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© 2000 American Society for Clinical Oncology Phase I Trial of Recombinant Immunotoxin Anti-Tac(Fv)-PE38 (LMB-2) in Patients With Hematologic MalignanciesByFrom the Laboratories of Molecular BiologyLaboratory of Clinical Pathology, Metabolism Branch, Medicine Branch, and Biopharmaceutical Development Program, Science Applications International Corporation Frederick, National Cancer Institute, National Institutes of Health, Bethesda, MD. Address reprint requests to Ira Pastan, MD, Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Building 37/4E16, 9000 Rockville Pike, Bethesda, MD 20892.
PURPOSE: To evaluate the toxicity, pharmacokinetics, immunogenicity, and antitumor activity of anti-Tac(Fv)-PE38 (LMB-2), an anti-CD25 recombinant immunotoxin that contains an antibody Fv fragment fused to truncated Pseudomonas exotoxin. PATIENTS AND METHODS: Patients with CD25+ hematologic malignancies for whom standard and salvage therapies failed were treated with LMB-2 at dose levels that ranged from 2 to 63 µg/kg administered intravenously over 30 minutes on alternate days for three doses (QOD x 3). RESULTS: LMB-2 was administered to 35 patients for a total of 59 cycles. Dose-limiting toxicity at the 63 µg/kg level was reversible and included transaminase elevations in one patient and diarrhea and cardiomyopathy in another. LMB-2 was well tolerated in nine patients at the maximum-tolerated dose (40 µg/kg QOD x 3); toxicity was transient and most commonly included transaminase elevations (eight patients) and fever (seven patients). Only six of 35 patients developed significant neutralizing antibodies after the first cycle. The median half-life was 4 hours. One hairy cell leukemia (HCL) patient achieved a complete remission, which is ongoing at 20 months. Seven partial responses were observed in cutaneous T-cell lymphoma (one patient), HCL (three patients), chronic lymphocytic leukemia (one patient), Hodgkin’s disease (one patient), and adult T-cell leukemia (one patient). Responding patients had 2 to 5 log reductions of circulating malignant cells, improvement in skin lesions, and regression of lymphomatous masses and splenomegaly. All four patients with HCL responded to treatment. CONCLUSION: LMB-2 has clinical activity in CD25+ hematologic malignancies and is relatively nonimmunogenic. It is the first recombinant immunotoxin to induce major responses in cancer. LMB-2 and similar agents that target other cancer antigens merit further clinical development.
IN 1988, HUSTON ET AL1 and Bird et al2 separately reported that the variable domains of antibodies could be linked together to make 25-kd recombinant proteins capable of binding antigen. Approximately 1,000 published reports have since described single-chain Fv molecules, which have been engineered in most cases to target malignant disease. Because small proteins have been shown to be much more efficient at distributing through tumors than large proteins,3,4 attempts have been made to use Fvs instead of larger antibodies to direct radionuclides or toxins to treat tumors. So far, radiolabeled single-chain Fvs have been shown to successfully image tumors in patients,5 but no major responses have yet been reported in cancer therapy trials. This has been attributed to the large number of radiolabeled molecules needed to kill a tumor cell and the short lifetime of radiolabeled Fvs in the circulation. In 1989, Chaudhary et al6 reported the construction of the first recombinant immunotoxin, which contained a single-chain Fv fragment of the anti-Tac monoclonal antibody (mAb) to the interleukin-2 receptor (IL-2R) alpha subunit (also referred to as Tac, p55, or CD25) fused to a truncated form of the bacterial toxin Pseudomonas exotoxin (PE). This recombinant toxin was universally cytotoxic toward malignant cells from patients with adult T-cell leukemia (ATL).7-9 An equally cytotoxic and slightly smaller derivative of this immunotoxin, anti-Tac(Fv)-PE38 (LMB-2), was developed for clinical testing.10,11 LMB-2 does not require the beta and gamma subunits of the IL-2R to be present for cytotoxicity.10,11 Based on structural12,13 and functional14 studies, intoxication by LMB-2 has been shown to require binding to CD25, internalization and processing of the toxin within its translocation domain,15-17 binding of the 35-kd carboxyl terminus of the toxin to the intracellular Lys-Asp-Glu-Leu (KDEL) receptor that carries it to the endoplasmic reticulum,18,19 translocation of the toxin into the cytoplasm,20,21 and finally catalytic adenosine diphosphate ribosylation of elongation factor 2, which leads to apoptosis and cell death.22,23 When LMB-2 was administered to mice bearing CD25+ human tumors, the immunotoxin rapidly accumulated in the tumors and produced complete regressions of the solid tumors.11,24 Toxicology studies showed that blood levels causing tumor regression in mouse xenografts are well tolerated by monkeys,25 with dose-dependent reversible transaminase elevations being the major side effect. Techniques were developed to allow efficient large-scale production of the immunotoxin from inclusion bodies after expression in Escherichia coli,18 which allowed sufficient clinical material to be made for treating patients with CD25+ hematologic malignancies. CD25 is present on the malignant cells of patients with a variety of hematologic malignancies. Although it is consistently present at very high density on ATL cells (103 to 104 sites/cell),26 lower but significant levels are detectable on malignant cells from approximately 50% of patients with B-cell chronic lymphocytic leukemia (CLL),27,28 up to 100% of anaplastic large-cell lymphomas,29 33% to 55% of B-cell non-Hodgkin’s lymphomas (NHL),30,31 20% to 50% of peripheral T-cell lymphomas (PTCL),32,33 40% to 50% of cutaneous T-cell lymphomas (CTCL),30,34 75% to 90% of patients with Hodgkin’s disease (HD),30,35 and 80% of patients with hairy cell leukemia (HCL).36 To determine the toxicity, pharmacokinetics, immunogenicity, and therapeutic activity of LMB-2 in humans, we performed a phase I trial in patients with CD25+ hematologic malignancies who experienced treatment failure with chemotherapy.
Immunotoxin Production Clinical-grade LMB-2 was produced by the Monoclonal Antibody and Recombinant Protein Production Facility, National Cancer Institute (NCI), Frederick, MD. The investigational new drug application is held by the Cancer Therapy and Evaluation Program of the NCI. The protein was produced by methods previously published,37 in which LMB-2 is isolated from E coli cell paste by detergent washing of inclusion bodies, followed by denaturing and reducing the immunotoxin in guanidine hydrochloride with dithioerythritol, refolding, and purification by anion exchange and sizing chromatography. For this trial, two clinical lots were produced that each had identical properties. The plasmid expressed for the first clinical lot was pRK79,11 and the E coli were grown in the presence of ampicillin. For the second clinical lot, the gene fragment encoding LMB-2 was placed into a vector that contained a chloramphenicol resistance gene.
Eligibility Criteria
Antibody Assays
Treatment Plan and Study Design
Immunotoxin Administration
Plasma Pharmacokinetics
Response Evaluation
Thirty-five patients with chemotherapy-refractory hematologic malignancies were treated with LMB-2. The patient characteristics are listed in Table 1. The age range of patients was 24 to 79 years, and the median age was 47 years. There were slightly more than twice as many males as females (24 v 11). Eleven patients had HD, eight had CLL, four had HCL, six had B-cell NHL, and the remaining six had T-cell leukemias or lymphomas. The patients were heavily pretreated, with up to 19 prior regimens (mean, 5.3 regimens; median, four regimens), and nearly one half of the patients (16) had experienced treatment failure with high-dose chemotherapy and bone marrow or peripheral-blood stem-cell transplantation. Thus the patients were heavily pretreated and often had significant underlying myelosuppression.
Toxicity Drug-related toxicity with one cycle of LMB-2, as listed in Table 2, was seen at  10 µg/kg QOD x 3 and was reversible. As noted in Table 3, five patients had incomplete cycles. Patient no. 13 missed the last dose of cycle 1 because of pericardial effusion and had less than one dose of cycle 2 because of an allergic reaction. Patient no. 14 received only one dose because of the diagnosis of acute culture-positive influenza B pneumonia, patient no. 27 received only one dose because of allergic reaction, and patient nos. 31 and 32 received two doses and one dose, respectively, because of DLT.
The most common toxic effect was an elevation of the AST and ALT levels, usually in the grade 1 or 2 range. At dose levels of 10 to 63 µg/kg QOD x 3, 26 (90%) of 29 patients had transaminase elevations. Grade 3 transaminase elevations were observed in one, three, and two patients at dose levels of 30, 40, and 50 µg/kg QOD x 3, respectively, but were not associated with impaired liver function as assessed by prothrombin time and fibrinogen and bilirubin levels. Several patients who received dose levels of 40 to 63 µg/kg QOD x 3 experienced transient grade 1 nausea and (less frequently) vomiting, but could not be correlated with the degree of transaminase elevations. It was therefore decided that grade 4 but not grade 3 transaminase elevations would be dose-limiting. Patients who were re-treated (see re-treatment schedule; Table 3) had transaminase elevations, except patient nos. 10 and 35. Of patients treated with a total of two cycles, patient nos. 10, 11, and 34 experienced grade 1 transaminase elevations on both cycles, patient no. 13 experienced grade 2 elevations followed by no elevations, and patient no. 33 experienced grade 3 followed by grade 1 elevations. Patient no. 30 was the only one of these patients with substantially more toxicity during the second (grade 3) compared with the first cycle (grade 1); this was attributed to induction of a CR between these two cycles, which led to higher plasma levels on the second cycle. Of patients treated with three cycles, patient no. 8 experienced grade 1 elevations only during the latter two cycles, and patient no. 26 had grade 3 elevations for all three cycles. Patient no. 1 had a grade 1 elevation during only the third of four total cycles, patient no. 7 had a grade 1 elevation during only the last of five cycles (although the last dose was twice as high as the previous four), and patient no. 17 had grade 1 elevations during each of six cycles. Thus patients did not tend to have cumulative hepatic toxicity with re-treatment, and in all cases transaminase elevations resolved to normal (grade 0) levels before patients began a subsequent cycle of LMB-2. The transaminase elevations were never observed to increase after day 8 of each cycle in any patient, and in all patients with grade 3 to 4 elevations, the AST and ALT levels resolved to no greater than 40 U/L. Fever occurred in most of the patients who were treated at dose levels greater than 10 µg/kg QOD x 3. The fever typically occurred within hours of the first dose and did not recur after the second and third doses. This pattern would typically recur with subsequent cycles. The fever occasionally remitted with antipyretics or anti-inflammatory agents, and in many patients, fever was not symptomatic enough to treat.
Classic severe vascular leak syndrome (VLS), which consisted of hypoalbuminemia, systemic and pulmonary edema, hypotension, tachycardia, and myalgia, was not observed with LMB-2. As listed in Table 2, 18 of the 22 patients with normal pretreatment albumin levels ( Drug-related renal toxicity was observed infrequently, was always reversible and of grade 1 severity, and in all three patients was associated with underlying predisposing factors. These included congenital absence of a kidney in one patient, Bence-Jones proteinuria in another patient, and the initiation of trimethoprim/sulfamethoxazole for prophylaxis of Pneumocystis carinii pneumonia in a third patient. Nevertheless, grade 1 renal toxicity seemed to be at least partly drug-related (Table 2) due to the transient appearance of abnormal renal epithelial cells in the urine after treatment. DLT was observed in two of three patients at the 63 µg/kg QOD x 3 dose level. The first patient (no. 31) had an AST level of 858 U/L (grade 4) on day 5 with a grade 3 ALT level. Interestingly, this patient was asymptomatic with normal appetite and food intake. The second patient (no. 32) with DLT had HCL and experienced grade 2 fever, nausea, vomiting, and grade 3 diarrhea after the first dose and therefore did not receive subsequent doses. This patient on day 5 developed low output cardiac failure with diffuse cardiomyopathy but no evidence of myocardial infarction or necrosis. The patient’s cardiac function returned to normal by day 7. FACS of peripheral blood on day 9 indicated a large number of dead tumor cells in the peripheral blood. Although it has not been determined whether the transient cardiomyopathy was directly due to toxicity of LMB-2 on the heart or was cytokine-mediated,45,46 perhaps due to tumor response, the 63 µg/kg QOD x 3 dose level was considered to be clearly dose-limiting. The 50 µg/kg QOD x 3 dose level was dose-limiting in only one of six patients (no. 27) due to an allergic reaction. However, because this dose level was only 20% less than the 63 µg/kg QOD x 3 level, we decided to define 40 µg/kg QOD x 3 as the MTD. Three additional patients were treated at this dose level. Table 3 also indicates how many patients at each dose level had circulating malignant cells and the maximum toxicity grade in patients with and without circulating malignant cells. In both groups of patients, higher grades of toxicity were seen with higher doses. Although with most dose levels the maximum toxicity grade was higher in patients without circulating malignant cells, the difference was never more than one grade. Table 4 lists in detail the LMB-2–related toxicities at the MTD, at which a total of nine patients were treated. Toxicities were well tolerated and were usually of grade 1 severity, except in patients who experienced a transient grade 2 fever after the first but not subsequent doses of LMB-2, and in patients with transient grade 3 non–dose-limiting transaminase elevations. As was found at other dose levels, nausea and fever could not be correlated directly with the degree of transaminase elevations in these patients who were treated at the MTD.
Pharmacokinetics To determine the amount of bioactive drug in the plasma of treated patients, we quantitated LMB-2 levels by cytotoxicity assay. The plasma disappearance was usually monoexponential and the mean half-life (T1/2) for each dose level varied from 173 to 494 minutes, as shown in Fig 1C. The variability within each dose level was partly related to disease burden in the peripheral blood and spleen. For example, patient no. 33, who was treated at the MTD for low-volume HD, had a soluble CD25 (sCD25) serum level of 1,150 U/mL (3.5 ng/mL) and a peak LMB-2 level of 1,040 ng/mL with a T1/2 of 177 minutes. In contrast, patient no. 34, who had ATL involving spleen and peripheral blood, an sCD25 serum level of 73,600 U/mL, and a peak LMB-2 level of 457 ng/mL, had a longer T1/2 of 272 minutes. The areas under the curve (AUC) were similar for these two patients (158 µg · min/mL v 179 µg · min/mL, respectively). Table 5 stratifies patients with and without malignant peripheral-blood lymphocytes (PBLs) according to AUC, sCD25 serum level, and peak LMB-2 level. Significantly lower AUCs were observed at dose levels of 10 and 20 µg/kg QOD x 3, and significantly lower peak levels were observed at dose levels of 10, 20, and 40 µg/kg QOD x 3. Moreover, at 10 and 63 µg/kg QOD x 3, AUC was inversely proportional to PBL (r2 = 0.80 and 0.91, respectively), and at 6 and 63 µg/kg QOD x 3, peak levels were inversely proportional to sCD25 concentration (r2 = 0.77 and 0.99, respectively). However, we observed significant variability within each group regardless of the level of PBLs. Thus a significant decrease in pharmacokinetic results with increasing disease burden was a frequent but inconsistent finding, and significant variability remained within each dose group after stratifying patients for the presence of PBLs.
To quantitate the level of total LMB-2 in the plasma, including degraded LMB-2 or LMB-2 bound to neutralizing serum proteins, we developed an ELISA using plate-bound monoclonal mouse anti-PE and polyclonal HRP-conjugated rabbit anti-PE. We found no significant difference in pharmacokinetic parameters obtained from the cytotoxicity assay and ELISA. For example, using the ELISA, patient no. 30 had a 2-minute plasma level of 1,200 ± 640 ng/mL and an AUC of 215 µg · min/mL, compared with 1,200 ± 50 ng/mL and 200 µg · min/mL, respectively, using the cytotoxicity assay. Similarly, patient no. 31 had a 2-minute plasma level of 1,200 ± 230 ng/mL and an AUC of 189 µg · min/mL by ELISA, compared with 1,600 ± 120 and 260 µg · min/mL, respectively, by cytotoxicity assay. Thus ELISA did not indicate that a large percentage of LMB-2 in the plasma was inactive and unable to be detected by the cytotoxicity assay. The latter assay was found to be more sensitive.
Immunogenicity
Several patients had allergic reactions associated with production of neutralizing antibodies. Patient no. 27 was previously treated with radiolabeled humanized anti-Tac (HAT) mAb, and it was determined in retrospect that he had made nonneutralizing antibodies to the anti-Tac idiotype before being treated with LMB-2. Because HAT and murine anti-Tac(Fv) do not share framework regions, the anti-Tac idiotype constitutes the only amino acid residues that HAT and LMB-2 have in common. This patient developed a transient anaphylactic reaction to the first dose of LMB-2 and subsequently produced neutralizing antibodies. Another patient (no. 13) had an allergic reaction consisting of reversible bronchospasm (grade 3) immediately after the first dose of the second cycle, and this patient subsequently produced neutralizing antibodies. A third patient (no. 34) had chest tightness and dyspnea without wheezing (grade 2) immediately after receiving the first dose of the second cycle, despite usual pretreatment with oral hydroxyzine 25 mg and ranitidine 150 mg. This allergic reaction did not recur after the addition of 50 mg of IV diphenhydramine to the pretreatment regimen. However, this patient was found to have neutralized LMB-2 after the first dose of the second cycle with antitoxin, antimurine IgG, and anti-idiotype antibodies. Thus significant allergic reactions were always associated with the formation of neutralizing antibodies against LMB-2, but most seroconversions were not associated with allergic reactions.
Responses Major responses were observed in four of four patients with HCL, all of whom had experienced treatment failure with at least 2-chlorodeoxyadenosine and interferon. A CR was induced in patient no. 30, who had pancytopenia with pretransfusion hemoglobin concentrations as low as 3.8 g/dL, platelet count of 47,000/µL, ANC of 360/µL, and also an enlarged spleen and precarinal lymph nodes. The response after 63 µg/kg QOD x 3 included rapid (2 days) disappearance of circulating HCL cells by morphology; resolution of HCL cells from the bone marrow biopsy by 1 week; resolution of thrombocytopenia, splenomegaly, and precarinal nodes by 1 month; and resolution of granulocytopenia and anemia by 2 months. By 6 to 8 months, circulating HCL cells had decreased by 5 logs from pretreatment, and then the peripheral blood FACS was no longer diagnostic of HCL. This patient met criteria for CR based on findings from large HCL trials42,43 but had minimal residual disease in the bone marrow aspirate (0.15% contamination by HCL) as defined by FACS, without relapse after 20 months. Three PRs were observed in the three other patients with HCL who were treated. The maximum reduction of malignant cells in the peripheral blood varied from 98% to 99.8%. These three patients did not relapse during a median of 44 days of follow-up after first demonstration of a PR. Figure 2 shows responses of malignant cells in patients with CLL, CTCL, and ATL. The CLL patient with a WBC count of 230,000/µL had experienced treatment failure with chlorambucil, chlorambucil/prednisone, cyclophosphamide/vincristine/prednisone, fludarabine, and splenectomy. This patient had Coombs-positive hemolytic anemia, Bence-Jones proteinuria, and mildly high creatinine associated with gamma light chains, cervical adenopathy, multiple opportunistic infections, and multiple pulmonary emboli that required warfarin. The first cycle of LMB-2 at 50 µg/kg QOD x 3 was complicated by fever, which led to the diagnosis by bronchoscopy of Pneumocystis carinii pneumonia. As shown in Fig 2A, the malignant count decreased by 50% during the first cycle and did not progress before the next cycle. The patient’s abnormal enlargement of the cervical lymph nodes resolved after the first cycle. The next two cycles of LMB-2 were well tolerated, without further opportunistic infections. A cumulative response of 70% to 80% was achieved (Fig 2A), after which the patient became asymptomatic. He therefore elected to be followed-up off therapy, and as shown in Fig 2A, the malignant count increased very slowly during the ensuing 5 months. The CTCL patient treated with LMB-2 had stage IVB disease with 930 Sézary cells/µL in the peripheral blood and diffuse erythroderma. The patient had been unsuccessfully treated with interferon, 2-chlorodeoxyadenosine, prednisone, azathioprine, and psoralen and ultraviolet A radiation and had opportunistic infections, including disseminated herpes zoster that caused unilateral blindness. She received LMB-2 at 30 µg/kg QOD x 3 and, as shown in Fig 2B, had a rapid (7 days) reduction in malignant circulating cells as assessed by FACS analysis. She was retreated with LMB-2 for five additional cycles, which maintained an 80% to 95% response that was associated with improvement in erythroderma. The patient’s sixth cycle was delayed because of reduced LMB-2 supply before completion of the second clinical lot, and despite regrowth of the malignant cells during the 77 days after cycle 5, a sixth PR was induced. Most ATL patients screened for the LMB-2 phase I trial were not eligible because of very poor performance status and active severe infections. Two patients with ATL were eligible to be treated. The first was treated with 50 µg/kg QOD x 3 and had an 80% response in circulating cells by day 8 (data not shown). However, between days 12 and 24, the malignant count returned and the patient when restaged was found to have PD. As shown in Fig 2C, the second ATL patient, who was treated with LMB-2 at 40 µg/kg QOD x 3, had a rapid response with more than 95% reduction of malignant circulating cells by day 8. Re-treatment was begun on day 26, when the malignant count had returned to nearly 50% of its original level, and as shown, the second cycle succeeded at lengthening the response duration. Splenomegaly resolved almost completely in this patient by day 8.
Recombinant single-chain immunotoxins at 63 kd are approximately one third the size of conventional immunotoxins prepared by chemical conjugation of a mAb to a toxin. Potential benefits that were recognized during their preclinical development included homogeneity in composition, more rapid tumor penetration, and relatively low cost of production.24,25 Before the testing of these immunotoxins in humans, however, it was not clear whether their plasma lifetimes would be long enough to allow clinical efficacy, or whether Fv residues, which are not protected by antibody constant domains, would target toxins to normal tissues and make the protein more immunogenic. To determine the clinical behavior of a recombinant immunotoxin in humans, we tested LMB-2 in patients with CD25+ hematologic malignancies. We found this agent to be well tolerated in patients at 40 µg/kg IV QOD x 3 and also observed a relatively long half-life, low immunogenicity that permitted re-treatment, and a significantly high response rate. The deglycosylated ricin A-chain (dgA)–containing immunotoxins IgG-RFB4-dgA, IgG-HD37-dgA, and IgG-RFT5-dgA, which target CD22, CD19, and CD25, respectively, were associated with VLS as the most common toxicity.47-52 The MTDs (total dose/cycle) of IgG-RFB4-dgA, IgG-HD37-dgA, and IgG-RFT5-dgA were approximately 800, 200, and 375 µg/kg, similar to that of LMB-2 (total dose/cycle, 120 µg/kg) given its approximately three-fold lower molecular weight. VLS in these trials consisted of decreased serum albumin, pulmonary and systemic edema, weight gain, hypotension, tachycardia, and myalgia. As listed in Table 2, a minority of patients treated with LMB-2 had significant weight gain consistent with third spacing. The transient hypotension that was seen with LMB-2 was associated with fever that followed the first dose, but this usually led to decreased body weight because of insensible weight losses, which were usually corrected with volume repletion and usually did not lead to significant weight gain. It has previously been shown that dgA alone but not LMB-2 is cytotoxic toward human umbilical vein endothelial cells,53 and this may at least partially explain the low incidence of significant VLS in patients who were treated with LMB-2. More recently, a variety of in vivo and in vitro models of VLS have suggested that many different toxins at high enough doses, including PE derivatives, may induce VLS, and that the etiology may be more complicated than simple cytotoxicity toward endothelial cells.54-59 Like the current trial, some evidence of third spacing of fluid is commonly reported in clinical trials of chimeric toxins in which the DLT is not determined by VLS. Examples include DAB389IL260 and anti-B4 blocked Ricin (anti-B4-bR).61 Although elevated cytokine levels were not observed in patients treated with IgG-RFB4-dgA,47 it is possible that the third spacing that was seen with LMB-2 is cytokine-mediated or related to liver toxicity; this is currently under investigation. The toxicity of LMB-2 in patients most commonly consisted of transient transaminase elevations and fever. Transaminase elevations were common in patients treated with the recombinant IL-2–fusion toxins DAB486IL262-66 and DAB389IL2,60,67,68 and also with anti-B4-bR.61 Although DLT in several of these trials was determined by transaminase elevations, patients did not have significant impairment in hepatic function, which is similar to our experience with LMB-2. The MTDs of DAB389IL2 and anti-B4-bR were similar to that of LMB-2 (135 v 200 v 120 µg/kg/cycle, respectively) on a molar basis.60,61 Interestingly, fever and transaminase elevations were not related to quantity of malignant cells in patients who were treated with LMB-2 and has not been observed with a PE38-containing immunotoxin that targets an antigen (Lewis Y) on solid tumors.40 Thus it is possible that these toxic effects are mediated by the Fv of anti-Tac binding to normal cells, possibly inducing cytokine production.69 The T1/2 of LMB-2 was much more prolonged than that which was expected based on monkey and mouse studies, in which the T1/2 was 83 to 100 minutes in monkeys and 48 minutes in mice24 compared with a median T1/2 of 257 minutes in humans. DAB389IL2, which is smaller in molecular weight than LMB-2 (55 v 63 kd, respectively), had a shorter T1/2 in patients (72 minutes overall).67 The larger conventional immunotoxins Fab-RFB4-dgA and anti-B4-bR also were excreted more rapidly than LMB-2.61,70,71 Several immunotoxins, including IgG-HD37-dgA, IgG-RFB4-dgA, and anti-B4-bR, have been administered as short infusions and also by continuous infusion, with results showing modest increases in MTDs with continuous infusion but little improvement in responses.47,49,72 These data, along with the relatively long T1/2 for LMB-2, support the continued use of short infusions in future clinical studies of this agent. Although plasma levels were quantitated by cytotoxicity assay, the plasma levels listed in Table 5 would not be underrepresented artifactually because of LMB-2 binding in vitro to sCD25 instead of the cell line SP2/Tac. Because this cell line is so sensitive to LMB-2 (concentration that inhibits 50% = 0.01 ng/mL), the plasma samples have to be diluted to very low, insignificant sCD25 concentrations. For example, patient no. 34, whose sCD25 level of 73,600 U/mL (220 ng/mL) was the highest observed in our study, had plasma that was significantly cytotoxic even by 24 hours so that it required more than 500-fold dilution to quantitate the 11 ng/mL concentration of LMB-2. At this dilution, the sCD25 concentration would be less than 0.5 ng/mL (< 12 pmol/L), which is too low to interfere with the cytotoxicity of LMB-2.9 It remains possible that high sCD25 levels in patients, which are often associated with high tumor burden, could affect the pharmacokinetics of LMB-2 by increasing the removal of LMB-2 from the plasma. There are many other factors that might decrease plasma LMB-2 levels, including (1) the concentration of malignant PBLs; (2) the presence of malignant cells in lymph nodes, bone marrow, and spleen; (3) the density of CD25 on the surface of the malignant cells; (4) the presence of normal CD25+ cells; (5) the presence of nonneutralizing antibodies occurring before or during treatment; and (6) variations in renal or hepatic excretion of LMB-2. We believe that differences among patients in some or all of these factors causes variability in pharmacokinetic parameters (Fig 1 and Table 5). Additional trials with LMB-2 will be necessary to more accurately predict pharmacokinetics and possibly toxicity on the basis of clinical and immunophenotypic variables. Most of the patients who were treated with LMB-2 did not produce neutralizing antibodies after the first cycle, which allowed most patients who were responding to be treated with several cycles. Interestingly, none of the eight patients with CLL ever developed any evidence of neutralizing antibodies to LMB-2. Although our assay of immunogenicity is based on cytotoxicity that only detects neutralizing antibodies, the low rate of immunogenicity seen with LMB-2 is consistent with the low (20% to 30%) immunogenicity rates seen in other trials of immunotoxins in patients with hematologic malignancies47-49,61,70 and is much lower than the 90% to 100% immunogenicity rates seen in immunotoxin trials against solid tumors.40,73 The anti-CD25 immunotoxin IgG-RFT5-dgA produced antibodies in nearly one half (seven of 15) of the HD patients treated, which is intermediate between the high rates for solid tumors and the low rates for B-cell malignancies.50,51 Three of 11 patients with HD produced high levels of neutralizing antibodies to LMB-2 (Table 3). LMB-2 may be slightly less immunogenic than conventional immunotoxins because of the smaller size of the molecule and the absence of immunogenic epitopes on the constant domain of murine antibodies. With the conventional immunotoxin LMB-1, which is composed of mAb B3 conjugated to PE38, more immunogenicity was to the murine antibody rather than to the toxin,40 and this was not the case with LMB-2. Patients with hematologic malignancies treated with DAB389IL2 had high rates of seroconversion as detected by ELISA, but these antibodies did not prevent responses in some patients.60,67 In contrast, in the few patients who did develop high levels of neutralizing antibodies to LMB-2 (patient nos. 34 and 35; Table 3), responses that were observed in the first cycle were noticeably blunted in subsequent cycles. Efforts are underway to decrease the incidence of immunogenicity further for both hematologic and solid tumors. Patients who are treated with corticosteroids seem to seroconvert to immunotoxins less frequently,48 but significant results from clinical trials are so far lacking. Both patients treated with LMB-2 who were receiving chronic corticosteroids had CLL and no neutralizing antibodies after three to five cycles (patients nos. 7 and 26; Table 3). A clinical trial is now being performed in which solid tumor patients are being treated with the immunotoxin LMB-1 in conjunction with the anti-CD20 mAb rituximab74 to deplete B cells that might mediate a humoral immune response (Pai-Scherf and Pastan, unpublished data). LMB-2 induced one CR and seven PRs in 34 assessable patients, all occurring among the 20 patients who received more than 60 µg/kg (total dose/cycle) of LMB-2. Responses were evident within 1 week in most patients. Malignant cells from leukemic patients were sensitive ex vivo to LMB-2 but not to mutants of LMB-2 that were defective in either binding or adenosine diphosphate–ribosylating activity (data not shown). This indicates that clinical responses were due to the direct cytotoxic effect of LMB-2 and not simply to CD25 binding alone or to nonspecific internalization of LMB-2 into cells. The responses were often quite durable and long lasting, with one patient still in CR 20 months after beginning LMB-2 and several other responses lasting more than 6 months. Interestingly, we have not yet observed CD25- tumor cells causing relapses in patients who respond to LMB-2. We have shown previously that LMB-2 does not require other subunits of the IL-2R for cytotoxicity.10,11 All four cases of HCL were responsive to LMB-2, regardless of how many prior regimens with which patients had experienced treatment failure, which makes LMB-2 a new, potentially effective form of salvage therapy in this disease. DAB389IL2 is effective against CTCL, with response rates of 30%.60,67 The durable PR that we observed in the one CTCL patient who was treated with LMB-2 indicates the need to test additional patients with CTCL, particularly those whose malignant cells express CD25 but not other subunits of the IL-2R. In conclusion, the acceptable toxicity and responses seen with LMB-2 serve as proof-of-principle of the potential clinical utility of recombinant immunotoxins and validate efforts to further develop LMB-2 and similar biologic agents. Other antigens, including CD22, are currently being targeted clinically with recombinant immunotoxins.75
We recognize the contributions of Dr Toby Hecht and Daniel Coffman at the Monoclonal Antibody and Recombinant Protein Production Facility; Drs Catherine Laurencot, Jay Greenblatt, and Thomas Davis at the NCI Cancer Therapy and Evaluation Program; and Dr David Waters and Vickie Marshall at the Science Applications International Corporation, Frederick, MD. We thank Inger Margulies for technical assistance. Drs Cary Queen, Vijay Chaudhary, and Q.C. Wang made important early contributions in the construction and purification of LMB-2. We also thank clinical personnel at the National Institutes of Health clinical center, including research nurses Deborah Pearson, Valerie Dyer, Miranda Raggio, and Denise Motok, pharmacists Barry Goldspiel, David Kohler, and George Grimes, and finally the medical oncology fellows, attendings, nurses, and consultants.
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ABSTRACT
INTRODUCTION
