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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3693-3701
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Departments of Hematology, Nephrology, Pathology,
Gastroenterology, and Pharmacology and Toxicology, University Nijmegen,
Nijmegen, The Netherlands; Department of Immunology, Dutch Cancer
Institute, Amsterdam, The Netherlands.
This study evaluated the anti-graft versus host disease (GVHD)
potential of a combination of immunotoxins (IT), consisting of a murine
CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to
deglycosylated ricin A. In vitro efficacy data demonstrated that these
IT act synergistically, resulting in an approximately 99% elimination
of activated T cells at 10
Stem cell transplantation forms a widely accepted
method for restoration of normal hematopoiesis of patients treated for
a hematologic malignancy or otherwise suffering from a defective hematopoietic or immunologic system. For a successful engraftment, a
minimal number of donor T cells in the graft appears to be a prerequisite. The underlying mechanism by which these cells promote engraftment is not fully understood, but probably includes the creation
of an immunologically tolerogenic environment by eliminating the
remainder of the patient's immune system.
In treatment of a malignancy, the cotransplantation of
donor T cells confers additional benefit because they contribute to the
so-called graft versus leukemia (GVL) effect, which involves the
elimination of residual malignant cells.1 The basis of GVL
forms the recognition by donor T cells of (minor or major) histocompatibility antigens expressed by the malignant recipient cells.
Unfortunately, this antigenic disparity may also lead to graft versus
host disease (GVHD), a major cause of morbidity and mortality after
allogeneic stem cell transplantation.2 GVHD is thought to
be initiated by alloactivation of donor T cells resulting in the
production of cytokines (interleukin [IL]-2 and interferon
[INF]- When prophylaxis has failed (typically cyclosporine, often combined
with methotrexate), severe GVHD is usually treated with low-dose
corticosteroids. If the reaction progresses, the dose may be increased
(up to levels of 10-20 mg/kg/d) or, alternatively, polyclonal
antithymocyte/antilymphocyte globulin (ATG/ALG) or experimental
immunosuppressive drugs may be applied. An example of such an
experimental reagent is Xomazyme-CD5 Plus, a murine CD5 monoclonal
antibody (MoAb) conjugated to the A chain of the phytolectin ricin.
Especially in the initial reports, Xomazyme-CD5 Plus demonstrated
substantial efficacy in treating steroid-resistant acute
GVHD.4,5 In more recent comparative trails, Xomazyme-CD5 Plus was not more effective than high-dose corticosteroids or ATG.6,7 Encouraged by its initial success, we developed a therapy based on the use of a combination of 2 anti-T-cell immunotoxins (IT): murine MoAb SPV-T3a (CD3) and WT1 (CD7), both conjugated to
deglycosylated ricin A (dgA). We present the preclinical efficacy data
and preliminary clinical results, both of which suggest that this IT
combination has the potential for helping to control severe acute GVHD.
Immunotoxins
Peripheral blood mononuclear cells (PBMC)
Flow cytometric quantification of in vitro cell kill
Reduction of cytotoxic T-cell toxicity by SPV-T3a Cytotoxic T-cell (CTL) toxicity was assayed in vitro using an EBNA3C12-reactive CTL clone. The CTL clone was incubated with MoAb SPV-T3a (10 8 mol/L) or isotype-matched
control MoAb MOPC-141 at 37°C for 24 hours. Subsequently, cells
were washed and cultured in culture medium for another 72 hours.
Remaining cytolytic activity was assayed with an autologous
Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line
(EBV-LCL), labeled with 100 µCi 51Cr (Amersham, Bucks,
UK) at 37°C for 2 hours. Labeled EBV-LCL were plated in triplicate
(103/well) in V-bottom microtiter plates (Greiner,
Fickenhausen, Germany) and saturated with endogenous EBNA3C (5 µmol/L
for 1 hour at 37°C) to enhance T-cell receptor (TCR)-mediated
lysis. Subsequently, varying numbers of MoAb-treated CTL cells were
added to each well in a final volume of 150 µL culture medium. Plates
were centrifuged (50g, 1 minute) and further incubated at
37°C. After 4 hours, 100 µL supernatant was collected from each
well and counted in a gamma counter. Specific lysis was expressed as
percentage maximal lysis by detergent, both corrected for spontaneous
51Cr release.
Modulation of the CD3-antigen by SPV-T3a Surface CD3 expression of SPV-T3a-treated CTL was determined by indirect fluorescence staining with a saturating amount of SPV-T3a (10 µg/mL, 4°C 30 minutes), followed by a fluorescein isothiocyanate (FITC)-conjugated F(ab')2 goat-antimouse IgG (American Qualex International, La Mirada, CA). CD3 antigen still bound by SPV-T3a used for treatment was identified by staining with the FITC-conjugated antibody only. Expression was indicated as percentage relative to untreated control cells.In vitro reduction of NK activity The PBMC (106/mL) were incubated with 108 mol/L MoAb or IT for 24 hours. Subsequently,
cells were washed and cultured for 3 additional days without MoAb/IT
(to enable full exposure of toxicity). PBMC were serially diluted in
96-well U-bottomed plates, and a fixed concentration of
51Cr-labeled K562 cells was added (104/well) to
yield effector/target ratios of 10:1 to 0.37:1 in a final volume of 150 µL culture medium. Plates were centrifuged (50g, 1 minute)
and further incubated at 37°C. After 4 hours, 100 µL supernatant
was collected from each well and counted in a gamma counter. NK
activity was expressed as percentage maximum lysis by detergent, both
corrected for spontaneous 51Cr release. Recombinant
IL-2 (500 U/mL) (Cetus, Emeryville, CA) was present during
the entire assay to increase NK activity.
Clinical pilot study The first patients were treated in a single center, nonrandomized, open-labeled, dose-escalating study (ongoing) with the aim of obtaining estimates of the safety and efficacy of the IT combination administered to patients with life-threatening GVHD. Patients with acute GVHD were eligible if they had received second-line high-dose corticosteroid therapy (methylprednisolone 1000 mg/d) for at least 3 days without a decrease in the severity of clinical symptoms. Patients were not eligible if they had evidence of intrapulmonary disease (which might aggravate the clinical severity of vascular leak syndrome [VLS]13), or had allergy or antibodies to mouse Ig or dgA. Before entering the trial, all patients gave informed consent in accordance with the institutional review board and ethics committee. For assessment of safety and responses, patients were evaluated daily during hospitalization, and weekly thereafter, with a physical examination and by obtaining serum chemistries, complete blood counts, and leukocyte differential. In addition, blood samples were collected for determination of the pharmacokinetics and immunogenicity of the IT combination. Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC; version 2.0).Administration of the IT combination Patients were supposed to receive 4 doses of the IT combination administered intravenously in 4-hour infusions at 48-hour intervals. Prior to therapy, an intravenous test dose of 200 µg IT combination was administered to rule out anaphylactic reactions. Immunosuppressive agents used for prophylaxis and initial treatment of GVHD were allowed to remain unchanged.Pharmacokinetics Plasma levels of intact IT were measured with an enzyme immunoassay. Affinity-purified rabbit antiricin antibody (Sigma) in carbonate buffer (pH 9.6) was adsorbed to 96-wells maxisorp plates (Nunc, Roskilde, Denmark), and residual binding sites were blocked with 4% bovine serum albumin (BSA). Patient plasma samples were serially diluted in 50% PHS in the absorbed microtiter plates and incubated at 37°C for 1 hour. Subsequently, plates were washed and alkaline-phosphatase conjugated goat antimouse IgG2b or IgG2a (SBA, Birmingham, MI) was added at 37°C for 1 hour to bind captured SPV-T3a-dgA or WT1-dgA, respectively. Plates were washed and the reaction was developed using p-nitrophenylphosphate. Optical densities were read at 450 nm, and levels of circulating IT were calculated from standard curves obtained with a known concentration of IT combination diluted in pretreatment plasma. The detection limit was 0.02 µg/mL for both SPV-T3a-dgA and WT1-dgA. Plasma concentration versus time data were analyzed using the nonlinear least square regression program WinNonlin version 1.1 (Scientific Consulting, Apex, NC).Measurement of human antimouse antibodies (HAMA) and human antiricin antibodies (HARA) An enzyme immunoassay was used for detection of antibody responses to components of the IT combination. Patient plasma samples were serially diluted (undiluted to 1:2048) in phosphate-buffered saline with 1% human serum albumin in 96-well maxisorp plates (Nunc) containing either adsorbed SPV-T3a, WT1, or dgA. Bound human antibodies were probed using alkaline phosphatase-conjugated goat antihuman IgG/IgM (H+L) antibodies (Jackson, West Grove, PA), and the reaction was developed with p-nitrophenylphosphate. Optical densities were read at 450 nm, and the serum titer was expressed as the end-point dilution. Positive titers were considered to be at least 3 times background.Flow cytometry Lymphocyte phenotyping was performed by multicolor flow cytometry using directly fluorescent-labeled MoAb according to a whole blood lysis method (FACS lysing, Becton Dickinson, San Jose, CA). T cells and NK cells were identified simultaneously with a mixture of fluorescence-labeled CD2 and CD5 MoAb (MT910-PE and DK23-PE, respectively) (DAKO, Copenhagen, Denmark). The percentage T/NK cells relative to all leukocytes was determined by costaining with a CD45-FITC MoAb (J33-FITC) (Immunotech, Marseille, France), and converted to absolute numbers based on the leukocyte count. B cells were quantified accordingly, using a PE-conjugated CD19 MoAb (HD37-PE) (DAKO).Staging of GVHD and definitions of clinical responses Each organ system was staged grade 1 through 4 acute GVHD according to established criteria.14 Patients were also given an overall grade of acute GVHD (I through IV) based on severity of organ involvement.14 Responses to therapy were defined as follows:
In vitro elimination of T cells T cells may become more susceptible to dgA-IT on activation.15 This would be beneficial for the treatment of immunologic disorders. Ideally, such treatment inhibits ongoing or beginning immune responses without affecting the resting T-cell pool with its ability to counter future hazardous infections. To address this issue, nonactivated and PHA-activated PBMC were treated with IT for 24 hours and analyzed by flow cytometry for the number of surviving cells (Figure 1). Without prior PHA activation, treatment with SPV-T3a-dgA and WT1-dgA, either alone or in combination, resulted in a 2.0- to 2.7-fold reduction of viable cells at 108mol/L (about 1.8 µg/mL, the highest nontoxic
concentration in vitro). After 48 hours of PHA activation, SPV-T3a-dgA,
WT1-dgA, and the combination demonstrated an approximate 3-, 11-, and
34-fold increase in maximal killing capacity, respectively. The
combination appeared very effective at concentrations above
109 mol/L, resulting in the elimination of 99% of
activated T cells at 108 mol/L. Costaining with
lineage-specific markers revealed no difference in vulnerability
between CD4+ and CD8+ T-cell subsets (data not
shown). Neither incubation with isotype-matched control IT, nor with
MoAb SPV-T3a or WT1, significantly reduced the number of viable cells
(< 1.15-fold reduction at 108 mol/L; n = 3, data
not shown).
Immunosuppressive activity of native MoAb SPV-T3a SPV-T3a may deliver an immunosuppressive effect, irrespective of its conjugation to dgA, by modulation of the T-cell receptor/CD3 complex (TCR/CD3 complex) or by induction of Fas-mediated apoptosis according to the process described as activation-induced cell death (AICD).16 To mimic the alloactivation induced in a transplantation setting, a CTL clone reactive with minor histocompatibility antigen, EBV-peptide EBNA3C, was stimulated with an autologous EBV-LCL. Incubation of this CTL with MoAb SPV-T3a for 24 hours abrogated almost completely its cytolytic activity as measured in a 51Cr-release assay (Figure 2). Flow cytometric analysis (Figure 3) revealed that this may be attributed to both (1) AICD of the CTL clone (to 16% of the untreated control) and (2) modulation of the CD3 antigen (to 20% of the untreated control). In addition, about half of the nonmodulated CD3 antigen was still occupied by SPV-T3a.
NK activity after IT treatment Although initiated by CTL, GVHD is thought to be aggravated by less specific cytokine-stimulated "bystander cells" like NK cells.3,17,18 The capacity of the described MoAb or IT to reduce NK activity was assayed by inhibition of lysis of 51Cr-loaded K562 cells. Figure 4 shows the NK activity of PBMC incubated with IT for 24 hours. As expected, treatment with SPV-T3a-dgA did not affect the NK activity (NK cells being CD3). In
contrast, NK activity was almost completely abolished 3 days after
incubation with WT1-dgA. Similar results were obtained with cell line
Daudi, which is predominantly vulnerable for lymphokine-activated killer cells (data not shown). Neither nonconjugated MoAb WT1, nor
isotype-matched control IT UPC-10-dgA, impaired the lysis of K562 or
Daudi cells (data not shown, n = 3).
Pilot study participants So far, 4 patients, all white men, have been enrolled in the study. The clinical features of these patients are summarized in Table 2. Patients 1 and 2 have been treated at the lowest dose level (2 × 2 mg/m2 followed by 2 × 4 mg/m2) and patients 3 and 4 at the second dose level (4 × 4 mg/m2). Due to their early deaths, patients 1 and 4 received only 3 of the 4 planned infusions.
Pharmacokinetics The plasma clearance curves best fitted a 1-compartment model with a constant rate infusion and a first-order elimination rate for both SPV-T3a-dgA and WT1-dgA individually and given in combination. The pharmacokinetic parameters determined over the entire courses are listed in Table 3. The mean T1/2 was 6.5 ± 2.4 hours for SPV-T3a-dgA, 7.5 ± 1.7 hours for WT1-dgA, and 6.7 ± 2.1 hours for the IT combination. Peak plasma levels were attained directly after each infusion and decreased (nearly) to baseline level in about 48 hours (Figure 5). The maximum plasma concentration (Cmax) for the IT combination ranged from 258 ng/mL after the first dose for patient 2 (2 mg/m2) to 3210 ng/mL after the third dose for patient 1 (4 mg/m2). The latter seemed to be an exception because the maximum peak plasma level in the other patients fell within the relative narrow range of 1220 to 1650 ng/mL (all attained after a dose of 4 mg/m2). The 2-fold higher level in patient 1 may be explained by his aggravating multiorgan failure interfering with the IT plasma clearance. When comparing the separate infusions, the mean T1/2 of the IT combination almost doubled over the complete treatment course (Table 4). This may be explained by a reduction of available target antigens, which act as an antigen sink especially during the first infusion(s).
Safety The adverse events noted during the first 6 weeks following initiation of therapy are listed in Table 5. Because of the advanced disease of the patients, the etiology of the observed clinical events was obscured by multiple medical complications and concomitant medications. Patients 1 and 4 died during therapy due to worsening of complications already existing before start of the treatment, patient 1 from progressive multiorgan failure and patient 4 from generalized aspergillosis in combination with a cytomegalovirus (CMV) infection. Symptoms thought to be related to the IT combination were mild and transient. Patient 1 demonstrated edema in his shoulder not associated with weight gain, potentially due to limited capillary leakage. Patient 3 experienced episodes of fever during and between the IT combination infusions. This fever may have been caused by a viral infection as well (see biologic responses). Patient 4 demonstrated a rise of plasma CK levels (not accompanied by myalgia) to 280 U/L on day 9 of therapy, being 1.45 times the upper limit of the normal range for men. The increase was not accompanied by a rise in the heart muscle isomer creatine kinase MB (CK-MB). Patient 4 had aphasia of short duration (< 1 hour) 1 day after the first infusion of the IT combination. At that time he was taking cyclosporine and had hemolysis with high reticulocyte counts, progressive thrombocytopenia, and fragmented red cells in the blood smear. It was concluded that aphasia was associated with cyclosporine-induced thrombotic thrombocytopenic purpura (TTP) and was not caused by the IT combination (aphasia has been reported as a side effect of other immunotoxins19-21). Cyclosporine was withdrawn and aphasia did not recur despite the further administration of the IT combination.
Immunogenicity Patient plasma samples were measured frequently before, during, and after the trial for HAMA and HARA. No antibodies against SPV-T3a nor WT1 were detected in any of the cases. Patient 4 demonstrated a weakly positive HARA titer, 8 times the pretreatment value, on day 8 (1 day before his death).Biologic responses Biologic responses were monitored by flow cytometric evaluation of circulating T cells or NK cells or both. The concentrations of NK cells were too low to enable a reliable quantification with NK-specific markers. Therefore, NK cells and T cells were identified simultaneously using a mixture of a fluorescent-labeled CD2 (binding T cells and NK cells) and a CD5 MoAb (binding T cells). Figure 6 shows the amount of circulating T/NK cells expressed as a percentage relative to the concentration at start of therapy. Patient 2 could not be adequately monitored due to the low initial number of circulating T/NK cells (about 6 × 107/L). All 3 evaluable patients already demonstrated a remarkable reduction of T/NK cells during the first administration of the IT combination. Immediately after the 4 hours of infusion, the number of circulating T/NK cells of patients 1, 3, and 4 had dropped to 17%, 8%, and 24% of the pretreatment level, respectively. For patients 1 and 4, this number gradually declined to 1% to 3% after the third infusion. Patient 3, in contrast, showed signs of a rebound of NK/T cells between the infusions. After the last infusion, a progressive expansion of T/NK cells was observed resulting in a peak concentration of about 45 times the pretreatment value at day 14. Flow cytometric analysis with CD4/CD8 MoAb and a T-cell receptor V -panel pointed out that these cells were virtually all
CD8+ and oligoclonal in origin. At day 18, the number of T
cells had decreased again to about 5 times the pretreatment level
(probably by apoptosis as suggested by annexin staining, data not
shown). These observations, combined with the episodes of fever patient 3 experienced during the first week, are suggestive of a T-cell response after a viral infection.
Clinical responses
The in vitro efficacy data demonstrate that simultaneous application of SPV-T3a-dgA and WT1-dgA results in a synergistic cytotoxicity, leading to an approximately 99% elimination of activated T cells. A comparable synergism has been described for other IT combinations as well.15,22-28 The most obvious advantage above single IT treatment is that fewer target cells will be negative for multiple antigens than for a single antigen. In addition, the cells that express substantial levels of multiple target antigens may be loaded with more IT molecules. When the respective IT follow a different intracellular routing, the chance to escape the cytotoxic activity of the IT may be further reduced. With respect to anti-T-cell IT, reports addressing the combination approach are thus far focused on in vitro applications, including the purging of bone marrow grafts.15,22-24 In this resport, we state that the combination of SPV-T3a-dgA and WT1-dgA appears appropriate for the in vivo elimination of unwanted T cells as well. This particular combination affords important benefits that surpass the "common synergism" as observed with the combinations of anti-T-cell IT described so far.
We are grateful to Dr Harry Dolstra who generously provided the EBNA3C-reactive CTL-cell clone, to Dr Elly van de Wiel-van Kemenade for helpful advice throughout the project, and to Arie Pennings, Cathy Maass, Mary Leenders, and Jacky Greene for technical assistance.
Submitted October 20, 1999; accepted February 14, 2000.
Supported in part by a grant from the Technology Foundation STW, Nieuwegein, The Netherlands (NGN55.3949).
Reprints: Ypke van Oosterhout, Department of Hematology, University Hospital St Radboud, Geert Grooteplein 8, 6525 GA Nijmegen, The Netherlands; e-mail: y.vanoosterhout{at}chl.azn.nl.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
1.
Horowitz MM, Gale RP, Sondel PM, et al.
Graft-versus-leukemia reactions after bone marrow transplantation.
Blood.
1990;75:555-562 2. Ferrara JL, Deeg HJ. Graft-versus-host disease [see comments]. N Engl J Med. 1991;324:667-674[Medline] [Order article via Infotrieve].
3.
Antin JH, Ferrara JL.
Cytokine dysregulation and acute graft-versus-host disease.
Blood.
1992;80:2964-2968
4.
Kernan NA, Byers V, Scannon PJ, et al.
Treatment of steroid-resistant acute graft-vs-host disease by in vivo administration of an anti-T-cell ricin A chain immunotoxin.
JAMA.
1988;259:3154-3157
5.
Byers VS, Henslee PJ, Kernan NA, et al.
Use of an anti-pan T-lymphocyte ricin a chain immunotoxin in steroid-resistant acute graft-versus-host disease.
Blood.
1990;75:1426-1432 6. Hings IM, Severson R, Filipovich AH, et al. Treatment of moderate and severe acute GVHD after allogeneic bone marrow transplantation. Transplantation. 1994;58:437-442[Medline] [Order article via Infotrieve].
7.
Martin PJ, Nelson BJ, Appelbaum FR, et al.
Evaluation of a CD5-specific immunotoxin for treatment of acute graft-versus-host disease after allogeneic marrow transplantation.
Blood.
1996;88:824-830 8. Spits H, Keizer G, Borst J, Terhorst C, Hekman A, de Vries JE. Characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned CTL lines. Hybridoma. 1983;2:423-437[Medline] [Order article via Infotrieve]. 9. Tax WJ, Greaves MF, Willems HM, Leeuwenberg HF, Capel PJ, Koene RA. WT1: a monoclonal antibody reactive with T-ALL but not with other leukemias. Hamatol Bluttransfus. 1983;28:139-141[Medline] [Order article via Infotrieve]. 10. Tax WJ, Tidman N, Janossy G, et al. Monoclonal antibody (WT 1) directed against a T cell surface glycoprotein: characteristics and immunosuppressive activity. Clin Exp Immunol. 1984;55:427-436[Medline] [Order article via Infotrieve]. 11. Ghetie V, Thorpe P, Ghetie MA, Knowles P, Uhr JW, Vitetta ES. The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and a hindered disulfide bond. J Immunol Methods. 1991;142:223-230[Medline] [Order article via Infotrieve].
12.
Burrows SR, Misko IS, Sculley TB, Schmidt C, Moss DJ.
An Epstein-Barr virus-specific cytotoxic T-cell epitope present on A- and B-type transformants.
J Virol.
1990;64:3974-3976
13.
Amlot PL, Stone MJ, Cunningham D, et al.
A phase I study of an anti-CD22-deglycosylated ricin A chain immunotoxin in the treatment of B-cell lymphomas resistant to conventional therapy.
Blood.
1993;82:2624-2633 14. Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation. 1974;18:295-304[Medline] [Order article via Infotrieve]. 15. Preijers FW, Tax WJ, Wessels JM, Capel PJ, De Witte T, Haanen C. Different susceptibilities of normal T cells and T cell lines to immunotoxins. Scand J Immunol. 1988;27:533-540[Medline] [Order article via Infotrieve]. 16. Kabelitz D, Pohl T, Pechhold K. Activation-induced cell death (apoptosis) of mature peripheral T lymphocytes. Immunol Today. 1993;14:338-339[Medline] [Order article via Infotrieve]. 17. Rhoades JL, Cibull ML, Thompson JS, et al. Role of natural killer cells in the pathogenesis of human acute graft-versus-host disease. Transplantation. 1993;56:113-120[Medline] [Order article via Infotrieve]. 18. Hill GR, Krenger W, Ferrara JL. The role of cytokines in acute graft-versus-host disease. Cytokines Cell Mol Ther. 1997;3:257-266[Medline] [Order article via Infotrieve]. 19. Frankel AE, Laver JH, Willingham MC, Burns LJ, Kersey JH, Vallera DA. Therapy of patients with T-cell lymphomas and leukemias using an anti-CD7 monoclonal antibody-ricin A chain immunotoxin. Leuk Lymphoma. 1997;26:287-298[Medline] [Order article via Infotrieve].
20.
Stone MJ, Sausville EA, Fay JW, et al.
A phase I study of bolus versus continuous infusion of the anti-CD19 immunotoxin, IgG-HD37-dgA, in patients with B-cell lymphoma.
Blood.
1996;88:1188-1197
21.
Vitetta ES, Stone M, Amlot P, et al.
Phase I immunotoxin trial in patients with B-cell lymphoma.
Cancer Res.
1991;51:4052-4058
22.
Vallera DA, Ash RC, Zanjani ED, et al.
Anti-T-cell reagents for human bone marrow transplantation: ricin linked to three monoclonal antibodies.
Science.
1983;222:512-515 23. Uckun FM, Azemove SM, Myers DE, Vallera DA. Anti-CD2 (T, p50) intact ricin immunotoxins for GVHD-prophylaxis in allogeneic bone marrow transplantation. Leuk Res. 1986;10:145-153[Medline] [Order article via Infotrieve]. 24. Katz FE, Janossy G, Cumber A, et al. Elimination of T cells from human peripheral blood and bone marrow using a cocktail of three anti-T cell immunotoxins. Br J Haematol. 1987;67:407-411[Medline] [Order article via Infotrieve]. 25. Crews JR, Maier LA, Yu YH, et al. A combination of two immunotoxins exerts synergistic cytotoxic activity against human breast-cancer cell lines. Int J Cancer. 1992;51:772-779[Medline] [Order article via Infotrieve].
26.
Ghetie MA, Tucker K, Richardson J, Uhr JW, Vitetta ES.
The antitumor activity of an anti-CD22 immunotoxin in SCID mice with disseminated Daudi lymphoma is enhanced by either an anti-CD19 antibody or an anti-CD19 immunotoxin.
Blood.
1992;80:2315-2320 27. Engert A, Gottstein C, Bohlen H, et al. Cocktails of ricin A-chain immunotoxins against different antigens on Hodgkin and Sternberg-Reed cells have superior anti-tumor effects against H-RS cells in vitro and solid Hodgkin tumors in mice. Int J Cancer. 1995;63:304-309[Medline] [Order article via Infotrieve].
28.
Flavell DJ, Noss A, Pulford KA, Ling N, Flavell SU.
Systemic therapy with 3BIT, a triple combination cocktail of anti-CD19, -CD22, and -CD38-saporin immunotoxins, is curative of human B-cell lymphoma in severe combined immunodeficient mice.
Cancer Res.
1997;57:4824-4829 29. Holtrop S, Rijke-Schilder GP, Koene RA, Tax WJ. A polymorphic Fc receptor for mouse IgG2b on human B cells and monocytes. Immunology. 1991;74:613-620[Medline] [Order article via Infotrieve]. 30. Frenken LA, Koene RA, Tax WJ. The role of antibody isotype in IFN-gamma and IL-2 production during anti-CD3-induced T cell proliferation. Transplantation. 1991;51:881-887[Medline] [Order article via Infotrieve]. 31. Conry RM, Khazaeli MB, Saleh MN, et al. Phase I trial of an anti-CD19 deglycosylated ricin A chain immunotoxin in non-Hodgkin's lymphoma: effect of an intensive schedule of administration. J Immunother Emphasis Tumor Immunol. 1995;18:231-241[Medline] [Order article via Infotrieve].
32.
Sausville EA, Headlee D, Stetler-Stevenson M, et al.
Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: a phase I study.
Blood.
1995;85:3457-3465
33.
Senderowicz AM, Vitetta E, Headlee D, et al.
Complete sustained response of a refractory, post-transplantation, large B-cell lymphoma to an anti-CD22 immunotoxin.
Ann Intern Med.
1997;126:882-885
34.
Engert A, Diehl V, Schnell R, et al.
A phase-I study of an anti-CD25 ricin A-chain immunotoxin (RFT5-SMPT-dgA) in patients with refractory Hodgkin's lymphoma.
Blood.
1997;89:403-410
35.
Vallera DA, Carroll SF, Snover DC, Carlson GJ, Blazar BR.
Toxicity and efficacy of anti-T-cell ricin toxin A chain immunotoxins in a murine model of established graft-versus-host disease induced across the major histocompatibility barrier.
Blood.
1991;77:182-194
36.
Onda M, Kreitman RJ, Vasmatzis G, Lee B, Pastan I.
Reduction of the nonspecific animal toxicity of anti-Tac(Fv)-PE38 by mutations in the framework regions of the Fv which lower the isoelectric point.
J Immunol.
1999;163:6072-6077
37.
Greco B, Bielory L, Stephany D, et al.
Antithymocyte globulin reacts with many normal human cell types.
Blood.
1983;62:1047-1054
38.
Raefsky EL, Gascon P, Gratwohl A, Speck B, Young NS.
Biological and immunological characterization of ATG and ALG.
Blood.
1986;68:712-719 39. Rebellato LM, Gross U, Verbanac KM, Thomas JM. A comprehensive definition of the major antibody specificities in polyclonal rabbit antithymocyte globulin. Transplantation. 1994;57:685-694[Medline] [Order article via Infotrieve]. 40. Bell L, Girardin C, Sharma A, Goodyer P, Mazer B. Lymphocyte subsets during and after rabbit anti-thymocyte globulin induction in pediatric renal transplantation: sustained T cell depletion. Transplant Proc. 1997;29:6S-9S[Medline] [Order article via Infotrieve]. 41. Muller TF, Grebe SO, Neumann MC, et al. Persistent long-term changes in lymphocyte subsets induced by polyclonal antibodies [see comments]. Transplantation. 1997;64:1432-1437[Medline] [Order article via Infotrieve]. 42. Wesselborg S, Janssen O, Kabelitz D. Induction of activation-driven death (apoptosis) in activated but not resting peripheral blood T cells. J Immunol. 1993;150:4338-4345[Abstract]. 43. Owen-Schaub LB, Yonehara S, Crump WL, Grimm EA. DNA fragmentation and cell death is selectively triggered in activated human lymphocytes by Fas antigen engagement. Cell Immunol. 1992;140:197-205[Medline] [Order article via Infotrieve]. 44. Chatenoud L, Baudrihaye MF, Kreis H, Goldstein G, Schindler J, Bach JF. Human in vivo antigenic modulation induced by the anti-T cell OKT3 monoclonal antibody. Eur J Immunol. 1982;12:979-982[Medline] [Order article via Infotrieve].
45.
Bertram JH, Gill PS, Levine AM, et al.
Monoclonal antibody T101 in T cell malignancies: a clinical, pharmacokinetic, and immunologic correlation.
Blood.
1986;68:752-761 46. Parlevliet KJ, ten Berge IJ, Yong SL, Surachno J, Wilmink JM, Schellekens PT. In vivo effects of IgA and IgG2a anti-CD3 isotype switch variants. J Clin Invest. 1994;93:2519-2525. 47. Anasetti C, Martin PJ, Storb R, et al. Treatment of acute graft-versus-host disease with a nonmitogenic anti-CD3 monoclonal antibody. Transplantation. 1992;54:844-851[Medline] [Order article via Infotrieve]. 48. Land W, Hillebrand G, Illner WD, et al. First clinical experience with a new TCR/CD3-monoclonal antibody (BMA 031) in kidney transplant patients [letter]. Transpl Int. 1988;1:116-117[Medline] [Order article via Infotrieve].
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Abstract
Introduction
), which in turn activate additional effector cells, like
monocytes, macrophages, and natural killer (NK) cells, to produce
inflammatory proteins (IL-1, tumor necrosis factor [TNF]-
, and
IL-6).
Complete response (CR)
the disappearance of symptoms in all
organ systems
) and WT1-dgA (
), applied individually and in
combination (
) (half a dose each). Nonactivated and PHA-activated
PBMC were incubated with various concentrations IT for 24 hours at
37°C. Following treatment, cells were washed and cultured for an
additional 4 days in the presence of PHA to enable full exposure of IT
toxicity. Subsequently, cells were stained with viability markers and
analyzed by flow cytometry for the number of viable T cells relative to
the untreated control. Data represent the mean ± SD as obtained
with the PBMC of 3 healthy individuals.
) or culture medium (
) for 24 hours at 37°C. Subsequently
cells were washed and cultured for another 72 hours in culture medium.
CTL cytotoxicity was then assayed by specific lysis of a
51Cr-loaded autologous EBV-LCL and expressed as percentage
relative to maximum lysis by detergent. Data represent the mean ± SD of 3 experiments performed in triplicate.
5 (grade
3). When repeated at day 31, biopsy demonstrated a marked reduction of
lymphocyte infiltrates without further signs of active GVHD. In patient
4 only a postmortem liver sample could be taken. Microscopic analysis
revealed extensive cholestasis. Notably, no infiltrating lymphocytes
could be detected.
