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Garden State Cancer Center, Belleville, New Jersey 07109
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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107 Ab molecules are taken up per cell per day (2
, 3)
. The internalized Abs are rapidly delivered to lysosomes and catabolized, but if "residualizing" radiolabels are used (which are trapped inside the cell, usually within lysosomes, after catabolism of the Ab; Refs. 4
and 5
), a large amount of radioactivity accumulates intracellularly. We demonstrated recently that B-lymphoma cells in vitro were efficiently and specifically killed by LL1 conjugated to various radiolabels, including 111In, 125I, 99mTc, 67Ga, 131I, and 90Y (6
, 7)
. Total killing was obtained (greater than 6 logs) under conditions in which a nonreactive Ab labeled in the same way produced no significant toxicity. Many of these radionuclides (111In, 99mTc, and 67Ga) are considered "imaging" radionuclides, but these, as well as 125I, emit low-energy electrons, including both Auger and conversion electrons, which are believed to be the toxic agents. Dosimetry calculations indicated that the amount of radiation delivered by the low-energy electrons was sufficient to produce the toxicity observed. The ß-particle emitters 131I and 90Y also effectively killed target cells in vitro when linked to anti-CD74 (7) . Such ß-particle emitters, which are most commonly used for RAIT, differ markedly from Auger electron emitters in their depth of tissue penetration. Greater tissue penetration is both an advantage and a disadvantage; it allows nontargeted cells (such as antigen-negative tumor cells) to be killed, but it also increases nonspecific toxicity. Circulating ß-particle emitters will deliver a substantial radiation dose to the bone marrow, whereas Auger electron-emitting radionuclides are much less toxic to nontargeted cells. Accordingly, much higher µCi doses of Auger electron emitters can be administered to mice (8) . Our in vitro experiments similarly demonstrated that ß-particle emitters had much greater nonspecific toxicity than Auger electron emitters (7) . Thus, it seems plausible that with Auger electron emitters, tumor therapy might be achieved at doses at which little toxicity occurs. The present study was undertaken to determine whether specific therapeutic effects could be demonstrated in vivo, in a mouse xenograft model, with LL1 conjugates of 111In, 67Ga, and 90Y.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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10 mCi/mg. 90Y conjugation used the chelator 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), as described (9)
, with a specific activity of 5 mCi/mg. Labeled Abs were analyzed by either gel filtration high-performance liquid chromatography on a Bio-Sil SEC-250 column (Bio-Rad, Hercules, CA) or by instant TLC on silica gel strips (Gelman Sciences, Ann Arbor, MI), or both, and >90% of the counts were associated with Ab (usually >95%). Immunoreactivity was determined on representative radiolabeled LL1 preparations, by binding to a large excess of Raji cells, and was 53–65%, with no evident differences between the radiolabels used.
Mice and Immunotherapy.
Female SCID/NCr mice were obtained from the Animal Production Program of the National Cancer Institute (Frederick, MD). Their care was in accord with institutional guidelines. At 4–6 weeks of age, mice were injected with 2.5 x 106 Raji cells i.v. into the tail vein, following the model discovered and characterized by Ghetie et al. (10)
. Although these authors used the Daudi cell line, Raji cells grow similarly (11)
. The mice were monitored daily for hind-leg paralysis, which occurred in 16–24 days in control mice, and were sacrificed when paralysis was observed. For therapy, groups of 9–10 mice were injected at various times with radiolabeled Abs, i.v. into the tail vein. Although virtually all of the untreated control mice became paralyzed, some of the Ab-treated mice were found dead, without having paralysis detected. This tended to occur after 2–4 months of tumor growth, i.e., 1–3 months after all control mice were found paralyzed. Such deaths can probably be attributed to tumor growth at sites other than the spinal canal, as described previously (10
, 12)
, but in this study we did not attempt to determine the cause of death in such mice. Experiments were terminated after 6 months, and all remaining mice at this time were normal and apparently tumor free. Statistical comparisons between treatment groups used the log-rank test.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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shows the results in terms of survival. Many of the surviving mice treated with near-lethal doses had a substantial loss of body weight 2–4 weeks after injection. We define the MTD as the highest dose that does not kill any of the mice. The MTD for all radionuclides was relatively low, much lower than would be expected for normal mice; this is primarily because of the SCID mutation, as discussed below. The MTD for 111In-labeled LL1 was between 300–400 µCi; we used 350-µCi doses in the experiments described below, with no radiation-induced deaths. With 67Ga, the MTD was 225–300 µCi, apparently slightly lower than with 111In (although the difference was not statistically significant). In a preliminary therapy experiment, 250 µCi of 67Ga were more effective than 300 µCi, and approximately half of the mice treated with 300 µCi died without paralysis or other indications of tumor growth at days 25–30. It seems reasonable to assume that these deaths were attributable to radiation toxicity, perhaps combined with effects of tumor growth. The dose of 250 µCi also appeared to kill mice occasionally, despite its therapeutic effectiveness; therefore, we used maximum doses of 240 µCi in the experiments described below. 90Y was much more toxic than either of the other two radionuclides, with 30 µCi still slightly above the MTD. We used doses of 25 µCi in the experiments described below, with no sign of toxicity.
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demonstrates the effective and specific Ab therapy of Raji B-lymphoma cells that were injected i.v. into SCID mice. In this experiment, the 111In-labeled Ab was injected i.v. 5 days after injection of the tumor cells. The dose injected was 350 µCi/mouse (the MTD), and the specific activity was very similar for both the specific (LL1) and nonspecific (MN-14) Ab. As shown, LL1 greatly reduced the tumor growth rate in all of the mice and produced a complete cure (6 months without evidence of tumor growth) in 40% of the mice. Some mice succumbed to the tumor after 2–3 months, which was three to four times longer than the mean survival time of untreated mice (19.2 ± 3.1 days). The nonspecific Ab had a very slight, nonsignificant effect relative to the group of untreated control mice.
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shows a dose-response experiment in which varying doses of 111In-labeled LL1 were injected. The maximum dose injected in this experiment was 275 µCi, slightly less than in the preceding experiment, but having a similar protective effect. With the 275-µCi dose, all of the mice showed delayed paralysis, and 70% were still alive at 10 weeks after tumor injection. However, some of these mice later developed tumors, and there were only 40% long-term survivors. The 92-µCi dose also produced a significant therapeutic effect, but only 20% of the mice survived for >50 days; these 2 mice remained tumor free for >6 months. The 31-µCi dose had only a small therapeutic effect, which however was still statistically highly significant (P < 0.005). A dose of 10 µCi was also tested in the same experiment, although it is omitted from Fig. 1B
for the purpose of clarity; the therapeutic effect of this dose was slightly less than that of the 31 µCi dose but was still statistically significant (P < 0.05). Thus, a dose of 275–350 µCi appears to be necessary to maximize the therapeutic effect. To demonstrate that the therapeutic effect was attributable to the radioactivity and not to the Ab itself, unlabeled LL1 was included as a control in two experiments, used at the same concentration as in the radiolabeled preparation. The protein dose injected ranged from 6 to 14 µg/mouse in the separate experiments. This treatment had no effect relative to the control untreated group (data not shown).
All experiments described above used a 5-day interval between tumor injection and Ab administration. Experiments in which this interval was varied are shown in Fig. 1C
. With an interval of 9 days, there was only a slight therapeutic effect, which however was still statistically significant (P < 0.025). With an interval of 3 days, tumor growth was totally prevented in essentially all of the mice. The only mouse not surviving for 6 months was found dead at day 155, without paralysis, and may have died from causes other than tumor growth.
The nonspecific toxicity of this therapeutic treatment was monitored by weekly determination of body weight for 6 weeks after Ab injection, as well as by visual inspection of the animals. This was included in the experiment shown in Fig. 1C
, with therapy at day 3 after tumor inoculation, in which all of the mice survived for >154 days. Mice cured of tumor growth, by 350 µCi of 111In-labeled LL1, displayed very little toxicity, with only a small loss in body weight of 5.1 ± 2.5% (mean ± SD, n = 9), which was not a statistically significant weight loss. The nadir was at day 12 after Ab injection.
Therapy with 67Ga-labeled and 90Y-labeled LL1.
Therapy with 67Ga-labeled LL1, at a dose of 240 µCi/mouse (the MTD), is shown in Fig. 2
, together with the effect of a control Ab conjugated similarly. The delay between tumor inoculation and therapy was again 5 days. A strong therapeutic effect was observed, similar to the results with 111In conjugates. Also shown are results with a lower dose of 75 µCi of 67Ga; this dose had a relatively small effect that was still statistically significant (P < 0.005). Fig. 3
shows results with 90Y-labeled LL1, also used at the MTD, 25 µCi. This dose had only a slight therapeutic effect; all of the treated mice became paralyzed, and the mean time to paralysis was 29 days, compared with 18 days for the control mice. Although this effect was statistically significant (P < 0.01), it was much less effective than the other radionuclides tested. Therefore, we have not analyzed its specificity by testing a nonreactive Ab.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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30 µCi/ml for 111In conjugates and 3-fold lower for 67Ga conjugates. With an injected dose of 300 µCi/mouse, the expected blood concentration after equilibration with interstitial fluid is 60 µCi/ml (13)
, assuming a blood volume of 2.0 ml, and the expected concentration in interstitial fluid is 30 µCi/ml (14)
. Thus, the dose-responses in vivo and in vitro are consistent. The very high cellular uptake of LL1 is unusual, but it seems possible that a similar approach may be effective with other Abs that are not internalized at such a high level. We showed recently that Abs binding in large amounts to the cell surface, without a high rate of internalization, were also able to deliver toxic amounts of 111In or 125I to cells in vitro (15)
. 111In and 67Ga are primarily used for imaging and are infrequently used for therapy. However, they emit Auger and conversion electrons that will kill cells at a sufficiently high dose (16) . Although this is the first description of therapy with an 111In-labeled Ab, therapy with 111In-labeled octreotide was reported previously (17) . However, it is very unlikely that cellular uptake of octreotide is at a level comparable with that of LL1. It has been suggested that the therapeutic effect of 111In-labeled octreotide is attributable to nuclear localization (18) . Ab conjugates of 125I, also an Auger electron emitter, have been used previously for tumor therapy in animal models (8 , 19) and in clinical studies (20 , 21) . These conjugates displayed some cell killing in vitro (19 , 21) , although much less than that obtained with LL1. The conventional iodine label that was used in these studies is not useful with LL1, as we have shown previously in vitro (6) , because the radioactive catabolic products are rapidly released from the target cell (5) .
The comparison of Auger electron emitters with ß-particle emitters is an important issue. In vitro, ß-particle emitters (131I and 90Y) killed B-lymphoma cells efficiently and specifically when conjugated to Ab LL1 (7)
. Such toxicity is predicted by the intracellular S-values of the radionuclides used (16)
. But ß-particle emitters produced greater nonspecific toxicity in vitro than Auger electron emitters (7)
, and our in vivo studies with 90Y demonstrated this same difference. The MTD of the Ab conjugate was only 25 µCi for 90Y, 10-fold lower than for 111In or 67Ga. At the MTD, 90Y conjugates produced a very weak therapeutic effect. Again, these in vivo results were consistent with the in vitro dose-response. That is, the 25-µCi dose is expected to produce a peak concentration in interstitial fluid of 2.5 µCi/ml; this concentration, in vitro, produced a small but significant level of cell kill, 90%. However, the conclusions for 90Y may or may not be applicable to lower-energy ß-particle emitters such as 131I. Lower energy ß-particles will have greater potency in single-cell kill and possibly less nonspecific toxicity than 90Y. Thus, 131I appeared preferable to 90Y in our in vitro studies. Unfortunately, the various residualizing forms of 131I that we have tested in vitro are not suitable for in vivo experiments because of low specific activity and/or high accumulation in the kidney or liver (22)
. Improved residualizing iodine labels are currently under development. However, on the basis of our in vitro data, it seems unlikely that any of the ß-particle emitters will display the therapeutic specificity seen with the Auger electron emitters. Even so, ß-particle emitters may have an advantage in treating solid tumor masses because of the ability of ß-particles to kill cells at some distance away from the site of emission. This factor may constitute a major advantage in cases in which full penetration of the tumor by the Ab is not readily achieved or where antigen expression on cells is heterogeneous. An attractive approach is to combine two radionuclides, on different or the same Abs, to adequately attack both microscopic disease and large tumor masses.
The apparent limitation of this method to treatment of a low disease burden should be noted. Although effective at day 5 after tumor inoculation, it had little effect at day 9. It is possible that better control of larger tumors may be achieved by administering multiple doses, or including ß-particle emitters, as noted above. On the other hand, the rapid growth of this tumor xenograft (control mice normally become paralyzed at days 16–24) makes it a challenging model for therapy. In any case, this strategy seems most directly applicable to patients with suspected microscopic tumors.
A comparison of 125I (Auger electron emitter) with 131I (ß-particle emitter) for RAIT was reported recently by Behr et al. (8)
. The MTD was 3.0 mCi/nude mouse for 125I and 10-fold lower for 131I, and at the MTD 125I was more potent than 131I in tumor therapy using a nude mouse model with a s.c. carcinoma xenograft. These results, together with ours, suggest the possibility that Auger electron emitters may be generally preferable to ß-particle emitters for RAIT. We are not aware of any case in which the inverse was found, with both radionuclides used at their MTDs. But much additional work is required to determine whether this speculation is correct. Behr et al. (8)
also demonstrated that the dose-limiting toxicity for 125I-labeled Abs was bone marrow toxicity, as it is for Ab conjugates with ß-particle emitters. Work with various radiometals, such as 90Y or 67Cu, which are residualizing labels, demonstrate that the bone marrow is also the dose-limiting organ with these labels (23
, 24)
, at least if stable chelators are used. Although we have not examined toxicity to other organs, the bone marrow is likely to be the dose-limiting organ for radioconjugates of LL1.
In vitro, 67Ga appeared to be better than 111In as a toxic conjugate because of its greater level of specific killing (7) . This difference was not confirmed in these therapy experiments, in which 111In was equivalent to 67Ga or better. Although the reason for this discrepancy is not known, the most likely explanation is the difference in specific activity, which was up to 8-fold higher for the 111In conjugates. It may therefore be useful to develop methods of conjugating 67Ga to Abs with higher specific activity.
Effective RAIT in animal models, with human tumor xenografts, has been reported many times, using tumors of various histological types (25) . However, we are not aware of previous reports of effective RAIT of disseminated B-cell lymphoma using the SCID mouse model, as used herein. Using nude mouse s.c. xenograft models of B-cell lymphoma, effective RAIT was described with 131I-labeled anti-CD37 (26) and 67Cu-labeled Lym-1 (anti-MHC class II; Ref. 24 ). Recently, Wei et al. (12) described effective RAIT of s.c. lymphomas in immunodeficient mice, with 131I-labeled anti-CD22 or anti-CD19. The therapy was much more effective in nude mice than in SCID mice because of the greater radiation sensitivity of the latter. These authors also attempted to treated disseminated tumors; RAIT alone was ineffective, but combined treatment with RAIT and an immunotoxin was very potent. We are currently evaluating the ability of 111In-labeled LL1 to provide therapy for s.c. lymphoma xenografts. The ability of LL1 to specifically localize to s.c. xenografts was demonstrated recently (9) .
The SCID mouse model used herein can be considered to be a particularly challenging model for RAIT, because SCID mice are known to be unusually sensitive to radiation (27 , 28) . Cells from SCID mice are approximately 2–3-fold as sensitive to ionizing radiation as cells from normal mice. This was reflected by the low MTDs obtained with all of the radionuclides tested, which were 2–3-fold lower than the MTDs in normal or nude mice.4 Similarly, Wei et al. (12) found that the MTD of 131I-labeled Abs was 3–5-fold lower in SCID mice than in nude mice. Accordingly, the ratio of the MTD to the effective therapeutic dose, although quite low in this SCID mouse model, would be 2–3-fold greater in normal animals.
The key difference between the mouse model and the human situation is, of course, the lack of antigen expression on normal tissues of the mouse. CD74 is present on normal B cells and macrophage-lineage cells, including Kupffer cells in the liver. It is uncertain how such normal tissue expression would impact on RAIT with this Ab. Because most bone marrow stem cells, including the most primitive population, are negative for the MHC class II antigen (29) and presumably for CD74, they will not be targeted. The lysis of normal B cells, as a side effect of cancer therapy, appears to be acceptable (30 , 31) , and the lysis of differentiated macrophage-lineage cells may also be acceptable. RAIT results with Ab Lym-1 (32) bear directly on this issue; this Ab reacts with the mature MHC class II antigen and thus has essentially the same normal tissue distribution as CD74. Although some liver uptake has been observed with Lym-1, it has been reduced by preinjection of unlabeled Ab and has not prevented the treatment of patients with high mCi doses of ß-particle emitters linked to Lym-1. Moreover, the toxicity of LL1 linked to Auger emitters should be less than that of Lym-1 linked to ß-particle emitters, because the toxicity of the Auger emitter is largely limited to cells which directly bind and internalize the Ab, as noted above. Thus, the delivery of 111In-labeled LL1 to Kupffer cells would be expected to have little effect on adjacent hepatocytes, although this is not the case for ß-particle emitters.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grants CA87059 (to M. J. M.) and CA39841 (to D. M. G.). 
2 To whom requests for reprints should be addressed, at Garden State Cancer Center, 520 Belleville Avenue, Belleville, NJ 07109. Phone: (973) 844-7013; Fax: (973) 844-7020; E-mail: mjmattes.gscancer{at}worldnet.att.net
3 The abbreviations used are: Ab, antibody; RAIT, radioimmunotherapy; SCID, severe combined immunodeficient; MTD, maximum tolerated dose.
Received 11/ 8/00; revised 1/18/01; accepted 3/ 9/01.
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) or 111In-labeled MN-14, a nonreactive control antibody (
). A control group of 10 mice had no antibody injected (
). The effect of LL1 was statistically significant, with P < 0.001 compared with either of the other two groups. In B, groups of 10 mice were treated on day 5 with 275 (
), 92 (
) or 67Ga-labeled MN-14, a nonreactive control antibody (