|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 737-745
By
From the Division of Clinical Research, Fred Hutchinson Cancer
Research Center, Seattle, WA; the Departments of Pediatrics
and Medicine, University of Washington, Seattle, WA; and the
Pacific Northwest National Laboratory, Richland, WA.
Targeted hematopoietic irradiation delivered by
131I-anti-CD45 antibody has been combined with conventional
marrow transplant preparative regimens in an effort to decrease
relapse. Before increasing the proportion of therapy delivered by
radiolabeled antibody, the myeloablative and immunosuppressive effects
of such low dose rate irradiation must be quantitated. We have examined the ability of 131I-anti-CD45 antibody to facilitate
engraftment in Ly5-congenic and H2-mismatched murine marrow transplant
models. Recipient B6-Ly5a mice were treated with
30F11 antibody labeled with 0.1 to 1.5 mCi 131I
and/or total body irradiation (TBI), followed by
T-cell-depleted marrow from Ly5b-congenic
(C57BL/6) or H2-mismatched (BALB/c) donors. Engraftment was achieved
readily in the Ly5-congenic setting, with greater than 80%
donor granulocytes and T cells after 0.5 mCi 131I
(estimated 17 Gy to marrow) or 8 Gy TBI. A higher TBI dose (14 Gy) was
required to achieve engraftment of H2-mismatched marrow, and
engraftment occurred in only 3 of 11 mice receiving 1.5 mCi 131I delivered by anti-CD45 antibody. Engraftment of
H2-mismatched marrow was achieved in 22 of 23 animals receiving 0.75 mCi 131I delivered by anti-CD45 antibody combined
with 8 Gy TBI. Thus, targeted radiation delivered via
131I-anti-CD45 antibody can enable engraftment of congenic
marrow and can partially replace TBI when transplanting
T-cell-depleted H2-mismatched marrow.
BONE MARROW transplantation has been used
for more than two decades to treat hematologic malignancies and
aplastic anemia and, more recently, genetic diseases such as
thalassemia and sickle cell anemia.1-6 However, the success
of marrow transplantation has been limited by the high relapse rates
seen in patients transplanted for advanced malignancies, by the
toxicity of preparative regimens, and by the lack of HLA-identical
family member donors for most patients. In principle, these limitations
could be addressed by the use of radiolabeled monoclonal antibodies
(MoAbs) to increase the irradiation to lymphohematopoietic tissues
while minimizing the exposure of normal organs. First, delivery of more
irradiation to sites of leukemic involvement should decrease the risk
of relapse. Second, targeted irradiation of lymphohematopoietic organs
should decrease the need for high-dose chemotherapy or total body
irradiation (TBI) to prevent rejection of the allogeneic graft.
Alternatively, adding targeted irradiation to full-dose systemic
therapy could enhance the level of immunosuppression, thereby
decreasing the risk of rejection of marrow from HLA-mismatched or
unrelated donors, especially when the marrow has been depleted of T
cells to prevent graft-versus-host disease (GVHD).
We have shown that infusion of radiolabeled antibodies against CD45
delivers irradiation selectively to lymphohematopoietic tissues.7-9 Significant supplemental doses of radiation
have been delivered to bone marrow and spleen in combination with
conventional marrow transplant preparative regimens without excessive
toxicity, and the relapse rates after these novel preparative regimens
have been low in preliminary studies. In the initial clinical studies, treatment with radiolabeled antibody was combined with standard preparative regimens containing cyclophosphamide and either
TBI9,10 or busulfan,11 because it is unknown
whether the relatively low dose rate radiation delivered by antibody
has sufficient antileukemic efficacy to prevent relapse and sufficient
immunosuppressive activity to prevent rejection of allogeneic marrow.
Radiolabeled antibody delivers radiation at an exposure rate that is
one to two orders of magnitude lower than the rate normally delivered
from an external beam source. Several studies have demonstrated the
influence of exposure rate on the amount of irradiation required to
achieve engraftment.12-15 van Os et
al14,15 compared exposure rates ranging from 0.5 to 40 cGy/min in a variety of murine transplant models. In a congenic donor
and recipient strain combination that differed by a single
nonimmunogenic marker, 5.5 Gy TBI delivered at 40 cGy/min was
sufficient to allow 80% engraftment in 50% of the recipients. In the
same model with TBI delivered at 2 cGy/min, a 7 Gy exposure was needed
to achieve the same degree of engraftment.15 In an
H2-compatible strain combination with disparity for multiple minor
histocompatibility antigens, 6 Gy TBI delivered at 40 cGy/min was
sufficient to allow engraftment. In the same model with TBI delivered
at 2 cGy/min, an 8 Gy exposure was needed.14 With T-cell-depleted marrow from H2-mismatched donors, the difference between low and high dose rate was at least 6 Gy, because full engraftment at the high-dose rate (40 cGy/min) occurred after 10 Gy,
but long-term engraftment did not occur after 16 Gy (the highest dose
tested) delivered at 2 cGy/min.14 These results suggest
that hematopoietic stem cells and precursors of cellular immunity have
a substantial ability to repair irradiation damage.
Little is known about the relative biologic effects of low dose rate
radiation resulting from the administration of radiolabeled antibody.
To address this issue, we initiated studies using
131I-labeled anti-CD45 antibody, with or without external
beam TBI, as a preparative regimen in two models of murine
transplantation. To measure marrow ablation,
B6-Ly5a recipients were transplanted with marrow
from congenic Ly5b donors.16,17 In this
model, the donor and recipient disparity is limited to CD45 allotype
and does not evoke a cellular immune response.18 In an
approach designed to test the immunosuppressive effects of the regimen,
the same recipients (H2b) were transplanted with
T-cell-depleted marrow from H2-mismatched (H2d) BALB/c
donors. Our results demonstrate that irradiation delivered solely by
131I-anti-CD45 antibody allows engraftment of marrow in
Ly5-congenic recipients but not in H2-mismatched recipients, where both
natural killer (NK) cells and T cells are known to cause
rejection. However, engraftment of T-depleted H2-mismatched marrow
could be accomplished by adding low-dose external beam TBI to the
radiation delivered by 131I-anti-CD45 antibody.
Mice.
Male B6-Ly5a mice were bred at the Fred Hutchinson
Cancer Research Center (Seattle, WA) and housed under specific
pathogen-free conditions with acidified water and autoclaved chow.
C57BL/6 and BALB/c donors were purchased from Jackson Laboratory (Bar
Harbor, ME) and were 6 to 12 weeks old at the initiation of each
experiment.
MoAbs.
MoAbs GK1.5 (rat IgG2b anti-CD4), 30-H12 (rat
IgG2b anti-thy1.2), and 2.34 (rat IgG2b
anti-CD8) were prepared from culture supernatants of cells lines
obtained from ATCC (GK1.5 and 2.34) or Dr Jeffrey Ledbetter
(Bristol Meyers Squibb, Seattle, WA; 30-H12). A hybridoma cell line
secreting MoAb 30F11 (rat IgG2b), which recognizes all
murine CD45 isoforms, was the gift of Dr Ledbetter. Hybridoma cell
lines secreting MoAbs A20 (murine IgG2a), which recognizes
the Ly5.1 epitope encoded by the Ly5a allotype of
murine CD45, and 104 (murine IgG2a), which recognizes the
Ly5.2 epitope encoded by the Ly5b allotype of
murine CD45, were the gift of Dr Shoji Kimura (Sloan Kettering
Institute, New York, NY). Ascites containing each MoAb was produced in
BALB/c mice under specific pathogen-free conditions. Batch extraction
and purification of antibodies from ascites was performed using
Abx exchange resin (J.T. Baker, Phillipsburg, NJ) and
high-pressure liquid chromatography (Biosys 510 Beckman, Fullerton,
CA).
Iodination and characterization.
Antibodies were iodinated with Na125I or Na131I
(ICN, Irvine, CA) using the Iodogen method19 for trace
labeling and the chloramine T-labeling method for high specific
activity labeling.20 The immunoreactivity (percentage of
counts able to bind at antigen excess) of each preparation was
determined by incubating antibody at low concentration (5 ng/mL) with
1.0 × 106 to 3 × 107
B6-Ly5a splenocytes for 1 hour at room temperature
and measuring unbound antibody as previously described.21
Preparations with less than 70% immunoreactivity were not used.
Antibody localization and estimation of radiation absorbed doses.
The double isotope labeling method of Pressman22 was used
to determine the biodistribution of 30F11 antibody as previously described.8 Animals were injected via tail vein with 200 µL volume containing 100 µg of antibody labeled with
131I (specific activity, 1 µCi/µg) and an equal amount
of rat IgG (Sigma Immunochemical, St Louis, MO) labeled with
125I. Groups of 5 mice were killed at multiple time points
from 1 to 96 hours after injection, and multiple tissues were sampled. The 125I and 131I contents of weighed samples
and standard samples of the injection mix were determined by
multichannel gamma counting (Packard 5000 Autogamma counter; Packard
Instrument Co, Downers Grove, IL). Counting data were adjusted for
cross-over from the 131I channel to the 125I
channel and were corrected for decay.
Bone marrow transplantation.
Four days before marrow infusion, groups of 6 recipient
B6-Ly5a mice were treated with 100 µg of 30F11
antibody labeled with varying amounts of 131I at a maximum
specific activity of 1.5 mCi 131I/100 µg. Cages were
changed every other day for 1 week to decrease the irradiation from
excreted urine in the cage bedding. External beam TBI was administered
on the day of marrow infusion and was delivered at 0.2 Gy/min from dual
60Cobalt sources (J.L. Shepherd Co, San Fernando, CA).
Assessment of engraftment.
Mice were examined daily for survival and for signs of GVHD or other
illness. At 4, 8, and 12 weeks posttransplant, orbital blood samples
were stained with 50 µL of biotinylated A20 (anti-Ly5.1) antibody or
biotinylated 104 (anti-Ly5.2) antibody at 100 µg/mL for 30 minutes
followed by phycoerythrin-streptavidin (PharMingen, San Diego, CA) and
fluorescein isothiocyanate (FITC)-conjugated CD3-specific antibody
(PharMingen). Control samples consisted of cells from unmanipulated
mice of both donor and host strains. Samples were analyzed by flow
cytometry using a FACScan (Becton Dickinson, San Jose, CA). Lymphocyte
and granulocyte populations were defined by forward and 90° scatter
characteristics. The lymphocyte population was analyzed separately
using two colors to distinguish CD3+ T cells and
CD3 Biodistribution of 131I-anti-CD45 antibody and estimation
of radiation absorbed doses.
B6.Ly5a mice were injected with a 100 µg dose of
trace 131I-labeled anti-CD45 MoAb 30F11 or
125I-labeled rat IgG and the amount of isotope in major
organs was determined. The resulting time-activity curves demonstrated
greater uptake and retention of radiolabeled anti-CD45 antibody in
spleen, lymph nodes, and marrow as compared with the lung, the normal nontarget organ with the highest concentration
(Fig 1A). The corresponding time-activity
curves for 125I-labeled rat IgG (ie, IgG purified from sera
of normal rats), used as a nonspecific control, did not show any
specific retention in spleen, lymph nodes, or marrow (Fig 1B).
Anti-CD45 antibody was cleared from the blood far more rapidly than
control rat IgG, presumably by rapid binding of circulating antibody to
CD45 on readily accessible cells in spleen and marrow (Fig 1C).
Engraftment of congenic marrow after 131I-30F11
antibody or TBI.
In the initial experiments with C57BL/6 donors and congenic
B6-Ly5a recipients, we administered a 100 µg dose
of 30F11 antibody labeled with 0 (ie, unlabeled antibody), 0.5, 1.0, 1.25, and 1.5 mCi of 131I, followed 4 days later by
injection of T-cell-depleted marrow or medium alone. All mice
receiving marrow survived, whereas 2 of 6 mice treated with 1.25 mCi
131I and 4 of 6 treated with 1.5 mCi 131I and
no marrow died between 11 and 15 days after radiolabeled antibody
injection (data not shown). Although postmortem examinations were not
performed, death at these time points after delivery of radiation is
consistent with death from marrow aplasia. All mice receiving at least
0.5 mCi 131I-anti-CD45 antibody demonstrated successful
engraftment (Table 2). Myeloid engraftment
occurred within 4 weeks posttransplant, but full T-cell engraftment did
not occur until 12 weeks, presumably reflecting delayed disappearance
of recipient T cells.
Engraftment of T-cell-depleted H2-mismatched marrow after
131I-anti-CD45 antibody alone or with external beam TBI.
In the initial experiments with BALB/c donors and MHC-mismatched
B6-Ly5a recipients, we administered either a 100 µg dose of antibody 30F11 labeled with 0.5 to 1.5 mCi of
131I on day
We asked if radiation delivered by 131I-labeled anti-CD45
antibody could enable engraftment of congenic marrow and of
H2-incompatible marrow. Using congenic marrow,
131I-anti-CD45 antibody alone enabled engraftment, with
The authors are indebted to Minna Zheng, Jennifer Smith, and Carol Dean
for their expert technical assistance.
Submitted April 30, 1998;
accepted September 22, 1998.
Address reprint requests to Dana C. Matthews, MD, Fred Hutchinson
Cancer Research Center D1-100, 1100 Fairview Ave N, PO Box 19024, Seattle, WA 98109; e-mail: dmatthew{at}fhcrc.org.
1.
Christiansen NP:
Allogeneic bone marrow transplantation for the treatment of adult acute leukemias.
Hematol Oncol Clin North Am
7:177, 1993[Medline]
[Order article via Infotrieve]
2.
Appelbaum FR:
Indications for bone marrow transplantation (BMT) in the treatment of acute myeloid leukemia (AML).
Leukemia
7:1081, 1993[Medline]
[Order article via Infotrieve]
3.
Brochstein JA, Kernan NA, Groshen S, Cirrincione C, Shank B, Emanuel D, Laver J, O'Reilly RJ:
Allogeneic bone marrow transplantation after hyperfractionated total-body irradiation and cyclophosphamide in children with acute leukemia.
N Engl J Med
317:1618, 1987[Abstract]
4.
Storb R, Anasetti C, Appelbaum F, Bensinger W, Buckner CD, Clift R, Deeg HJ, Doney K, Hansen J, Loughran T, Martin P, Pepe M, Petersen F, Sanders J, Singer J, Stewart P, Sullivan KM, Witherspoon R, Thomas ED:
Marrow transplantation for severe aplastic anemia and thalassemia major.
Semin Hematol
28:235, 1991[Medline]
[Order article via Infotrieve]
5.
Lucarelli G, Galimberti M, Polchi P, Angelucci E, Baronciani D, Giardini C, Andreani M, Agostinelli F, Albertini F, Clift RA:
Marrow transplantation in patients with thalassemia responsive to iron chelation therapy.
N Engl J Med
329:840, 1993
6.
Walters MC, Patience M, Leisenring W, Eckman JR, Scott JP, Mentzer WC, Davies SC, Ohene-Frempong K, Bernaudin F, Matthews DC, Storb R, Sullivan KM:
Bone marrow transplantation for sickle cell disease: A multicenter collaborative investigation.
N Engl J Med
334:369, 1996
7.
Matthews DC, Appelbaum FR, Eary JF, Hui TE, Fisher DR, Martin PJ, Durack LD, Nelp WB, Press OW, Badger CC, Bernstein ID:
Radiolabeled anti-CD45 monoclonal antibodies target lymphohematopoietic tissue in the macaque.
Blood
78:1864, 1991
8.
Matthews DC, Badger CC, Fisher DR, Hui TE, Nourigat C, Appelbaum FR, Martin PJ, Bernstein ID:
Selective radiation of hematolymphoid tissue delivered by anti-CD45 antibody.
Cancer Res
52:1228, 1992
9.
Matthews DC, Appelbaum FR, Eary JF, Fisher DR, Durack LD, Bush SA, Hui TE, Martin PJ, Mitchell D, Press OW, Badger CC, Storb R, Nelp WB, Bernstein ID:
Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled anti-CD45 antibody, combined with cyclophosphamide and total body irradiation.
Blood
85:1122, 1995
10.
Matthews DC, Appelbaum FR, Eary JF, Mitchell D, Press OW, Bernstein ID:
Phase I study of 131I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome.
Blood
90:417a, 1997 (abstr, suppl 1)
11.
Matthews DC, Appelbaum FR, Eary JF, Mitchell D, Press OW, Bernstein ID:
131I-anti-CD45 antibody plus busulfan/cyclophosphamide in matched related transplants for AML in first remission.
Blood
88:142a, 1996 (abstr, suppl 1)
12.
Gengozian N, Carlson DE, Allen EM:
Transplantation of allogeneic and xenogeneic (rat) marrow in irradiated mice as affected by radiation exposure.
Transplantation
7:259, 1969[Medline]
[Order article via Infotrieve]
13.
Down JD, Tarbell NJ, Thames HD, Mauch PM:
Syngeneic and allogeneic bone marrow engraftment after total body irradiation: Dependence on dose, dose rate, and fractionation.
Blood
77:661, 1991
14.
van Os R, Konings AW, Down JD:
Compromising effect of low dose-rate total body irradiation on allogeneic bone marrow engraftment.
Int J Radiat Biol
64:761, 1993[Medline]
[Order article via Infotrieve]
15.
van Os R, Thames HD, Konings AW, Down JD:
Radiation dose-fractionation and dose-rate relationships for long-term repopulating hemopoietic stem cells in a murine bone marrow transplant model.
Radiat Res
136:118, 1993[Medline]
[Order article via Infotrieve]
16.
Morse HC 3d, Shen FW, Hammerling U:
Genetic nomenclature for loci controlling mouse lymphocyte antigens.
Immunogenetics
25:71, 1987[Medline]
[Order article via Infotrieve]
17.
Seldin MF, D'Hoostelaere LA, Steinberg AD, Saga Y, Morse HDI:
Allelic variants of Ly-5 in inbred and natural populations of mice.
Immunogenetics
26:74, 1987[Medline]
[Order article via Infotrieve]
18.
Sykes M, Chester CH, Sundt TM, Romick ML, Hoyles KA, Sachs DH:
Effects of T cell depletion in radiation bone marrow chimeras. III. Characterization of allogeneic bone marrow cell populations that increase allogeneic chimerism independently of graft-vs-host disease in mixed marrow recipients.
J Immunol
143:3503, 1989[Abstract]
19.
Badger CC, Krohn KA, Peterson AB, Shulman H, Bernstein ID:
Experimental radiotherapy of murine lymphoma with 131I-labeled anti-Thy-1.1 monoclonal antibody.
Cancer Res
45:1536, 1985
20.
Hunter WM, Greenwood FL:
Preparation of I131-labeled human growth hormones of high specific activity.
Nature
194:495, 1942
21.
Badger CC, Krohn KA, Bernstein ID:
In vitro measurement of avidity of radiodinated antibodies.
Nucl Med Biol
14:605, 1987
22.
Pressman D:
Radiolabeled antibodies.
Ann NY Acad Sci
69:644, 1957
23.
Nelson WR, Hirayama H, Rogers DWO:
The EGS4 Code System, Report 265. Palo Alto, CA, Stanford Linear Accelerator Center, 1985.
24.
Hui TE, Fisher DR, Kuhn JA, Williams LE, Nourigat C, Badger CC, Beatty BG, Beatty JD:
A mouse model for calculating cross-organ beta doses from Yttrium-90-labeled immunoconjugates.
Cancer
73:951, 1994[Medline]
[Order article via Infotrieve] (suppl 3)
25.
Berger MJ:
Distribution of aborbed dose around point sources of electrons and beta particles in water and other media.
J Nucl Med
12:5, 1971
26.
Lapidot T, Terenzi A, Singer TS, Salomon O, Reisner Y:
Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts.
Blood
73:2025, 1989
27.
van Os R, de Witte T, Dillingh JH, Mauch PM, Down JD:
Increased rejection of murine allogeneic bone marrow in presensitized recipients.
Leukemia
11:1045, 1997[Medline]
[Order article via Infotrieve]
28.
Vallera DA, Taylor PA, Sprent J, Blazar BR:
The role of host T cell subsets in bone marrow rejection directed to isolated major histocompatibility complex class I versus class II differences of bm1 and bm12 mutant mice.
Transplantation
57:249, 1994[Medline]
[Order article via Infotrieve]
29.
Ferrara J, Lipton J, Hellman S, Burakoff S, Mauch P:
Engraftment following T-cell-depleted marrow transplantation. I. The role of major and minor histocompatibility barriers.
Transplantation
43:461, 1987[Medline]
[Order article via Infotrieve]
30.
Uharek L, Gassmann W, Glass B, Steinmann J, Loeffler H, Mueller-Ruchholtz W:
Influence of cell dose and graft-versus-host reactivity on rejection rates after allogeneic bone marrow transplantation.
Blood
79:1612, 1992
31.
Martin PJ:
Influence of alloreactive T cells on initial hematopoietic reconstitution after marrow transplantation.
Exp Hematol
23:174, 1995[Medline]
[Order article via Infotrieve]
32.
Lapidot T, Singer TS, Salomon O, Terenzi A, Schwartz E, Reisner Y:
Booster irradiation to the spleen following total body irradiation. A new immunosuppressive approach for allogeneic bone marrow transplantation.
J Immunol
141:2619, 1988[Abstract]
33.
Rao SR, Peters SO, Crittenden RB, Stewart FM, Ramshaw HS, Quesenberry PJ:
Stem cell transplantation in the normal nonmyeloablated host: Relationship between cell dose, schedule and engraftment.
Exp Hematol
25:114, 1997[Medline]
[Order article via Infotrieve]
34.
Martin PJ:
Donor CD8 cells prevent allogeneic marrow graft rejection in mice: Potential implications for marrow transplantation in humans.
J Exp Med
178:703, 1993
35.
Stewart FM, Crittenden RB, Lowry PA, Pearson-White S, Quesenberry PJ:
Long-term engraftment of normal and post-5 fluorouracil murine marrow into normal nonmyeloablated mice.
Blood
81:2566, 1993
36.
Tomita Y, Sachs DH, Sykes M:
Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow.
Blood
83:939, 1994
37.
Salomon O, Lapidot T, Terenzi A, Lubin I, Rabi I, Reisner Y:
Induction of donor-type chimerism in murine recipients of bone marrow allografts by different radiation regimens currently used in treatment of leukemia patients.
Blood
76:1872, 1990
38.
Storb R, Raff RF, Appelbaum FR, Graham TC, Schuening FG, Sale G, Pepe M:
Comparison of fractionated to single-dose total body irradiation in conditioning canine littermates for DLA-identical marrow grafts.
Blood
74:1139, 1989
39.
Tarbell NJ, Amato DA, Down JD, Mauch P, Hellman S:
Fractionation and dose rate effects in mice: A model for bone marrow transplantation in man.
Int J Radiat Oncol Biol Phys
13:1065, 1987[Medline]
[Order article via&nbs |
Top
Abstract
Introduction
-particle energy for
131I were calculated by applying electron transport theory
(EGS4
B cells.
4 or 2 to 16 Gy TBI on day 0, followed
by infusion of T-cell-depleted marrow or medium alone. With external
beam TBI and no marrow infusion, the LD50 was approximately
10 Gy, and no recipients survived exposures of 
14 Gy (data not
shown). With infusion of T-cell-depleted H2-incompatible marrow,
engraftment (>80% donor T cells at 3 months posttransplant) was
observed in all recipients prepared with 
