Synergistic antitumor activity of anti-CD25 recombinant immunotoxin LMB-2 with chemotherapy

Abstract

Purpose

Although anti-CD25 recombinant immunotoxin LMB-2 is effective against CD25+ hairy cell leukemia, activity against more aggressive diseases like adult T-cell leukemia (ATL) is limited by rapid progression between cycles. Our goal was to determine in vivo if rapid growth of CD25+ tumor is associated with high levels of tumor interstitial soluble CD25 (sCD25) and if chemotherapy can reduce tumor sCD25 and synergize with LMB-2.

Experimental design

Tumor xenografts expressing human CD25 were grown in mice, which were then treated with LMB-2 and chemotherapy either alone or in combination, and sCD25 and antitumor activity were measured.

Results

CD25+ human xenografts growing rapidly in nude mice had intratumoral sCD25 at levels 21–2200 (median 118)-fold higher than in serum, suggesting that interstitial sCD25 interacts with LMB-2 in tumors. Intratumoral sCD25 levels were 21–157 (median 54) ng/ml without treatment and 0.95–6.1 (median 2.6) ng/ml (p<0.0001) one day after gemcitabine. CD25+ xenografts too large to regress with LMB-2 alone were minimally responsive to gemcitabine alone but completely regressed with the combination. Ex vivo, different ratios of gemcitabine and LMB-2 were cytotoxic to the CD25+ tumor cells in an additive but not synergistic manner.

Conclusions

Gemcitabine is synergistic with LMB-2 in vivo unrelated to improved cytotoxicity. Synergism therefore appears related to improved distribution of LMB-2 to CD25+ tumors, and is preceded by decreased sCD25 within the tumor due to chemotherapy. To test the concept of combined treatment clinically, patients with relapsed/refractory adult T-cell leukemia are being treated with fludarabine plus cyclophosphamide prior to LMB-2.

Introduction

Recombinant immunotoxins are fusion proteins containing a bacterial toxin and a targeting Fv fragment of an antibody, and due to the high potency of the toxin, one or a few molecules in the cytosol are sufficient for catalytic activity (ADP ribosylation of elongation factor 2) leading to apoptotic cell death (1–3). Recombinant immunotoxins have shown potent activity in leukemias including hairy cell leukemia (HCL) without causing immunosuppression like chemotherapy (4–8). The first recombinant immunotoxin described was anti-Tac(Fv)-PE40 (9–10), containing a single-chain Fv fragment of anti-Tac (11), a MAb directed against CD25, the alpha subunit of the interleukin-2 (IL2) receptor. The toxin was Pseudomonas exotoxin A containing a deletion of the binding domain, so that the recombinant immunotoxin would bind selectively to CD25, internalize, and induce cell death in CD25+ cells. A derivative of this molecule, called anti-Tac(Fv)-PE38 or LMB-2, containing the toxin fragment PE38, was tested in patients with relapsed and refractory hematologic malignancies, and major responses were observed in HCL, chronic lymphocytic leukemia (CLL), Hodgkin’s lymphoma (HL), cutaneous T-cell lymphoma (CTCL) and adult T-cell leukemia (ATL) (4–5). Important limitations of clinical benefit included immunogenicity and rapid progression, both observed particularly in ATL. Another recombinant immunotoxin, BL22, targeting CD22, as a single-agent demonstrated significant activity against hairy cell leukemia (6, 8), but more limited activity in less indolent leukemias and lymphomas (7), particularly acute lymphoblastic leukemia (12).

A solid tumor antigen, mesothelin, was also targeted with MAb therapy (13), including an anti-mesothelin immunotoxin containing PE38 (14). The anti-mesothelin recombinant immunotoxin SS1P, also containing PE38, has limited clinical activity as a single agent (15–16), and was reported in murine xenografts to exhibit synergistic activity with either taxol (17–18), or gemcitabine (19). In the tumor model, there was no evidence of synergy ex vivo by cytotoxicity assay. It was later shown that chemotherapy depleted the levels of soluble (shed) mesothelin in the interstitial space of the tumors, resulting possibly in decreased interference with the distribution of immunotoxin molecules from binding with the tumor cells (18, 20). We hypothesized that LMB-2, particularly with its high affinity toward CD25 (9, 21), might interact with shed or soluble CD25 (sCD25) accumulating in tumors, prior to reaching the tumor cells. To determine whether chemotherapy could deplete sCD25 in tumors and improve targeting by LMB-2, we studied the combined use of LMB-2 and chemotherapy in a xenograft model and measured both sCD25 and antitumor activity.

Materials and Methods

Cell lines and cytotoxicity assays

Cell lines tested for sCD25 expression included the Hodgkin’s cell lines L540 (22) and L540cy (23), and cell lines transfected with human CD25, including murine plasmacytoma SP2/Tac (24) and the human epidermoid carcinoma line ATAC-4 (25). All cell lines grew in suspension except for ATAC-4. Suspension cells were cultured at 4 × 10⁵ cells/ml (4 × 10⁴/well), while ATAC-4 was plated at 1 × 10⁴/well in 96-well plates. Cytotoxicity assays were performed as described (21, 25).

Determination of interstitial sCD25

As previously reported (20), tumor extracellular fluid was isolated by excising tumors and centrifuging at 4°C over 297 um mesh (Spectra/Mesh, Spectrum, Houston, TX) in a 1.5 ml microfuge tube. An initial centrifugation for 10 min at 1500 RPM was performed to remove fluid on the outside of the tumor, followed by a second centrifugation for 10 min at 5000 RPM to obtain the tumor extracellular (or interstitial) fluid. Additional interstitial fluid with similar sCD25 could be obtained in mouse tumors with a 3^rd 10 min centrifugation at 15,000 RPM, although in human tumors, the sCD25 level in the 3^rd centrifugation was higher than in the 2^nd. sCD25 levels of serum and tumor fluid were determined using the high sensitivity kit obtained from R&D systems (Minneapolis, MN).

Antitumor experiments

Animal experiments were conducted under an approved protocol. ATAC-4 cells (1 × 10⁶ cells in 0.1 ml DMEM) were injected subcutaneously in ~20g athymic nude mice (Frederick, MD) and established tumors usually appeared by day 4. Mice were treated with LMB-2 and/or gemcitabine diluted in PBS containing 0.2% human serum albumin (HSA-PBS) at 10 uL/g body weight), intravenously (IV) via tail vein as described (21, 25) for LMB-2, and intraperitoneally (IP) for gemcitabine (19). When treated with gemcitabine and LMB-2 on the same day, the gemcitabine was administered first.

Statistics

Statistical analysis was performed by SAS version 8.02 using Wilcoxon non-parametric comparison of tumor sizes, or Fisher’s Exact comparison of regression rates. Combination index values to determine in vitro synergy were calculated using CalcuSyn 2.0 (Biosoft, Cambridge, UK).

Clinical trial data

Preliminary data are from a phase II clinical trial (NCT00117845) using fludarabine, cyclophosphamide and LMB-2 for adult T-cell leukemia. The trial and its consent forms were approved by the NCI investigators review board. Human samples tested were also covered by the protocol.

Results

While recombinant immunotoxins are markedly effective against HCL where most of the tumor burden is suspended in the peripheral blood or spleen (4–8), clinical activity in more aggressive malignancies with solid collections of tumor cells has been more limited. To determine whether sCD25, like soluble mesothelin (17, 19), can concentrate within tumors and potentially block immunotoxin distribution, we decided to measure sCD25 within CD25+ ATAC-4 xenografts before and after administration of gemcitabine chemotherapy. Mice bearing the xenografts were treated with LMB-2 and gemcitabine alone and in combination to establish whether these agents would display in vivo synergy. Finally, different combinations of gemcitabine and LMB-2 were incubated with the ATAC-4 cells in tissue culture to determine if these agents would display antagonistic, additive or synergistic cytotoxicity ex vivo.

Determination of sCD25 production from tumor cells

To determine the extent to which CD25+ tumors produce sCD25, the tumor cells were cultured ex vivo and supernants tested for sCD25. As shown in Table 1, ATAC-4 produced the highest levels of sCD25, 34 ng/ml. The second highest was SP2/Tac, 24 ng/ml, but this cell line did not grow reproducibly as subcutaneous tumors in nude mice. The Hodgkin’s lymphoma lines L540 and L540cy are able to grow in mice but had sCD25 levels in supernants < 1 ng/ml. Thus of the cell lines tested, ATAC-4 was best able to serve as a model for targeting ATL tumors with LMB-2, where the cells produce high levels of sCD25.

Table 1.

sCD25 levels in supernant of cell lines

Cell line	sCD25 (ng/ml)
SP2/Tac	24
L540	0.96
L540CY	0.525
ATAC-4	34

Levels of sCD25 in serum vs interstitial space of tumor xenografts

To determine the levels of sCD25 within the extravascular space of tumors, mice were injected subcutaneously with 1 × 10⁶ ATAC-4 cells on day 0, and subcutaneous tumors 126–1170 (median 330) mm³ were harvested. As shown in Figure 1A, sCD25 levels in the untreated tumor interstitial fluid were 21–157 (median 54) ng/ml, much higher than the serum levels shown in Figure 1B, < 0.02–1.6 (median 0.38) ng/ml (p<0.0001). For 11 mice with detectable serum sCD25, the ratios of tumor to serum sCD25 levels were 43–2200 (median 118). In the 10 mice with tumor sCD25 < 100 ng/ml, the tumor sCD25 correlated directly with serum sCD25 (r=0.81, p=0.004). No correlation was observed between tumor size and either tumor or serum levels of sCD25, indicating that other factors such as tumor architecture may influence levels of sCD25 in tumor and serum. Thus, like mesothelin (20), sCD25 was present in much higher levels in tumors than in serum of tumor-bearing mice. However, instead of the ~10-fold tumor to serum ratio observed with soluble mesothelin (20), this ratio with sCD25 was usually > 100.

Figure 1. Soluble CD25 (sCD25) measured in tumor bearing mice.

ELISA-determined sCD25 levels are shown in the interstitial space of ATAC-4 tumors (A) and in the serum (B) of mice. Tumors were excised from mice which were either untreated (Untx) or 1 or 2 days after gemcitabine 100 mg/Kg IP. Serum sCD25 levels shown as ≤0.001 ng/ml in B were undetectable (Undet).

Effect of gemcitabine on tumor interstitial sCD25

To determine whether gemcitabine would decrease concentrations of sCD25 within the interstitial space of the CD25+ tumors, nude mice bearing CD25+ ATAC-4 tumors were treated with gemcitabine 100 mg/Kg and interstitial concentrations of sCD25 were determined 1 and 2 days later. As shown in Figure 1A, just 1 day after gemcitabine, tumor sCD25 levels in 33 mice were 0.95–6.1 ng/ml, much smaller than tumor sCD25 in untreated mice (p<0.0001), and the median tumor sCD25 level of 2.6 ng/ml was < 5% of the 54 ng/ml median for untreated mice. In this period of time, there was no significant decrease in the tumor size. Tumors from 10 other mice were harvested 2 rather than 1 day after treatment with gemcitabine, and as shown in Figure 1A, the tumor interstitial sCD25 levels, 17–118 (median 50) ng/ml, were much higher than at 1 day (p < 0.0001) and no different from those of untreated mice (p=0.5). Thus, gemcitabine depleted sCD25 from the interstitial space of tumors within 1 day, but the interstitial sCD25 recovered after an additional day, possibly because of recovery of cellular and micro-environmental characteristics of the tumor. As shown in Figure 1B, serum levels of sCD25 were undetectable in most mice 1 day after gemcitabine, significantly lower than serum sCD25 in untreated mice (p<0.0001), but serum sCD25 levels 2 days after gemcitabine were similar to those of untreated mice (p=0.11).

Antitumor activity of LMB-2 given after gemcitabine in CD25+ tumor-bearing mice

To determine if gemcitabine is synergistic in vivo with LMB-2, nude mice were injected with 1–2 ×10⁶ ATAC-4 cells on day 0 and treated with each of these agents alone or combined, as detailed in Table 2. In the first experiment, mice were first treated with gemcitabine chemotherapy on day 4 and 6, followed by LMB-2 on days 6, 8 and 10. This design was modeled from an ongoing clinical trial in which ATL patients are treated with fludarabine plus cyclophosphamide (FC) on days 1, 2 and 3, followed by LMB-2 on days 3, 5 and 7. In the clinical trial, FC is given prior to LMB-2 not only to decrease tumor burden and sCD25 in ATL tumors, but also to decrease immunogenicity. In the mouse experiment shown in Figure 2A, LMB-2 and gemcitabine have similar activity alone, and are much more effective when combined. The combination of gemcitabine 80 mg/Kg IP x2 and LMB-2 100 ug/Kg IV x3 produced greater tumor regression than gemcitabine alone (p=0.009–0.04 days 13–14, p=0.001 days 16 and 19, p<0.001 days 17 and 20) or LMB-2 alone (p=0.015–0.024 days 13–20). In fact, even half of each agent combined was more effective than gemcitabine (p=0.003–0.04 days 13–14, p<0.001 days 16–20) or LMB-2 (p=0.03–0.045 days 12–17 and day 20) alone. Thus, LMB-2 given after 2 doses of gemcitabine was synergistic, since the antitumor activity was significantly greater than either LMB-2 or gemcitabine alone even when half the dose of each was combined. However, the group receiving LMB-2 alone may have had larger tumors because treatment in this group was delayed 2 days more than in the other groups.

Table 2.

Doses used for antitumor experiments

Experiment A:
Group	n	Gemcitabine (IP)	LMB-2 (IV)
1	9	None	100 ug/Kg d6, 8,10
2	9	80 mg/Kg d4, 6	None
3	9	80 mg/Kg d4, 6	100 ug/Kg d6, 8,10
4	10	40 mg/Kg d4, 6	50 ug/Kg d6, 8,10
5	10	20 mg/Kg d4, 6	25 ug/Kg d6, 8,10
Experiment B:
Group		Gemcitabine (IP)	LMB-2 (IV)
0	5	None	None
1	10	None	160 ug/Kg d6, 8,10
2	9	80 mg/Kg d6, 8,10	None
4	10	40 mg/Kg d6, 8,10	80 ug/Kg d6, 8,10
5	10	20 mg/Kg d6, 8,10	40 ug/Kg d6, 8,10
Experiment C:
Group		Gemcitabine (IP)	LMB-2 (IV)
0	5	None	None
1	10	None	120 ug/Kg d7, 9,11
2	10	60 mg/Kg d7, 9,11	None
3	10	60 mg/Kg d7, 9,11	120 ug/Kg d7, 9,11
4	10	30 mg/Kg d7, 9,11	60 ug/Kg d7, 9,11
5	10	15 mg/Kg d7, 9,11	30 ug/Kg d7, 9,11

Figure 2. Antitumor activity of LMB-2 and gemcitabine.

For experiments A, B and C, mice were injected subcutaneously with ATAC-4 cells (2×10⁶ in A, 1×10⁶ in B–C) on day 0, and treated with gemcitabine and LMB-2 at the doses and time points indicated in Table 2. Group 0 (◆) received neither, group 1 (❍) LMB-2 only, group 2 (●) gemcitabine only, group 3 (△) in A and C, both at full doses, and groups 4 (▲) and 5 (◊) received both at ½ and ¼ doses, respectively.

Antitumor activity of gemcitabine and LMB-2 given on the same days

To determine the synergistic activity of gemcitabine with LMB-2 more accurately, both agents were given on the same day. As shown in Table 2 and Figure 2B, mice receiving gemcitabine 40 mg/Kg IP plus LMB-2 80 ug/Kg IV on days 6, 8 and 10 (group 4) had smaller tumors than mice receiving twice this dose of either gemcitabine (p=0.025 day 9, p=0.001–0.007 days 10–12 and 16–20, p<0.001 days 13–15) or LMB-2 (p=0.003 day 12, p<0.001 days 13–20) alone. Moreover, even a ¼ dose of the combination of gemcitabine and LMB-2 (group 5) showed higher antitumor activity than either untreated mice (p=0.028–0.05 days 8–9, p=0.003–0.008 days 10–13, p<0.001 days 15–16), or mice receiving gemcitabine (p=0.02 days 8 and 18, p=0.0036–0.009 days 9, 10, 16 and 20, and p<0.001 days 12–15) or LMB-2 (p=0.003 day 12, p<0.001 days 13–20) alone. To evaluate synergy more accurately using larger tumors and lower doses, mice received gemcitabine 60 mg/Kg IP and/or LMB-2 120 ug/Kg IV days 7, 9 and 11, as well as ½ and ¼ doses of the combination (Table 2). As shown in Figure 2C, gemcitabine and LMB-2 were again synergistic, with complete regression in all mice receiving the full dose combination, persisting in 9 of 10 mice past day 60, compared to no complete regressions in mice receiving gemcitabine or LMB-2 alone (p<0.001). The time to reach a mean tumor size of 150 mm³ was 51 days for the full dose combination (group 3), compared to 7 days for untreated mice and 12 or 15 days for mice receiving LMB-2 (group 1) or gemcitabine (group 2) alone, respectively. Thus LMB-2 and gemcitabine each prolonged tumor progression by 5 and 8 days, respectively, making 13 days the expected prolongation of progression for additive antitumor activity. The observed prolongation by the combination of 44 (51 minus 7) days thus confirms in vivo synergy. Moreover, even when combined at ¼ the dose levels (group 5), gemcitabine and LMB-2 were more effective than either gemcitabine (p=0.016–0.04 days 9 and 26, p<0.001 days 11–20, p=0.005–0.008 days 22 and 24) or LMB-2 (p=0.002 days 11–20) alone. In vivo synergy relative to toxicity could also be expressed in this experiment by dividing days of prolongation of tumor progression by the ratios of dose used to the LD10 dose. By this definition, the tumor progression values were 37–47 for the combination vs 9 for LMB-2 vs 11 for gemcitabine alone.

Dose-response of gemcitabine combined with LMB-2

To determine dose-response, tumor sizes and complete regression rates in mice receiving 60 mg/Kg IP plus LMB-2 120 ug/Kg IV days 7, 9 and 11 (group 3) were compared with ½ (group 4) and ¼ (group 5) doses of the combination (Table 2, Figure 2C). Complete regression rates in these 3 respective groups of 10 mice (groups 3, 4 and 5 in Table 2) were 100%, 60% and 10% (p<0.001). Permanent complete regression rates in groups 3–5 were 90%, 30% and 10% (p=0.001), respectively. Tumor sizes in group 3 were smaller compared to either group 4 (p=0.03–0.05 days 12, 22 and 24, p=0.001–0.007 days 16–20, 28, and 30), or group 5 (p=0.001–0.007 days 11–16, 20–30, p<0.001 day 18), and the 2 smaller groups 4 and 5 also differed from each other with respect to tumor sizes (p=0.004–0.015 days 15–28, p=0.025 day 30). Thus by either measurement of regression rates or tumor sizes, a dose-response was observed in the 3 different dose levels of gemcitabine and LMB-2 combinations.

Assessment of synergy ex vivo

To determine if the synergy observed in vivo between gemcitabine and LMB-2 was due to synergistic cytotoxicity toward the cell line used, instead of other factors like improved immunotoxin distribution, different concentrations of LMB-2 and gemcitabine were studied in cytotoxicity assays ex vivo. Isobolograms are shown in Figure 3, where concave, linear and convex curves are expected for synergistic, additive and antagonistic cytotoxicity ex vivo, respectively (26–27). Both IC₅₀ and IC₇₅ isobolograms in Figures 3A and B, respectively, show additive cytotoxicity, with the regression most consistent with a straight line (r² = 0.78–0.9, p<0.001). Another method for determining synergy is by calculation of combination indices (CI) from the percent inhibition ex vivo at each combination of the 2 agents tested, with values < 1 consistent with synergy, particularly with CI <0.1 (28). Calculated CI values for the different ratios of LMB-2 and gemcitabine tested are shown in Table 3. The LMB-2 : Gemcitabine ratio used in vivo was usually 1:500 (Table 2, Figures 2B and 2C).

Figure 3. Cytotoxicity of LMB-2 and gemcitabine towards ATAC-4 cells.

Dilutions of different ratios of LMB-2 and gemcitabine were incubated with ATAC-4 cells for 20 hours, and the calculated concentrations needed for 50% (A) and 75% (B) inhibition of protein synthesis are shown.

Table 3.

Combination index (CI) values for LMB-2 and gemcitabine

LMB-2: gemcitabine ratio	IC25	IC50	IC75
(1:4)	1.033	1.018	1.049
(1:5)	1.205	0.972	1.499
(1:10)	2.269	1.463	3.538
(1:20)	0.841	0.899	0.791
(1:100)	1.504	0.896	2.554
(1:100)	1.028	0.659	1.643
(1:500)	0.598	0.695	0.520
(1:1000)	0.979	0.893	1.088
(1:2000)	0.515	0.874	0.306
(1:2500)	0.625	1.463	0.267
Median CI values:	1.004	0.897	1.069

Ratios are based on ng/ml of LMB-2 and gemcitabine added simultaneously to ATAC-4 cells ex vivo

IC₂₅, IC₅₀, and IC₇₅ are defined as the calculated concentrations necessary for 25, 50 and 75% inhibition, respectively.

Although some variability was observed ex vivo, CI values were usually >0.5 and medians were very close to 1.0. IC₇₅ CI values as low as 0.267 were calculated for LMB-2 : gemcitabine ratios of 1:500 to 1:2500 (Table 3). However, since the half-life of LMB-2 (α= 35 min, β= 192 min) is longer than that of gemcitabine (17 min) in mice (21, 29), the higher IC₇₅ CI values (0.791–3.538) from the higher LMB-2 : gemcitabine ratios (1:4 to 1:100) are more relevant. Thus the in vivo synergism between gemcitabine and LMB-2 observed in tumor bearing mice is not consistent with synergistic cytotoxicity against the ATAC-4 cells making up the tumor. Rather, the in vivo synergy may be due to other factors, such as improved distribution of LMB-2 to the tumor cells.

Discussion

Our goal was to determine whether sCD25, like soluble mesothelin, can concentrate within CD25+ tumors, and whether improved antitumor activity can be achieved by combining the anti-CD25 recombinant immunotoxin with chemotherapy. We measured sCD25 within CD25+ xenografts before and after administration of gemcitabine chemotherapy, and then determined whether gemcitabine and LMB-2 would show in vivo or in vitro synergy. We found that sCD25 levels within the interstitial space of tumors were much (median > 100-fold) higher than in the serum, and could be reduced >10-fold with gemcitabine. Moreover, gemcitabine and LMB-2 showed synergistic antitumor activity in vivo but only additive cytotoxicity in vitro, consistent with improved targeting of LMB-2 to tumors.

Synergy of chemotherapy with other immunotoxins

While SS1P targeting mesothelin is the only other recombinant immunotoxin to our knowledge which has been shown to exhibit synergistic in vivo antitumor activity with chemotherapy (17, 19–20), synergism with chemotherapy has been reported for other immunotoxins and recombinant growth-factor fusion toxins, either in vitro, in vivo, or both. Examples of in vitro synergism were reported using chemotherapeutic agents with an anti-p40 prostate antigen ricin A-chain immunotoxin conjugate (30), the immunotoxin 317G5-rRTA directed against ovarian cancer (31), the diphtheria toxin-containing fusion toxin DT388-GM-CSF toward acute myelogenous leukemia (26–27), the anti-CD20 immunotoxin containing rituximab and saporin-S6 (32), and the anti-erbB2 recombinant immunotoxin HEL-PE38KDEL against breast and gastric cancer (33). Additive toxicity was reported in vitro and in vivo with doxorubicin and anti-transferrin receptor-ricin A chain immunotoxin (34). Chemotherapy with anti-CD19 anti-B4-blocked ricin showed in vitro synergism (35–36), and improved antitumor activity (35). In vivo synergism was reported with cyclophosphamide and NRLU-10/PE, an anti-colon carcinoma conjugated with whole Pseudomonas exotoxin (37). Interferon and daunorubicin synergized in vivo with SN1-RA and SN2-RA, ricin A-chain immunotoxins targeting their respective T-cell antigens, TALLA and GP37 (38). The recombinant circularly permuted fusion toxin IL4(83-37)-PE38KDEL, targeting IL4-receptor-bearing carcinomas and lymphomas (39), was shown to be synergistic in vitro and in vivo with gemcitabine against pancreatic adenocarcinoma (40). Finally, bexarotene was found to upregulate CD25 in CTCL cells of patients treated with the anti-IL2-receptor recombinant fusion toxin denileukin diftitox, and the combination was well tolerated (41). Other than SS1P, we were unable to find any previous reports of chemotherapy-immunotoxin combinations shown in animals to have in vivo without in vitro synergy.

Synergy with recombinant immunotoxins having limited plasma lifetime

Compared to monoclonal antibodies, which have half-lives up to several weeks in duration, recombinant immunotoxins of ~63 kDa in size are much more limited in half-life, quantified in humans at ~8 hours for SS1P (15) and 4 hours for LMB-2 (5). Although the short half-life of LMB-2 may limit capillary leak syndrome (5), it may also limit distribution of the recombinant immunotoxin to the cells within a tightly packed tumor. Unlike conventional immunotoxins, smaller recombinant immunotoxins like LMB-2 may not remain in the plasma long enough for the immunotoxin molecules to bind to tumor cells, particularly if the immunotoxin encounters a block or delay in reaching the target cell. Thus synergistic combinations with chemotherapy or other agents may be particularly needed for these smaller targeting agents.

Mechanism of synergy in mice receiving LMB-2 and gemcitabine

The cytotoxicity experiment in Figure 3 excludes synergistic cytotoxicity in vitro as the mechanism for in vivo synergism of LMB-2 and gemcitabine, making improved distribution of LMB-2 to tumor cells the likely outcome of combined use with chemotherapy. Because murine CD25 and sCD25 do not bind anti-Tac MAb or LMB-2, it was not our goal to further define the exact mechanisms of synergy in our xenograft model. Improved tumor distribution of LMB-2 could be due to mechanisms in addition to or instead of sCD25 depletion, including decreased packing of tumor cells due to partial cytotoxicity of chemotherapy (42). In this latter case, the sCD25 depletion would be a marker of antitumor activity rather than a cause of synergism. Whether or not intratumoral sCD25 can in fact prevent distribution of LMB-2 to tumor cells might depend on the off-and on-rates of LMB-2 for sCD25, the stability of LMB-2 in tumor interstitial space vs time course of LMB-2 in the serum, the 3-dimensional relationships between sCD25 and tumor-associated CD25 molecules within the tumor, and complex interactions between LMB-2, sCD25, and other contents of the tumor interstitial microenvironment. It is unlikely that synergistic tumor regression was due to nonspecific toxicity of chemotherapy to the animals, resulting in decreased tumor size as animals lose weight, since animals remained healthy during the experiment. It is also unlikely that gemcitabine creates synergy by increasing CD25 on the malignant cells, since expression levels are already ~2 ×10⁵ sites/cell (25). Indirect mechanisms, for example involving cytokines liberated by either agent, are unlikely since murine factors would be inactive against human xenografts. LMB-2 can eliminate T-regulatory (Treg) cells expressing CD4, CD25 and Foxp3 of human but not of murine origin (43), and previous experiments indicate that this would be unlikely to lead to synergy in patients due to rapid reconstitution of Treg cells from CD25-negative T-cells (44). Thus we believe that gemcitabine induces in vivo synergy with LMB-2 by allowing better distribution of LMB-2 to tumor cells. While these animal experiments cannot further define the mechanism of synergy with LMB-2 and chemotherapy, they do suggest sCD25 as an important marker to follow in serum and tumors of patients being treated.

Time course of sCD25 decrease and synergy

We found that intratumoral and serum sCD25 were significantly decreased 1 but not 2 days after gemcitabine dosing in mice (Figure 1), but that synergy was achieved by dosing gemcitabine and LMB-2 every 2 days (Figures 2B and 2C). Biodistribution studies of LMB-2 using the ATAC-4 model previously reported persistence of LMB-2 in the tumor interstitial space at >2000 ng/ml for 15 minutes to 6 hours after IV injection, and 460 ng/ml at 24 hours (21). These earlier experiments suggest that significant levels of LMB-2 are present in the tumor 24 hours after gemcitabine dosing, the time point when sCD25 is depleted. Thus chemotherapy administered on the same day as LMB-2 could allow improved distribution of LMB-2 to tumor cells 24 hours later when the chemotherapy effect has occurred.

Relevance to clinical testing of LMB-2 in ATL

At this time, a clinical trial is ongoing in which FC chemotherapy is administered prior to LMB-2 to patients with aggressive (leukemic or lymphomatous) forms of ATL. Patients receive 3 daily doses of FC, and 2 weeks later begin cycles every 3 weeks in which FC is administered on days 1, 2 and 3, followed by LMB-2 on days 3, 5 and 7. The goal of this trial using FC is not only to improve LMB-2 distribution to solid ATL tumors by use of chemotherapy, but also to help prevent rapid progression of ATL between cycles and to block immunogenicity. FC was chosen for the clinical trial based on transplant data in humans showing safe reductions of normal T-and B-cells (45–46) and prevention of human anti-murine antibodies (47), but could not be used in mice due to unfavorable pharmacokinetics. Rather than the purine analog fludarabine, we therefore used the pyrimidine analog gemcitabine, which has activity in both hematologic and solid human tumors (48–49) and has shown synergistic antitumor activity in mice with recombinant immunotoxin SS1P (19). Since prevention of immunogenicity was not a goal of the xenograft model, gemcitabine was an appropriate substitute for FC in mice. In the clinical trial of FC/LMB-2, lack of immunogenicity has allowed up to 4 cycles of combination FC and LMB-2 to be administered, and in 9 evaluable patients a response rate of 56% has been observed, including 33% complete remissions. Although sCD25 data is limited from excisional biopsies, in one patient prior to treatment, the sCD25 was 32.9 ng/ml in the tumor vs 7.8 ng/ml in the serum. We believe that both clinical and laboratory data from combined use of chemotherapy and immunotoxins will lead to expansion of the successful use of these biological agents in the treatment of hematologic and solid tumors.

Statement of translational relevance.

Recombinant anti-CD25 immunotoxin LMB-2 has had limited efficacy in patients with rapidly growing tumors. In this study, we determined that CD25+ tumor xenografts contain levels of soluble CD25 (sCD25) within the tumor xenograft interstitial space which are >100-fold higher than in serum. We were able to reduce both intratumoral and serum sCD25 levels with chemotherapy and observed in vivo synergy with LMB-2. When the tumor cells were treated ex vivo with chemotherapy and LMB-2, no synergy was observed, indicating that the in vivo synergy was due to improved distribution of LMB-2 to the tumor, rather than improved cytotoxicity. Thus clinical results were brought to the lab for study, and combination treatment was developed which was in turn translated back to the clinic. At this time, a clinical trial using chemotherapy and LMB-2 is ongoing in adult T-cell leukemia, a rapidly growing and fatal leukemia/lymphoma with inadequate standard options.