Resimmune, an anti-CD3ε recombinant immunotoxin, induces durable remissions in patients with cutaneous T-cell lymphoma.

Resimmune is a second-generation recombinant immunotoxin composed of the catalytic and translocation domains of diphtheria toxin fused to two single chain antibody fragments reactive with the extracellular domain of CD3ε. We gave intravenous infusions of Resimmune 2.5 - 11.25 µg/kg over 15 minutes to 30 patients (25 with cutaneous T-cell lymphoma, 3 with peripheral T-cell lymphoma, 1 with T-cell large granular lymphocytic leukemia and 1 with T-cell prolymphocytic leukemia) in an inter-patient dose escalation trial. The most common adverse events were fever, chills, hypotension, edema, hypoalbuminemia, hypophosphatemia, and transaminasemia. Among the 25 patients with cutaneous T-cell lymphoma, there were nine responses for a response rate of 36% (95% CI, 18%-57%) including four complete remissions (16%, 95% CI, 5%-36%). The durations of the complete remissions were 72+, 72+, 60+ and 38+ months. There were five partial remissions lasting 3, 3, 3+, 6+ and 14 months. Of 17 patients with a modified skin weighted assessment tool score <50, 17 patients with stage IB/IIB, and 11 patients with both a score <50 and stage IB/IIB, nine (53%), eight (47%), and eight (73%) had responses, respectively. Further studies of Resimmune in patients with low tumor burden, stage IB-IIB cutaneous T-cell lymphoma are warranted. This trial is registered at clinicaltrials.gov as #NCT00611208.

Development of a diphtheria toxin based antiporcine CD3 recombinant immunotoxin.

Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.

A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin.

T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.

Stable multilineage chimerism without graft versus host disease following nonmyeloablative haploidentical hematopoietic cell transplantation.

BACKGROUND: Hematopoietic cell transplantation may offer the only cure for patients with hematological diseases. The clinical application of this therapy has been limited by toxic conditioning and lack of matched donors. Haploidentical transplantation would serve to extend the potential donor pool; however, transplantation across major histocompatibility complex barriers is often associated with severe graft-versus-host disease. Here we evaluate a novel protocol to achieve engraftment across mismatch barriers without toxic conditioning or significant posttransplant complications. METHODS: Nine major histocompatibility complex (MHC)-defined miniature swine received haploidentical hematopoietic cell transplantation following standard myeloablative conditioning. Nine additional animals received haploidentical hematopoietic cell transplantation following a minimally myelosuppressive regimen, consisting of 100 cGy total body irradiation, immunotoxin mediated T-cell depletion, and a short course of cyclosporine. Donor cell engraftment and peripheral chimerism was assessed by polymerase chain reaction and flow cytometry. Graft-versus-host disease was monitored by clinical grading and histology of skin biopsy specimens. RESULTS: All animals conditioned for haploidentical hematopoietic cell transplantation using myeloablative conditioning were euthanized within 2 weeks due to engraftment failure or graft-versus-host disease. All animals conditioned with the nonmyeloablative regimen developed multilineage peripheral blood chimerism during the first 2 months following transplantation. Six animals evaluated beyond 100 days maintained multilineage chimerism in the peripheral blood and lymphoid tissues, showed evidence of progenitor cell engraftment in the bone marrow, and had minimal treatment-related complications. CONCLUSIONS: Here we report that stable multilineage chimerism and engraftment can be established across haploidentical major histocompatibility complex barriers with minimal treatment-related toxicity and without significant risk of graft-versus-host disease.

Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion.

In a bioreactor culture of genetically engineered Pichia pastoris secreting a bivalent immunotoxin, 64% of the secreted immunotoxin was present in aggregate forms and this resulted in a loss of bioactivity. Biochemical analyses of the secreted immunotoxin and an in vitro aggregation study using purified monomeric immunotoxin suggested that aggregation was primarily an extracellular event. By employing limited methanol feeding at 0.75 mlmin(-1) per 10l initial medium, oxygen consumption was reduced, permitting a lowering of the bioreactor agitation speed from 800 to 400 rpm. By increasing the anti-foam reagent to 0.6 mll(-1), the thickness of the air/liquid interfacial foam layer was reduced by 80%. These steps reduced the immunotoxin aggregates from 64% to 5%. Consequently immunotoxin purification yield was increased from 53.0% to 73.8%. Simultaneously this methodology enhanced immunotoxin secretion to 120 mgl(-1) at 163 h of methanol induction in a toxin resistant production strain. We conclude that minimizing shearing force and reducing the air/liquid interfacial foam area are crucial factors in reducing hydrophobic protein aggregation upon secretory expression in yeast bioreactor cultures.

Vascularized thymic lobe transplantation in a pig-to-baboon model: a novel strategy for xenogeneic tolerance induction and T-cell reconstitution.

BACKGROUND: This laboratory has previously demonstrated the induction of allogeneic tolerance by vascularized thymic lobe (VTL) transplantation in miniature swine. We report here our initial attempt to induce tolerance by VTL transplantation in the clinically relevant, discordant, pig-to-baboon model of xenotransplantation. METHODS: Six baboons received xenografts of hDAF VTLs. Four of these baboons also received omental thymic tissue implants. All recipients were treated with an immunosuppressive conditioning regimen that included thymectomy, splenectomy, extracorporeal immunoadsorption of anti-alpha Gal antibodies, and T-cell depletion. Two control baboons received sham operations, of which one also received 5x10 hDAF porcine thymocytes/kg intravenously. RESULTS: Transplanted VTL grafts supported early thymopoiesis of recipient-type immature thymocytes, and facilitated engraftment of nonvascularized thymic omental implants. Recipients of the VTL grafts demonstrated donor-specific unresponsiveness in MLR assays, development of peripheral CD45RAhigh/CD4 double positive (DP) cells, and positive cytokeratin staining of thymic stroma in the grafts for 2 months following xenotransplantation. The control baboons did not show these markers of thymic reconstitution. The eventual return of Gal natural antibodies led to the destruction of graft epithelial cells and the rejection of all VTL grafts by 3 months posttransplantation. CONCLUSIONS: VTL transplantation from hDAF swine to baboons induced early thymopoiesis in the recipients and donor-specific cellular unresponsiveness in vitro. When coupled with additional strategies aimed at silencing humoral rejection, VTL transplantation may significantly prolong xenograft survival and result in long-term tolerance.

Expression and characterization of recombinant soluble monkey CD3 molecules: mapping the FN18 polymorphic epitope.

The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T cell depleting reagent when conjugated to binding site mutants of diphtheria toxin. This anti-CD3 antibody shares certain properties with its anti-human counterparts UCHT1, OKT3 and Leu-4 in that it precipitates two different CD3 chains from membrane detergent extracts. However, in contrast to human CD3, rhesus and cynomolgus monkeys display CD3 polymorphisms producing FN18 negative phenotypes. Using recently published sequence data, we have expressed the ectodomains of cynomolgus CD3-epsilon CD3-gamma and CD-delta chains in E. coli, and have refolded these chains separately and in pairs to produce CD3 homo- and heterodimeric proteins. These proteins were fractionated by anion exchange according to their differing isoelectric points and further identified by size differences on SDS gels. On the basis of ELISA, the FN18 epitope is restricted to the CD3-epsilongamma ectodomain heterodimer, CD-epsilondelta and CD-epsilonepsilon being non-reactive. Either of the two amino acid polymorphisms reported in the CD3-epsilon chain were sufficient to degrade the bioactivity of the CD3-epsilongamma towards FN18.

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.

Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine.

BACKGROUND: Tolerance to composite tissue allografts might allow the widespread clinical use of reconstructive allotransplantation if protocols to achieve this could be rendered sufficiently nontoxic. The authors investigated whether tolerance could be generated in miniature swine to composite tissue allografts across a major histocompatibility (MHC) barrier. A clinically relevant tolerance protocol involving hematopoietic cell transplantation without the need for irradiation or myelosuppressive drugs was tested. METHODS: Seven recipient animals were transiently T-cell depleted and a short course of cyclosporine was initiated. Twenty-four hours later, a donor hematopoietic cell transplant consisting of cytokine-mobilized peripheral blood mononuclear cells or bone marrow cells and a heterotopic limb transplant were performed. In vitro anti-donor responsiveness was assessed by mixed-lymphocyte reaction and cell-mediated lympholysis assays. Acceptance of the limb allografts was determined by gross and histologic appearance. Chimerism in the peripheral blood and lymphohematopoietic organs was assessed by flow cytometry. RESULTS: All seven experimental animals accepted the musculoskeletal elements but rejected the skin of the allografts. All but one of the animals displayed donor-specific unresponsiveness in vitro. The animals that received cytokine mobilized-peripheral blood mononuclear cells showed chimerism but had clinical evidence of graft-versus-host disease (GVHD). None of the animals that received bone marrow cells showed stable chimerism and none developed GVHD. CONCLUSIONS: This protocol can achieve tolerance to the musculoskeletal elements of composite tissue allografts across an MHC barrier in miniature swine. Stable chimerism does not appear to be necessary for tolerance and may not be desirable because of the risk of GVHD.

Xenogeneic thymus transplantation in a pig-to-baboon model.

BACKGROUND: We have tested whether fetal porcine thymic tissue transplantation can lead to tolerance across a discordant (pig-to-baboon) xenogeneic barrier. METHODS: Six baboons underwent a conditioning regimen with thymectomy, splenectomy, and anti-monkey CD3 antibody conjugated to a diphtheria toxin binding site mutant (FN18-CRM9). Porcine fetal or neonatal thymic tissue was transplanted into three baboons. Three control baboons received either no transplanted pig tissue (n=1) or adult pig lymph node (n=2). Cellular responses and skin xenografts were used to test for tolerance. RESULTS: After T-cell depletion and thymic transplantation, recovery of thymus-dependent naïve-type CD4 cells (CD4/CD45RA ) and in vitro xenogeneic hyporesponsiveness were observed. No sensitization of alpha-galactosyl antibody responses was observed. The thymic grafts survived up to 48 days. Porcine skin xenografts were performed in two of these animals with survival of 22 and 24 days. Only two of these animals were completely T-cell depleted, and both failed to recover thymus-dependent T cells (CD4/CD45RA ). In one animal, general in vitro hyporesponsiveness was observed, with subsequent death from infection. The second animal demonstrated delayed recovery of T cells and prolonged general hyporesponsiveness in vitro. Neither animal demonstrated prolongation of porcine skin grafts compared with allografts (both rejected by day 13). CONCLUSIONS: Porcine thymic tissue is able to induce xenogeneic hyporesponsiveness. More efficient thymic engraftment may allow this approach to induce xenograft tolerance.

Variable relationship between chimerism and tolerance after hematopoietic cell transplantation without myelosuppressive conditioning.

BACKGROUND: We have previously described a mixed chimerism protocol that avoids myelosuppressive conditioning and permits hematopoietic cell transplantation across MHC barriers without the need for whole body irradiation in miniature swine. Here, we report our current experience including animals conditioned without thymic irradiation, and we attempt to define the relationship between long-term chimerism and stable tolerance in these animals. METHODS: Recipient swine received in vivo T-cell depletion, with or without thymic irradiation on day -2. Cyclosporine was administered for 30 to 60 days beginning on day -1. A total of 1 to 2 x 10(10) /kg cytokine-mobilized donor hematopoietic cells were infused during 3 days. Chimerism was determined by flow cytometry. In vitro tolerance assays and donor-matched kidney transplantation were performed after cessation of cyclosporine. RESULTS: Most recipients maintained stable chimerism (26 of 35) and were specifically tolerant to donor-matched cells in vitro regardless of whether they received thymic irradiation. Donor-matched kidney transplantations performed in chimeric animals without in vitro antidonor immune responses were accepted without immunosuppression. Some animals developed in vitro evidence of antidonor MHC responsiveness despite the persistence of donor cells in the peripheral blood. Donor-matched kidney transplantations performed in the face of these responses were rejected. CONCLUSIONS: These data indicate that this nonmyelosuppressive protocol can induce stable chimerism and robust tolerance even in animals conditioned without thymic irradiation. However, the data also demonstrate that macrochimerism does not always correlate with tolerance. Lack of in vitro antidonor immune responses in chimeric animals is an important predictor of renal allograft acceptance in this model.

Separation of bivalent anti-T cell immunotoxin from Pichia pastoris glycoproteins by borate anion exchange.

A major problem encountered in the large-scale purification of the bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), from Pichia pastoris supernatants was the presence of host glycoproteins exhibiting similar charge, size, and hydrophobicity characteristics. We overcame this problem by employing borate anion exchange chromatography. The borate anion has an affinity for carbohydrates and imparts negative charges to these structures. We found that at a concentration of sodium borate between 50 and 100 mM, the nonglycosylated immunotoxin did not bind to Poros 50 HQ anion exchanger resin, but glycoproteins, including aggregates related to the immunotoxin, did. By using this property of the immunotoxin in the presence of sodium borate, we successfully developed a 3-step purification procedure: (i) Butyl-650M hydrophobic interaction chromatography, (ii) Poros 50 HQ anion exchange chromatography in the presence of borate, and (iii) HiTrap Q anion exchange chromatography. The final preparation exhibited a purity of greater than 98% and a yield of greater than 50% from the supernatant. Previously, boronic acid resins have been used to separate glycoproteins from proteins. However, combining borate anion with conventional anion exchange resins accomplishes the separation of the immunotoxin from glycoproteins and eliminates the need to evaluate nonstandard resins with respect to good manufacturing practice guidelines.

A pilot open-label trial of minocycline in patients with autism and regressive features.

BACKGROUND: Minocycline is a tetracycline derivative that readily crosses the blood brain barrier and appears to have beneficial effects on neuroinflammation, microglial activation and neuroprotection in a variety of neurological disorders. Both microglial activation and neuroinflammation have been reported to be associated with autism. The study was designed to evaluate the effects of minocycline treatment on markers of neuroinflammation and autism symptomatology in children with autism and a history of developmental regression. METHODS: Eleven children were enrolled in an open-label trial of six months of minocycline (1.4 mg/kg). Ten children completed the trial. Behavioral measures were collected and cerebrospinal fluid (CSF), serum and plasma were obtained before and at the end of minocycline treatment and were analyzed for markers of neuroinflammation. RESULTS: Clinical improvements were negligible. The laboratory assays demonstrated significant changes in the expression profile of the truncated form of brain derived neurotrophic factor (BDNF) (P = 0.042) and hepatic growth factor (HGF) (P = 0.028) in CSF. In serum, the ratio of the truncated BDNF form and α-2 macroglobulin (α-2 M), was also significantly lower (P = 0.028) while the mature BDNF/α-2 M ratio revealed no difference following treatment. Only the chemokine CXCL8 (IL-8) was significantly different (P = 0.047) in serum while no significant changes were observed in CSF or serum in chemokines such as CCL2 (MCP-1) or cytokines such as TNF-α, CD40L, IL-6, IFN-γ and IL-1ß when pre- and post-treatment levels of these proteins were compared. No significant pre- and post-treatment changes were seen in the profiles of plasma metalloproteinases, putative targets of the effects of minocycline. CONCLUSIONS: Changes in the pre- and post-treatment profiles of BDNF in CSF and blood, HGF in CSF and CXCL8 (IL-8) in serum, suggest that minocycline may have effects in the CNS by modulating the production of neurotrophic growth factors. However, in this small group of children, no clinical improvements were observed during or after the six months of minocycline administration. TRIAL REGISTRATION: NCT00409747.

A truncated diphtheria toxin based recombinant porcine CTLA-4 fusion toxin.

Targeted cell therapies are possible through the generation of recombinant fusion proteins that combine a toxin, such as diphtheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the fusion protein to the cell of interest, the diphtheria toxin is internalized which results in protein synthesis inhibition and subsequent cell death. We have recently expressed and purified the recombinant soluble porcine CTLA-4 both with and without N-glycosylation in yeast Pichia pastoris for in vivo use in our preclinical swine model. The glycosylated and non-N-glycosylated versions of this recombinant protein each bind to a porcine CD80 expressing B-cell lymphoma line (LCL13271) with equal affinity (K(D)=13 nM). In this study we have linked each of the glycosylated and non-N-glycosylated soluble porcine CTLA-4 proteins to the truncated diphtheria toxin DT390 through genetic engineering yielding three versions of the porcine CTLA-4 fusion toxins: 1) monovalent glycosylated soluble porcine CTLA-4 fusion toxin; 2) monovalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin and 3) bivalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin. Protein synthesis inhibition analysis demonstrated that while all three fusion toxins are capable of inhibiting protein synthesis in vitro, the non-N-glycosylated porcine CTLA-4 isoforms function most efficiently. Binding analysis using flow cytometry of the porcine CTLA-4 fusion toxins to LCL13271 cells also demonstrated that the non-N-glycosylated porcine CTLA-4 isoforms bind to these cells with higher affinity compared to the glycosylated fusion toxin. The monovalent non-N-glycosylated porcine CTLA-4 fusion toxin was tested in vivo. NSG (NOD/SCID IL-2 receptor γ(-)/(-)) mice were injected with porcine CD80(+) LCL13271 tumor cells. All animals succumbed to tumors and those treated with the monovalent non-N-glycosylated porcine CTLA-4 fusion toxin survived longer based on a symptomatic scoring system compared to the untreated controls. This recombinant protein may therefore provide a novel approach for in vivo depletion of porcine antigen presenting cells (APCs) for studies investigating the induction of transplantation tolerance, autoimmune disease and cancer treatment.

Pharmacology of anti-CD3 diphtheria immunotoxin in CD3 positive T-cell lymphoma trials.

Anti-CD3 recombinant diphtheria immunotoxin, A-dmDT(390)-bisFv(UCHT1), consists of the catalytic and translocation domains of diphtheria toxin fused to two single chain Fv fragments of an anti-CD3epsilon monoclonal antibody (UCHT1). A-dmDT(390)-bisFv(UCHT1) is capable of killing CD3(+) T-lymphoma cells and normal T cells specifically in the femtomolar concentration range. To study pharmacology of A-dmDT(390)-bisFv(UCHT1) in patients with CD3(+) T-cell lymphoma in a phase I clinical trial, (1) highly sensitive bioassay using Jurkat cells for measuring drug levels, (2) ELISA for measuring anti-DT antibody titer, and (3) 5-color FACS analysis method for measuring changes of subtype T-cell population were developed. In addition to evaluating drug efficacy and pharmacokinetics in patients, it is important to correlate pre-existing anti-DT antibody levels with maximum drug concentration in serum and extent of T-cell depletion because pre-existing anti-DT antibodies due to DPT (Diphtheria, Pertussis, and Tetanus) immunization can neutralize diphtheria immunotoxin. We observed that at the lowest treatment dose (2.5 microg/kg: twice daily for 4 days) A-dmDT(390)-bisFv(UCHT1) depletes greater than 99.0% of normal T cells in all six patients for a short period of time (2-3 days) and that there is no association of C (max) and extent of T-cell depletion with the pre-existing anti-DT antibody titer.

Preclinical studies in rats and squirrel monkeys for safety evaluation of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1).

The bivalent anti-human T cell immunotoxin A-dmDT390-bisFv(UCHT1) for treatment of patients with T cell malignancies is a single chain fusion protein composed of the catalytic domain and translocation domains of diphtheria toxin fused to two tandem sFv molecules reactive with human CD3 epsilon. This immunotoxin selectively kills CD3 epsilon positive T cells. To determine the maximum tolerated dose (MTD), pharmacokinetics and immunogenicity of A-dmDT390-bisFv(UCHT1), rat and squirrel monkey studies were performed. In both animal studies, animals received either 0, 2.5 (low), 25 (medium), or 56.25 microg/kg (high) of A-dmDT390-bisFv(UCHT1) intravenously twice daily for four consecutive days. Although transient elevation of liver transaminases in the high groups was observed, the A-dmDT390-bisFv(UCHT1) administration did not affect liver function, renal function, the hemogram, or produce serious organ histopathology. Adverse events included transient lethargy, inappetence and weight loss in high groups. A-dmDT390-bisFv(UCHT1) plasma half life was 26.95 min in rats and 18.33 min in squirrel monkeys. Immune responses to A-dmDT390-bisFv(UCHT1) were minimal in squirrel monkeys and mild in rats. In vitro cytokine release, T cell activation and CD3 epsilon receptor occupancy assays using human PBMC were further performed since rat and squirrel monkey T cells do not react with A-dmDT390-bisFv(UCHT1). A-dmDT390-bisFv(UCHT1) did not induce cytokine release or T cell activation. The A-dmDT390-bisFv(UCHT1) concentration for 50% CD3 epsilon receptor occupancy was 7.4 nM. The MTD of 200 microg/kg total provides a dose level sufficient for anti-tumor activity in vitro and in a rodent model. Therefore, we propose that this agent is a promising drug for patients with surface CD3+ T cell malignancies.

GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials.

The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical grade bivalent anti-T cell immunotoxin for phase I/II clinical trials, a single batch of 120 L bioreactor culture was performed using the Pichia pastoris mutEF2JC307-8(2) strain expressing the bivalent anti-T cell immunotoxin. After 162 h induction of the culture by methanol, the culture medium was harvested by a 0.1 microm hollow-fiber microfiltration step. The recombinant protein was purified by a 3-step purification procedure (Butyl 650 M capturing step, borate anion exchange step and final Poros anion exchange step). The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Expression level was 207 mg/L of culture supernatant and the final production yield was 69.6% or 144.2mg/L of culture supernatant. The final product was characterized by multiple assays. Vialed product was sterile. The drug concentration was 0.8 mg/mL in 150 mM NaCl, 5% glycerol, 1mM EDTA, and 5mM Tris (pH 8.0). Purity by SDS-PAGE was 98%. Aggregates by Superdex 200 HPLC were <1%. Potency revealed a 20 h IC(50) of 17f M on Jurkat cells. Endotoxin level was 0.02 U/mg. Chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The drug did not react with tested frozen human tissue sections except for T cells. LD(10) in mice was between 500 and 75 0microg/kg. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 1.5 year at -80 degrees C. The scalable synthesis of this protein drug should be useful for production for phase I/II clinical trials and can be applicable for other diphtheria toxin fusion drugs for clinical development.

Polymorphisms of CD3epsilon in cynomolgus and rhesus monkeys and their relevance to anti-CD3 antibodies and immunotoxins.

The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T-cell-depleting reagent when conjugated to diphtheria toxin that was mutated to prevent binding to non-targeted cells. The antibody recognizes a conformational epitope on the ectodomain of monkey CD3epsilon and displays a range of binding activity to the T cells from different rhesus and cynomolgus monkeys. Our quantitative fluorescence-activated cell sorting analysis of the FN18 reactivity to T cells from different rhesus and cynomolgus monkeys showed that there are at least three levels of FN18 reactivity in the monkeys tested: high, moderate and low. On the basis of available DNA sequence information, we determined the gene structure of rhesus CD3epsilon chain and designed primers that can be used to amplify and quickly sequence the ectodomain of monkey CD3epsilon. Our sequence analysis revealed that the extent of nucleotide sequence variation in this area is greater than that previously reported. In addition to the amino acids at positions 45 and 50, we demonstrated that position 35 of CD3epsilon was also important and substitution of amino acid A for V at this position greatly reduced T-cell reactivity to FN18. We found that T cells from monkeys with high FN18 reactivity all had V, E and R at positions 35, 45 and 50 in CD3epsilon, respectively; those having low FN18 reactivity were homozygous in CD3epsilon with at least one of the changes: V35 to A, E45 to G and R to 50Q, whereas members in the moderate group are heterozygous, having both V and A, E and G, R and Q at these locations. A cytotoxicity assay revealed that T cells from a heterozygous rhesus monkey with moderate FN18 reactivity were much (about 40 times) less sensitive to a FN18-derived immunotoxin than those from a homozygous rhesus monkey having high FN18 reactivity.