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Experimental Therapeutics |
University of Minnesota Cancer Center, Departments of Therapeutic Radiology, Section on Experimental Cancer Immunology [D. A. V., N. J., J. M. R. B.], and Pediatrics, Division of Bone Marrow Transplantation [A. P-M., B. R. B.], Minneapolis, Minnesota 55455, and Baylor College of Medicine, Houston, Texas 77030 [S-Y. C.]
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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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Cytokine fusion toxins are experimental pharmacological agents that are made by linking cytokines that specifically bind to cancer cells to potent catalytic toxins such as DT.3 Intact DT contains two fragments, A and B. The A fragment catalyzes the ADP-ribosylation of elongation factor 2 (EF-2) leading to protein-synthesis inhibition and cell death (5 , 6) . Although a single molecule of DT fragment A in the cytosol can be fatal to a cell, fragment A alone applied extracellularly is not highly toxic because the binding domain is located in fragment B. Thus, we included only the first 389 amino acids of DT that contained fragment A for our construct.
The major purpose of cytokine IT is to deliver therapy selectively to cancer cells that frequently overexpress cytokine receptors (7) . Although these agents selectively bind and kill cancer cells, clinically their use has been limited by: (a) their failure to penetrate and localize in adequate concentrations in cancer target tissue; and (b) their localization in nontarget organs limiting the tolerated dose and narrowing the therapeutic window (8) . We have explored a solution to this problem, reasoning that T cells that are highly suited for local cytokine delivery in the first place would be excellent vehicles for the delivery of cytokine fusion toxins if induced to secrete them by retroviral gene transduction. The secreted cytokine IT would preferentially destroy cancer cells displaying the appropriate cytokine receptor on their cell surface but would not kill normal tissue cells lacking the antigen. We chose a murine acute myeloid leukemia model because this leukemia is the most common form of adult leukemia (9) . IL-4 was chosen as the ligand for our IT because IL-4R is expressed on over 90% of acute myeloid leukemia cases (10) and IL-4 IT has been previously used in experimental cancer therapy studies (11, 12, 13) . DT was chosen as a toxin because it has extensive clinical use and has a history of use as a recombinant toxin (14, 15, 16) .
These studies show for the first time that gene therapy can be used to manipulate antigen-specific T cells into secreting cytokine fusion toxins with selective activity against myeloid leukemia cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, for transfection studies in NIH.3T3. For transduction, the
target gene was ligated into the retroviral expression vector LNCX
(Fig. 1B)
or a modified LNCX in which a fragment encoding
Neo was replaced with a gene fragment encoding human NGFR
(18
; Fig. 1C
). Successful integration of this
retrovirus resulted in the cell surface expression of NGFR,
which could be used as a quantitative marker. To produce a purifiable
IL-4 IT, a cytokine fusion toxin gene was assembled using DNA fragments
encoding IL-4 without signal peptide (amino acids 20–140) spliced to
DT390. The hybrid gene was ligated into a
commercial pET expression vector, pET21d (Novagen, Madison, WI).
Successful cloning of the designed gene was confirmed by sequencing.
Expression was induced and a Mr
60,000 recombinant protein was purified by ion exchange
chromatography as described previously (19)
.
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Genomic PCR.
To detect the DT provirus integrated into genomic host DNA, DNA from
transduced T15 cells was studied by PCR using Taq polymerase
(Perkin-Elmer, Foster City, CA) and primers 5'-GCGCTGATGATGTTGTTGAT-3'
and 5'-AAATGGTTGCGTTTTATG-3'. Amplification in a DNA thermal cycler
(Perkin-Elmer; 30 cycles at 94°C for 30 s, 55°C for 60 s,
and 72°C for 120 s), produced an 1170-bp product.
Transfection, Harvesting Viral Supernatants, and Viral
Transduction.
For transfection of IT genes into mammalian cells, target genes were
cloned into the pcDNA.3 mammalian expression vector (Invitrogen). Cells
at 2 x 105/well in DMEM/10% FCS
were seeded into 6-well Costar plates (Corning Inc., Corning, NY) and
incubated (10% CO2/90% air) until 70%
confluent. One ml of a solution of 2 µg of DNA mixed with 6 µl of
Lipofectamine (Life Technologies, Inc.) in 200 µl of DMEM on ice for
30 min was added to the cells. After 5 h of incubation at 37°C,
1 ml of DMEM plus 20% FCS was added, and the incubation continued
overnight.
For generation of viral supernatant, the embryo fibroblast line PA317 (American Type Culture Collection) was used as a packaging cell line and transfected by electroporation using Gene Pulser II (Bio-Rad, Hercules, CA). Cells were resuspended in electroporation buffer (272 mM sucrose, 7 mM K2HPO4, and 1 mM MgCl2) at 107 cells/800 µl electroporation buffer in an electroporation cuvette. Forty µg of plasmid was added for 10 min on ice and then electroporated at 200 V, 950 ufarads, 200 ohms for 80 msec. The cells were plated in a 100-mm dish containing 10 ml of DMEM/10% FCS and were incubated overnight at 37°C. The viral supernatants were collected and centrifuged at 2500 rpm for 10 min and were filtered and stored at -80°C.
For transductions, viral supernatants were collected from packaging cells and were used to transduce T15 cells. T15 cells were transiently transduced prior to each in vivo injection. One ml of supernatant was diluted in an equal volume of media and added to cells in 24-well plates plus 8 µg/ml polybrene, 100 units/ml mIL-2. The mixture was centrifuged at 2500 rpm at 32°C for 1.5 h and then was incubated at 32°C for 5 h. Cells were then transferred to a 100-mm dish and incubated in RPMI 1640/10% FCS for 24 or 48 h. Transduction frequency was quantitated by the cytometric analysis of NGFR-expressing transduced cells. Typically, 38–40% of cells expressed NGFR cell-surface marker after this transduction procedure. In one experiment, T15 cells transduced with sigIL4DT390/LNCX were selected in selection media containing G418 [600 µg/ml (Life Technologies, Inc. Co., Gaithersburg, MD)]. Cells were fed with new selection media every 3 days.
Flow Cytometric Analysis.
To assess the percentage of cells transduced with target gene,
transduced and nontransduced T15 cells were stained with mouse
anti-NGFR primary antibody (Boehringer Mannheim, Indianapolis, IN),
diluted 1:1000 for 15 min at room temperature, and FITC-labeled with
antimouse IgG secondary antibody diluted 1:100 (Chemicon, Temecula,
CA). The mixture was incubated for 15 min at room temperature, and
samples were analyzed on a FACScalibur (Becton Dickinson, Franklin
Lakes, NJ) as described previously (24)
. Forward- and
side-scatter settings were gated to exclude red cells and debris. Cells
(7,000–10,000) were analyzed for each determination. Irrelevant
monoclonal antibody control values were subtracted from values obtained
with relevant monoclonal antibodies.
Staining for Intracellular IT.
Cells were cultured on coverslips and transfected with
sigIL4DT390/pcDNA.3. After 30 h, coverslips
were washed twice with PBS and fixed with 95% ethanol/5% acetic acid
at -20°C for 5 min. Fixed cells were washed with PBS, incubated with
primary 11B11 Moab, diluted 1:50 in 5% BSA/PBS, and then incubated
1 h at room temperature. For DT staining, cells were incubated
with primary polyclonal rabbit anti-DT (generously provided by Dr. S.
Ramakrishnan, University of Minnesota, Minneapolis, MN) and secondary
FITC-labeled antibody. Coverslips were washed and incubated with
secondary FITC-rabbit antirat IgG (Sigma, St. Louis, MO) diluted 1:500
for 45 min at room temperature. Coverslips were washed again and then
mounted using a SlowFade Light Antifade Kit (Molecular Probes, Eugene,
OR). The cells were observed and digitally photographed using a Nikon
fluorescent microscope with Spot software (Diagnostic Instruments, Ann
Arbor, MI).
Viability Assays.
To assess retroviral IT killing, IL-4R+ C1498
cells were plated at 2 x 105/well
in 24-well plates (Costar). One ml of filtered supernatant from
cultured transfected or transduced T15 cells was added to each well.
Wells were sampled at 24, 48, and 72 h and diluted in trypan blue
dye/PBS solution, and the number of surviving cells was determined. To
assess selectivity, supernatants were simultaneously tested on
IL-4R- EL4 cells.
CTL Assay.
Cytotoxicity was measured by a modified JAM assay in which
target cell proliferation is assessed by thymidine incorporation
(25)
. Briefly, 2 x 105 C1498 or EL4 target cells are pulsed for
3.5 h with 10 µCi tritiated thymidine (Amersham Corp., Arlington
Heights, IL), washed, and then added to LAK or T15 effector cells in
96-well U-bottomed plates (Costar) at E:T ratios of 100, 50, 25, 12.5,
6.2, 3.1, and 1.5 to 1. Plates were centrifuged and incubated for an
additional 3.5 h at 5%CO2/95% air 37°C
and then were harvested and counted by standard scintillation counting
techniques. Cytotoxicity was calculated.
In Vivo Studies.
Two million C1498 cells were injected s.c. into the shaved flank of
C57BL/6 mice (5–6-week-old females purchased from The Jackson
Laboratory, Bar Harbor, ME) housed in a specific pathogen-free facility
at the University of Minnesota. Mice were given i.v. injections (via
caudal vein) of either transduced T15 or nontransduced T15. Because T15
is dependent on IL-2 for growth, 20,000 units/mouse mIL-2 was
administered i.p. at the time T15 was given. Five days after C1498,
three injections of transiently transduced 18–30 x 106 T15 cells were administered over a period of
11 days. Tumor volume was measured in centimeters and calculated as
w2 x l.
BUN, Creatinine, and ALT Assays.
All three of these assays were performed on Kodak EKTACHEM clinical
chemistry slides on a Kodak ETACHEM 950 by the Fairview University
Medical Center-University Campus (Minneapolis, MN). Mice were killed,
individual serum samples collected, and analysis was performed in a
coded fashion on the undiluted samples. Minimum specimen volume was 11
µl for each assay. The BUN assay is read spectrophotometrically
at 670 nm. The creatinine assay is read at 670 nM. In the
ALT assay, the oxidation of NADH is used to measure ALT activity at 340
nm.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows that inhibition was dose-dependent and 1.0
nM killed all of the cells by 48 h. Fig. 2B
shows that 20 µM anti-IL-4
monoclonal antibody blocked C1498 killing at DTIL-4 concentrations of 1
and 10 nM. The addition of anti-Ly5.2, an
irrelevant control antibody did not alter activity (Fig. 2C)
. In independent experiments, we found that DTIL-4 was
selectively toxic for another IL-4R+ murine
myeloid leukemia B162, for an IL-4R+ glioma, and
IL-4R+ neuroblastoma (data not shown). But it did not kill
the IL-4R- T cell thymoma EL4. Together, these
data indicate that IL-4 IT killing was selective and attributed to the
IL-4 moiety of the hybrid protein.
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To determine whether expressed protein was secreted, supernatants were
collected from an aliquot of these same transfected cells. Fig. 3
shows that supernatants collected from cultured NIH.3T3 cells
transfected with vector containing the target gene killed C1498 cells
but not control EL4 cells. Control supernatants from NIH.3T3 cells
transfected with empty vector did not inhibit either cell. Control
recombinant DTIL-4 at a concentration of 10-8
M inhibited C1498 in a similar manner, as did supernatants
from cells transfected with target gene. Together, these data show that
transfection with target gene results in the secretion of functional
fusion toxin that is selectively toxic.
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shows that T15 was not inhibited (Fig. 4B)
by concentrations
of DTIL-4 that otherwise killed C1498 (Fig. 4A)
. Also, the
fibroblast packaging line PA317 (Fig. 4C)
was not killed by
DTIL-4, which rendered them acceptable hosts for packaging virus
containing the sigDTIL-4 target gene. The in
vitro growth of T15 is dependent on IL-2 and antigen stimulation
by irradiated C1498 cells every 3 weeks. IL-2-dependent proliferation
of T15 cells measured by thymidine uptake was not inhibited by the
addition of DTIL-4 and other recombinant cytokine fusion toxins
described in other studies (17
, 19)
, including
DT390mIL-3,
DT390mGM-CSF, and
DT390 (data not shown). Proliferation was
inhibited by DT390IL-2. Fig. 5
shows that T15 is specific in its cytolytic activity because it kills
C1498 leukemia but has minimal activity against EL4 leukemia, although
killing of EL4 was measured at high E:T ratios in this experiment.
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, supernatants (collected from transfected cells) killed
target cells selectively; now we asked whether T15 T cells could be
transduced with viral supernatants that would induce them to express
and secrete IL-4 IT. T15 cells were transduced with viral supernatants
from PA317 packaging cells electroporated with the
sigIL4DT390/LNCX.NGFR retroviral vector. Fig. 6
shows T15 cells visualized by fluorescent microscopy that expressed
IL-4 (Fig. 6A)
or DT (Fig. 6C)
only after being
transduced with target gene. About 40–50% of cells were positive in
this experiment 30–40 h after transduction. Nontransduced T15 cells
were not positive for IL-4 or DT. As a tool for studying transduction
frequency, T15 cells were transduced with an LNCX vector in which neo
had been replaced with NGFR. Transduction with a gene encoding NGFR
results in the cell surface expression of the human NGFR protein that
can be detected with labeled antibody by flow cytometry and, thereby,
used to measure the transduction frequency. Transduction with viral
supernatants from sigIL4DT390/LNCX.NGFR-treated packaging cells
resulted in cell surface expression of NGFR on T15 cells (data not
shown).
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. EL4s continued to proliferate despite exposure to
supernatants from transduced or nontransduced T15 (Fig. 7B)
.
Control DTIL-4 inhibited C1498 but not EL4. Together, Figs. 3
, 6
, and 7
showed that the IT gene, when either transfected into
mammalian embryonic cells (NIH.3T3) or transduced into T15 T cells,
induced the expression and secretion of IL-4 IT that could selectively
destroy IL-4R-expressing target cells.
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shows the presence of the 1.2-kb DT390 gene in cultured T15
cells.
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. C1498-specific T15 T cells were transiently transduced with
sigIL4DT390/LNCX.NGFR virus and then administered
i.v. to tumor-infected mice. The transduction frequencies of the T15
cells at the time of injection were 38–40% by FACS analysis
and the viability of the transduced cells was 91–95%. Because the
cytolytic activity of T15 T cells is IL-2-dependent, these mice were
given 20,000 units of mIL-2 i.p. along with the T15 cells given i.v.
All of the groups, including controls, received mIL-2.
Twenty-seven x 106 T15 cells were
injected on day 5, 18 x 106 cells
were injected on day 10, and 30 x 106 cells were injected on day 16 after C1498
injection. Tumor size steadily increased in groups of mice given
nontransduced T15 cells or in groups of tumor mice that were not given
T15. In contrast, the rate of tumor growth remained very low until day
18 in groups of mice injected with transduced T15. After day 18, growth
began to steadily increase. These data show that transduced T cells
inhibited tumor growth, whereas nontransduced cells did not. Lack of
activity of nontransduced T15 could relate to inadequate amounts of
IL-2 or high tumor volume. Mice given transduced cells ultimately
developed tumor and metastases, which indicated that a greater level of
IT delivery is needed in vivo.
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. In a separate experiment, C1498 cells were administered s.c. in
a manner identical to that in Fig. 9
. Groups of mice were given the
maximum tolerated dose of IT (a total dose of 0.5/µg/day or 1.0
µg/day given b.i.d.) that was determined in a separate
experiment4
.Fig. 10
shows that systemic administration of recombinant IL-4 IT, which was
previously shown to be selectively toxic in nanomolar concentrations
in vitro (Fig. 2)
, had no effect on the growth of C1498
cells in vivo. Three of five of the mice in the group given
1.0 µg/day died of toxicity on day 9. There were no toxic deaths in
mice given 0.5 µg/day. Controls were not injected with IT.
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(n = 3/group). Fig. 11
shows that there were no significant differences in BUN or creatinine
levels in groups of mice given transduced T15 cells, which indicates
that retroviral IT did not damage organs despite its effect on C1498
cells. Tissue from mice given transduced T cells was collected between
the second and third administration of transduced T cells (days
10–15). Also, there was no difference in ALT levels, which indicates
that hepatic activity also was unaffected. These findings were
confirmed by histological analysis of tissues.
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DISCUSSION |
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An additional strength of the studies is the use of a syngeneic murine model to determine the feasibility of using antigen-specific T-cell lines for retroviral IT delivery in vivo. The CD8+CD4- MHC class I restricted T-cell line, T15, was produced by hyperimmunizing C57BL/6 mice with irradiated C1498 cells. Previous studies showed that T15 elicited an anticancer effect in C1498-infected mice (22) . In this report, transduction of T15 with sigIL4DT390 resulted in a significant (P < 0.05) anticancer effect in mice with s.c. tumors. Tumor growth was steadily held in check after injection of 18–30 x 106 transduced cells and then began to increase in size after 18 days. In contrast, the nontransduced control had no effect on the growing tumor. These findings, in which i.v. injected transduced T cells are used to treat s.c. tumors, indicate that T cells and therapy are finding their in vivo targets. Nontransduced T15 did not contribute an anti-C1498 effect as it did in past studies. This might be explained by the fact that we did not administer enough IL-2 to drive IL-2-dependent T15. In future studies, in vivo survival of injected T15 might be enhanced by the continuous administration of IL-2 injections or an additional transduction with IL-2 gene.
At the same time, these findings raise important issues. For example, although a significant anti-C1498 effect was measured, we do not know why efficacy was incomplete. Perhaps, more cells, stimulated to higher levels of cytotoxicity and higher transduction frequencies may be necessary for improved efficacy. It is also possible that only a small portion of i.v. injected T15 cells cleared the lungs and other internal organs and arrived at the tumor site.
This is also the first reported use of DT, a powerful inhibitor of protein synthesis (5) for retroviral IT production. Retroviral delivery of ITs has been theoretically limited by "cell suicide" expected to result from the delivery of the catalytic toxin into the cytosol. Although posttranslational protein transport in yeast does indeed permit translated toxins to react with their target, most mammalian cells undergo cotranslational protein transport in which the signal peptide directs the single-chain protein through the ER membrane directly into the ER lumen (29 , 30) . Data in this report indicate that toxin separation between the intracellular ER lumen and cytosolic compartments is remarkably conserved so that even minute amounts of toxin are prevented from gaining access to ribosomes. It is known that leader sequences are responsible for the passage of proteins into or through membranes, and retroviral ITs assembled in our laboratory without a leader sequence positioned upstream from the ligand were lethal to transfected cells. The leader peptide permits recognition by the signal recognition particle (SRP) and translocation of IT polypeptide directly into the ER lumen where it is sequestered (reviewed in 30 ). Whether this separation is maintained indefinitely is not known, but our data—in which transduced T15 cells, selected with G418, and then later examined for the presence of genomic DT390—indicate that cells are capable of surviving at least 2 weeks despite stable genomic integration of a lethal toxin provirus.
Another issue is the duration of secretion. Studies are in progress to measure retroviral IT production in vitro and in vivo. Although it will be useful to establish a correlation between retroviral IT levels and efficacy, it will be important to determine whether stable transductants can be generated in which provirus is stably integrated and retroviral IT is secreted. Currently, T15 must be transduced transiently prior to each injection, an approach that is neither labor-efficient nor cost-effective. The further generation of stable transductants via neo selection or flow cytometry sorting of NGFR+ transductants may address this point. Another issue to be considered is whether systemic leukemia will be an appropriate target for retroviral IT therapy. In these studies, C1498 was administered s.c. so that we could monitor tumor size on a daily basis. If C1498 is administered i.v., it will disseminate systemically with multiple growth sites. The production of retroviral IT at multiple sites by transduced T15 T cells may enhance the risk of systemic toxicity. Fortunately, all of these issues can be addressed in this model, and studies are currently underway.
LAK cells might be considered for retroviral IT delivery because they are more easily obtained than antigen-specific CTL. Investigators used transduced LAK cells to deliver an sFv IT recognizing Her-2/neu-expressing human breast cancer in severe-combined immunodeficient mice (31) . Because LAK cells are not antigen-specific, they may not be the best choice. Also, we transduced LAK cells in a manner identical to CTL and typically found that transduction of LAK cells with target gene resulted in at least half the frequency obtained with antigen-specific T15 cells.5 This might be attributed to the highly activated nature of T15 undergoing both antigen-stimulation and IL-2 expansion. Postmitotic cells cannot be transduced because mitosis is required for entry of the viral integration complex into the nucleus (32 , 33) , and perhaps there is a higher frequency of postmitotic cells in the LAK population. Certainly, differing transduction frequencies in the two populations are not strictly related to IL-2 because both T15 and LAK cells were transduced in the presence of IL-2. Perhaps, antigen-sensitized CTLs express a higher number of ectotropic receptors, and retroviral infection is dependent on virus binding to these specific receptors on the cell surface. The literature generally prefers the use of CTLs over LAK cells because antigen-specific T cells are more likely to penetrate to the site of tumor antigen (34 , 35) , and clinical adoptive immunotherapy studies with LAK cells have been limited by low response rates (36) .
Delivering IT using T cells may have advantages in reducing toxicity. The secretion of retroviral IT at the site of tumor may result in a higher saturation of target cells and less IT escaping to nontarget locations. Our data indicate that T-cell delivery resulted in efficacy combined with no hepatic or renal toxicity. In contrast, systemic delivery of IT at the maximum tolerated dose was not efficacious. Others have shown that the dose-limiting toxicity of IL-4 IT is due to hepatic toxicity (37) , and histological examination of tissues from our treated mice also revealed damaged livers (data not shown). Taken together, these data suggest that T cells are locally delivering IT at the site of tumor, and this is reducing systemic toxicity.
One appealing aspect of the retroviral IT approach is that T cells could be recruited as vehicles to deliver any cytokine fusion toxin. Although any cytokine could be used as a ligand for toxin delivery, we chose IL-4 because IL-4 receptors have been reported on most myeloid leukemias (10) , various lymphoid malignancies (11) , and nonlymphoid tumors (12) . Also, recombinant IL-4 fusion toxins have been effective in animal models because complete remission of human IL-4R-bearing carcinoma can be induced in nude mice by administering IL-4 IT (11) . Although expression of IL-4R on committed hematopoietic cells may be problematic, not all T cells express IL-4R as evidenced by our findings with transduced T15, and systemic exposure may be limited by local delivery to cancer cells. Also, although only a single molecule of toxin in the cytosol is capable of killing a cell, toxin must survive the endosomal journey to the cell’s interior, which may require the binding of hundreds or thousands of ITs and the avoidance of lysosomal compartments. Thus, low receptor expression and subsequent binding of low numbers of ITs may not result in cell killing. Normal hematopoietic cells that express low IL-4R levels may be spared. Other cytokine fusion toxins might work better than IL-4 IT. For example, studies show that IL-3 IT, which has been limited by systemic toxic effects, are highly selective and capable of destroying IL-3-receptor-expressing leukemias (18) . Because these studies show that IL-3 kills committed but not uncommitted bone marrow progenitor cells and IL-3 receptor is expressed on greater than 90% of myeloid leukemias, it is also an excellent candidate for the retroviral IT approach. IL-2 or any other cytokine with high level receptor expression on T cells are undesirable candidates for retroviral IT because the secretion of IL-2 IT, for example, would immediately result in the suicide of T cells expressing IL-2R.
In conclusion, we have used retroviral gene therapy of cancer-specific CTLs to deliver a cytokine fusion toxin to C1498 cancer cells in vivo. Retroviral transduction of T cells resulted in a significant, albeit temporary, antitumor effect with no organ toxicity. In contrast, systemic administration of this same IL-4 IT was not efficacious and was highly toxic. Delivering ITs using T cells in this manner could overcome the unfavorable physiology, vascularization, and high-tumor interstitial pressures that have limited the penetration of biologicals to cancer sites in vivo (38) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by USPHS Grants
RO1-CA36725, R01-CA82154, and R01-CA72669 awarded by the National
Cancer Institute and the National Institute of Allergy and Infectious
Diseases, Department of Health and Human Services, and by the Leukemia
Research Fund 
2 To whom requests for reprints should be
addressed, at University of Minnesota Cancer Center, Box 367 Mayo
Building, Harvard Street at East River Road, Minneapolis, MN 55455.
Phone: (612) 626-6664; Fax: (612) 624-3913; E-mail: valle001{at}tc.umn.edu
3 The abbreviations used are: DT,
diphtheria toxin; IT, immunotoxin; LAK, lymphokine-activated killer
(cell); IL, interleukin; IL-4R, IL-4 receptor; ER, endoplasmic
reticulum; NGFR, nerve growth factor receptor; ALT, alanine
transferase; BUN, blood urea nitrogen (assay); aa, amino acid.
5 D. A. Vallera, unpublished data.
Received 9/14/99. Accepted 12/14/99.
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