextrans was almost identical for conjugated and unconjugated GALA, suggesting that GALA forms the same pore structure regardless of conjugation state.

Therapy of human T-cell acute lymphoblastic leukaemia with a combination of anti-CD7 and anti-CD38-SAPORIN immunotoxins is significantly better than therapy with each individual immunotoxin.

Severe combined immunodeficient (SCID) mice injected i.v. with the human T-ALL cell line CCRF CEM (SCID-CEM mice) develop within 50 days life-threatening multi-organ growth of leukaemia cells. The development of leukaemia in SCID-CEM mice treated with three 10 μg i.v. doses of the anti-CD7 immunotoxin (IT) HB2-SAPORIN or the anti-CD38 IT OKT10-SAPORIN was significantly delayed compared with PBS sham-treated animals but 90% of animals treated with either IT eventually developed disseminated leukaemia cell growth. In contrast treatment of SCID-CEM mice with a combination of both ITs led not only to a significantly greater delay in time to leukaemia development but also in the numbers of animals remaining leukaemia free (60%). The native HB2 and OKT10 antibodies (both murine IgG 1 antibodies) exerted significant, though relatively weak therapeutic effects, probably mediated through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. Moreover, there was no in vivo additivity of therapeutic effect when both antibodies were used in combination. Apparent, however, was that the combination of HB2-SAPORIN IT with OKT10 antibody led to an intermediate therapeutic effect that was significantly greater than that obtained when either was used alone but significantly less than that obtained when the two IT combination was utilized. This was similarly the case for the combination of OKT10-SAPORIN IT with HB2 antibody though the effect was less pronounced in this instance. This result suggests that the therapeutic effect of IT + antibody treatment results from an additivity between antibody-mediated delivery of saporin combined with a SCID mouse NK cell-mediated ADCC attack on the target cell directed through target cell bound antibody Fc engagement with FcγRIII on the NK cell surface. The combination of both ITs however gave the best therapeutic outcome in SCID-CEM mice probably as the result of (i) delivery of greater amounts of saporin to target CEM cells positive for both CD7 and CD38, (ii) delivery of an effective dose of saporin to CEM cells downregulated or negative for one of the target antigens and (iii) through ADCC mechanisms that interact additively with IT action. We have previously proposed that combination IT therapy would be one means of overcoming the problem of heterogeneity of antigen expression within a global tumour cell population and these additional findings support this and provide a further strengthening of the rationale for employing cocktails of ITs for the treatment of human malignancies. © 2001 Cancer Research Campaign http://www.bjcancer.com