1. No category

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1995 86: 4367-4375
Therapy for ongoing graft-versus-host disease induced across the
major or minor histocompatibility barrier in mice with
anti-CD3F(ab')2-ricin toxin A chain immunotoxin
DA Vallera, PA Taylor, A Panoskaltsis-Mortari and BR Blazar
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Therapy for Ongoing Graft-Versus-Host Disease Induced Across the Major
or Minor Histocompatibility Barrier in Mice With Anti-CD3F(ab‘)2-Ricin
Toxin A Chain Immunotoxin
By Daniel A. Vallera, Patricia A. Taylor, Angela Panoskaltsis-Mortari, and Bruce R. Blazar
A new pharmacologic agent, anti-CDtF(ab‘ I2-ricin toxin A
chain (RTA), was synthesized for thepurpose of targeting T
cells and as a means of treating established graft-versushost disease (GVHD).The Fc region of anti-CD3 monoclonal
antibody (MoAbl was removedt o prevent its abilityt o activate T cells. The resulting F(ab’ l2 fragments wereconjugated
t o deglycosylated RTA (dgRTA), a catalytic and potent phytotoxin. The resulting immunotoxin (IT) was potent(greater
than 95% inhibition) and selective in inhibiting T-cell mitogenesis in vitro. In vivo, the ITdepleted 80% of l cells in mice
receiving bonemarrow (BM) transplants. Transplantation in
an aggressive acute GVHD model usingC57BL/6 donor cells
and H-2 disparate B1O.BR recipients resultedin an infiltration
of CD3-expressing cells and a median survival time (MST)
of 20 t o 30 days. A 5-day course of anti-CDSF(ab‘),-RTA (30
p g / d intraperitoneally) beginning 7 days after GVHD induction was beneficial in treating established GVHD in these
mice, as evidenced by significantly prolonged survival
(MST,
greater than 80days), superior mean weight values, and
improved clinical appearance. Neither intact anti-CD3, unconjugated anti-CD3 Flab‘ l2 fragments, nor a mixture of antiCD4 and anti-CD8 MoAbs (which are highly effective in prophylactic models) were as effective. F(ab’ lz fragments made
from anti-Lyt-l (themurine homologueof human anti-CD51
linked t o RTA were also not effective, despite the fact that
both anti-CDtF(ab’ ),-RTA and anti-Lyt-lF(ab’),-RTA had
similar half-lives of about 9 hours. The IT also increased MST
in two aggressive models of GVHD across non-H-2 minor
histocompatibility barriers, indicating that theusefulness of
anti-CDBF(ab’),-dgRTA is not limited to
a single-strain combination. This agent should be further investigated as an
alternative t o current strategies for treating steroid refractory GVHD.
0 1995 b y The American Society of Hematology.
G
CD5 functions as a T-cell costimulatory molecule recognizing the CD72 molecule on B cells.’’ Thus, MoAbs against
CD5” or murine Lyt-l l4 trigger T-cell activation. Our previous studies show that ITS made with intact anti-CD3 MoAb
that activate T cells have more nonspecific toxicity than ITS
made with F(ab’), ITS that do not activate T cells.15Therefore, in this study, we synthesized a nonactivating IT by
removing the Fc region of anti-CD~E
MoAb and then linked
it to RTA.
Several facts indicated that anti-CD3 would be an excellent choice for targeting toxin to target cells. ( 1 ) The CD3
portion of the T-cell receptor (TCR) complex is expressed
on all committed cytotoxic T cells and their precursors, and
immunohistology studies reported herein show significant
increases in CD3-expressing T cells in GVHD-target tissue.
(2) Other studies showed that anti-human CD3-RTA effectively inhibited mitogen-activated T cells in vitro.I6 (3) In
clinical studies, anti-CD3-RTA plus the potentiator NH4CI,
when used to eliminate GVHD-causing T cells from donor
RAFT-VERSUS-HOST disease (GVHD) results when
donor T lymphocytes react against the organs of the
immunosuppressed recipient.‘ It is a significant source of
morbidityand mortality after bone marrow (BM) transplantation. Despite advances in preventing GVHD using in
vivo chemotherapeutic prophylactic agents, ongoing GVHD
is generally difficult to treat and represents a major clinical
problem.’.’ Thus, better therapies for GVHD are needed.
Although numerous GVHD studies have addressed the
topic of prophylaxis, few studies have been directed at studying ongoing GVHD. Thus, we devised mouse models with
the express purpose of studying established GVHD. In the
model, donor marrow, as a source of stem cells, is mixed
with splenocytes, as a source of T cells, and transplanted
into H-2 or non-H-2 disparate recipients, resulting in lethal
GVHD directed against major (H-2) or minor (non-H-2)
histocompatibility disparities. The role of T cells in GVHD
in our H-2 disparate model has been established in previous
studies4,’
One approach for eliminating GVHD-causing T cells has
beentotarget
T cells with monoclonal antibody (MoAb)
linked to the potent catalytic phytotoxin, ricin toxin A chain
(RTA). RTA, the extractable A chain 30-kD component from
the toxin ricin (from the plant Ricinis communis), was chosen
for these studies because it requires only a single molecule
in the cytosol for cell death.6 It is an enzyme with first order/
single hit kinetics that inhibits the 28s RNA component of
the 60s ribosome, thereby inhibiting protein synthesis.’ RTA
was chemically deglycosylated before conjugation, as studies show that the removal of naturally occurring carbohydrates diminishes nonspecific reactivity without compromising activity.’
The first in vivo studies with immunotoxin (IT) and
GVHD therapy involved targeting of the CD5 differentiation
antigen inhumans’
and Lyt-l, the murine CD5 homologue.l0.” CD5 is a 65-kD glycoprotein that is broadly expressed on T cells. Transient responses at best were obtained
in both models. A problem with this approach may be that
Blood, Vol 86. No 11 (December l ) , 1995: pp 4367-4375
From the Section on Experimental Cancer Immunology, Department of Therapeutic Radiology, and the Division of Bone Marrow
Transplantation, Department of Pediatrics, University of Minnesota
Hospital and Clinics, Minneapolis, MN.
Submitted December 22, 1994; accepted July 21, 199.5.
Supported in part by US Public Health Service Grants No. ROICA31618, ROI-CA3672.5, POI-CA21737, R01-A13449.5, and POIAI35296 awarded by the National Cancer Institute and the National
Institute of Allergy and Infectious Disease, Department of Heatth
and Human Services. B.R.B. is a recipient of the Edward Mallinckrodt Jr Foundation Scholar Award.
Address reprint requests to Daniel A.Vallera,PhD, Box 367
UMHC, 420 SE Delaware St, Minneapolis, MN 554.55.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advettisement” in accordance with IS U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8611-0018$3.00/0
4367
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4368
grafts before bone marrow transplantation (BMT), achieved
aselective3-logdepletionof
CD3-expressingcells and
largely prevented lethal GVHD without the need for posttransplant imm~nosuppression.'~
Although it is more difficult tocontrol ongoing GVHDin vivo, these in vitro findings
wereencouraging.(4)Anti-CD3-RTAwas
morerapidly
andcompletely internalized ascompared with anti-CDSRTA and anti-CD2-RTA."
In a different study in which
internalization wasmeasured withradionucleotide-labeled
antibodies, anti-CD3 -RTA was internalized faster than antiCD5-RTA,anti-CD4-RTA,oranti-CD8-RTA."
( 5 ) In
mice, studies have confirmed that anti-CD3-RTA or antiCD3F(abr),-RTA can be administered in vivo and eliminate
the CD3+ cells responsible for BMgraft rejection, resulting
in an overall promotion of long-term BM engraftment.'' (6)
Anti-human CD3 has also been conjugated to intact diphtheria toxin" or to the diphtheria-binding site mutant CRM9,2'
resulting in selective and potent ITS.
In the current study, we show that anti-CD3 F(ab'),-RTA
has a depletionary effect
on CD3-expressing cells in vivo,
and in amodelof
aggressiveongoingGVHD, thatantiCD3F(ab'),-RTA is more effective than unconjugated antiCD3F(ab'), or anti-Lyt-IF(ab'),-RTA IT in the treatment
of lethal GVHD inducedby major orminor antigen disparate
BM transplants.
VALLERA ET AL
Phytohemagglutinin (PHAJ a s s y ~ s . PHA mitogenassays were
performed as described."
BMT. Our transplant protocol has been described in detail."
B 10.BR recipients were conditioned with 8.0 Gy total body irradiation (TBI) administered from a Philips
R T 250 Orthovoltage Therapy
Unit (PhilipsMedicalSystems,Brookfield,
WI) filtered through
0.35-mm Cu at a final absorbed dose rate of 0.41 Gylmin at 225 kV
and I7 mA. Donor BM was collected into RPMI 1640 medium by
flushing it from the shafts of femurs and tibias. Single-cell suspensions of donor splenocytes (as a source of GVHD-causing T cells)
were obtained by passing minced spleens through a wire mesh and
collecting them into RPMI 1640. Splenocytes were suspended with
BM at a concentration of 108/mL,and 0.5 mL, containing 25 x 10"
BMcellsplus 25 X IO" splenocytes,wasinfusedintothecaudal
vein. As an additional control, some recipients received donor splenocytes that were depleted of T cells before BMT
with anti-Thyl.2
+ C'' ' (hybridoma 30-H-12, a rat IgG2b, provided by Dr Ledbetter).
Pathologic examination of tissues. Mice were lulled and autopsied,andtissuesweretakenforhistopathologicanalysisasdescribed."' OrganswerescoredpositiveforGVHD
if there was
lymphocytic infiltrate with or without single-cell necrosis (skin. colon), crypt dropout (colon), periportal infiltrate with acute necrosis
(liver), or endothelialitis with a lymphocytic infiltrate (lung). These
features were present only in mice with active GVHD and
not in
normal mice or in irradiated recipients of syngeneic BMTs.
Flow cytometv analysis. MoAbs were directly labeled with biotin, fluorescein isothiocyanate (FITC), or phycoerythrin (PE) as previously described.I5
Single-cell
splenocyte
preparations
were
suspended in buffer (phospbate-buffered saline [PBS] + 5% colosMATERIALS ANDMETHODS
trum-free bovine serum + 0.015% sodium azide). Pelleted cells were
incubated for 15 minutes at 4°C with 0.4 p g of an anti-Fc receptor
Mice. BIO.BR/SgSnJ (H-2k,Mlslb/2b),C3H/HeJ
(H-2', MIS
MoAb (clone 2.462, provided by Dr Jay Unkless, Rockefeller Unilb/2a),CBA/J(H-2k, MIS ld2a), B10.D2(H-2d, MIS lb/2b),and
versity, New York. NY) to prevent Fcbinding.*' Optimal concentraDBA-2J (H-2d,MIS l d 2 a ) mice were purchased from Jackson Labotions of directly conjugated (biotin-.
PE-. or FITC-labeled) MoAb
ratory (BarHarbor,ME).C57BL/6(H-2b)micewerepurchased
were added to a total volume of 100 to 130 p L , and the mixture
through the Frederick Cancer Research and Development Center,
wasincubatedfor
1 hourat 4°C. For thebiotin-labeledMoAb,
NationalCancerInstitute(Frederick,MD).Donorsandrecipients
fluorescence was indirectly measured by adding streptavidin-labeled
were females. Donors were 4 to 6 weeks old, and recipients were 8
1 hourat
Red 613(GIBCO,GrandIsland,NY)foranadditional
to 10 weeks old at the time of BMT. The C57BL/6 and
BIO.BR
donodhost strain combination are mismatched at the H-2 major his- 4°C. After final washing, cells were fixed in I % paraformaldehyde.
ThefollowinglabeledMoAbsand
reagents, obtainedfromPhartocompatibility complex (MHC) and at several minor antigen
loci.
Mingen (San Diego, CA) unlessotherwiseindicated,were
used:
The Bl0.BR and CBA donorhost strain combination are matched
anti-CD3c (clone 145-2C11, hamster IgG), anti-CD8 (clone 53-6.72,
at H-2', but not atthe MIS minorantigen loci. TheB10.D2and
rat IgGZa, provided by Dr Ledbetter)," anti-CD4 (clone GK1.5, rat
DBA-2 donorhost strain combination are matched at H-2d, but not
IgG2b),anti-NKI.1(clonePK136,mouse
IgGZa, provided by Dr
at the Mls minor antigen loci.
Gloria Koo, Rahway, NJ)," and an irrelevant rat IgG2b anti-human
Reagents. Anti-CD3 is ahamster IgG MoAbproducedfrom
antibody (clone 3Ale).'x All samples were analyzed on a FACscan
hybridoma 345-2C1 I and was generously provided by Dr Jeffrey
(BectonDickinson,PaloAlto,
CA) usingconsort-30software. A
Bluestone (University of Chicago, Chicago, IL).It is reactive against
minimum of 20,000 events were examined. Background subtraction
the CD3 epsilon component of the murine TCR2* and was purified
usingadirectlyconjugatedirrelevantantibody
control was perby ammonium sulfate precipitation followed by ion exchange chroformed for each sample.
matography on DE52 (Whatman, Hillsboro, OR). F(ab')* fragments
Radioiodinafion. The labeling of IT with iodine-l 25
(lz51) was
were prepared as previously described' by incubating antibody at
performed using Iodo-beads according to the manufacturer's speci37°C for 90 minutes with a 1 :100 ratio of pepsin (Sigma Chemical
fications (Pierce, Rockford. 1L) and an adaptation of the
method first
CO, St Louis, MO) to antibody in 0.1 m o l L citrate buffer, pH 3.9.
described by Markwell.'" A 20% loss of protein content during the
The reaction was halted with the addition of 3.0
m o l L tris-HC1 (pH
8.6), and the mixture was dialyzed against borate-buffered saline, pH labeling procedure was assumed, based on previous studies, and all
concentrationswereadjustedusing
this value. Over82% of the
8.5. The F(ab'), fragments were purified on a protein A-Sepharose
countsoflabeledantibodywereprecipitablewithtrichloroacetic
column.*' ITwasproduced
by linkingthe purified fragmentsto
acid.
deglycosylated RTA (dgRTA; Inland Laboratories, Austin, TX) usBlood clearance of "'I-labeled IT. Blood clearance was detering N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP),asdemined as previously reported.'" Between 0.17 hours and 72 hours
scribed in detail elsewhere."' An anti-Lyt-1F(ab')2-RTA conjugate
afterinjectionintravenously(IV)
of 20pCiradiolabeled IT, five
was prepared similarly. Anti-Lyt-I from clone 53-7.313 (rat IgG2a)
was provided by Dr Jeffrey Ledbetter, Bristol-Meyer Squibb, Seattle, C3WHeJ mice in each group were anesthetized and bledby retroor(25
WA.' ' An irrelevant control IT was prepared from irrelevant hamster bital venipuncture at nine different time points. Whole bloodpL)
was aliquoted from each animal and countedin a liquid scintillation
fragments
(Rockland
Laboratory,
Gilbertsville,
PA) linked to
counter. Triplicate samples of the original injection standard were
dgRTA.
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4369
ANTI-CD3F(ab‘)2IMMUNOTOXIN FOR GVHD THERAPY
Table 1. Specific Activity of Anti-CDBF(ab‘)t-RTA Against
PHA-ActivatedMurine T Cells
CD3F(ab‘)2-RTA
CD3F(ab‘)>-RTA +
300 pg CD3F(ab’),
Hamster
F(ab’h-RTA
53.6 2 14.8
-t 14.9
21.1 5 3.2
2.7 2 0.4
64.1 2 10.1
65.4 f 3.6
59.5 2 2.2
42.8 f 18.6
43.2 f 8.9
56.8 2 5.2
59.6 2 3.5
48.0 2 18.6
Concentration
(pglmL)
0
50.40.1
1
10
Mouse splenocytes were incubated with IT for four hours at 37°C.
For blocking studies, cells were treated with 300 pg/mLunconjugated
anti-CD3F(abf),for 30 minutes before addition ofIT. After treatment,
cells were washed, and PHA was added. After 3 days, cells were
pulsed with tritiated thymidine and harvested. Data are presented as
cpm x lo-’ f one SD unit.
counted at the same time as tissues, allowing the determination of
the activity in tissue as a percentage of the injected dose, corrected
for the physical decay of the radionucleotide. The individual values
for the animals in each group were averaged for each time point.
The logarithms of the averages of the percentage of injected dose
per milliliter were plotted over the time after treatment. Linear regression by a least squares method yielded a straight line, allowing
an estimation of the biologic half-life of the radiolabeled antibodies.
Biodistribution studies. C3wHeJ mice (n = 5 ) were given intraperitoneal (IP) injections of 2 pCi of ‘ZSI-labeledIT. The animals
were killed 48 and 72 hours after injection of radiolabeled antibodies.
The spleen and thymus were removed and counted. Tissues were
counted on a Beckman 5500 gamma counter (Beckman, Arlington
Heights, IL)with a ‘*’I window.
Statistical analyses. Group comparisons of continuous data were
performed using Student’s t test. Survival data were analyzed by
lifetable methods using the Mantel-Peto-Cox summary of X’.’’ Probability (P)values less than .OS were considered significant.
RESULTS
IT composition. Purity of the IT was assessed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as de~cribed.~’Both anti-CD3F(ab’),-RTA and
anti-Lyt-lF(ab’),-RTA were heterogeneous, consisting of
one, two, or three toxin molecules linked to F(ab’),, but the
1:1 species generally predominated (data not shown). Less
than 13% unconjugated F(ab’), was present after purification, but no free toxin was detected.
Inhibitory effect of anti-CD3F(ab1),-RTA on mouse
splenocytes in vitro. To determine whether antiCD3F(ab’),-RTA selectively inhibited mouse T cells,
C57BL/6 splenocytes were preincubated with antiCD3F(ab’),-RTA for 4 hours at concentrations of 0, 0.1, I ,
and 10 p&mL (Table 1). Inhibition of PHA-induced activation was dose-dependent, and at 10 pg/mL, counts per minute (cpm) were reduced 95% (2.7 x lo” as compared with
53.6 X lo3 in the untreated control) without the benefit of
IT potentiators. Two control groups showed specificity: (1)
The addition of 300 pg/mL unconjugated anti-CD3F(ab’),
blocked the inhibitory effect of anti-CD3F(ab’),-RTA. (2)
An irrelevant control polyclonal hamster antibody conjugated to dgRTA did not inhibit PHA activation.
Depletionary effects of anti-CDJF(ab’)*-RTA. To determine whether anti-CD3F(abf),-RTA had a depletionary
effect, C57BL/6 mice were irradiated and then given synge-
neic BMTs to avoid complications of the sometimes immunosuppresive nature of GVHD occurring in allogeneic transplants. We studied the ability of anti-CD3F(abf),-RTA to
deplete CD3+ cells (Table 2). Irradiation was given on day
-1. Syngeneic BM and a single dose of anti-CD3F(ab’)*RTA were given on day 0. On day 2, mice were killed and
splenocytes studied by flow cytometry. Mice given 400 pg
unconjugated anti-CD3F(ab1), had 0.05 X lo6 CD3+ cells
in the spleen, while mice given less than half that dosage of
anti-CD3F(ab’),-RTA had 2.5-fold fewer CD3’ T cells.
Compared with PBS-treated controls, mice given antiCD3F(ab‘),-RTA had about fivefold fewer CD3’ cells (a
reduction of 82%). These findings show the depletionary
effect of anti-CD3F(abf),-RTA as compared with antiCD3F(ab’), in the BMT setting. Anti-CD3F(ab’),-RTA
treatment resulted in the same number of NK1.1+ cells as
in the PBS control group, further indicating that antiCD3F(ab’),-RTA selectively depleted CD3-expressing
cells. A significant reduction in these CD3’ T cells (42%)
was still observed in anti-CD3F(abf),-RTA-treated mice at
even 14 days postinjection of IT. A depletionary effect of
anti-CD3F(abf),-RTA was also observed in normal mice,
where a significant inhibition of both CD4-expressing and
CD8-expressing cells was observed (data not shown).
Effect of ar~ti-CDjlF(ab’)~-RTA
on ongoing GVHD across
the H-2 barrier. To determine whether anti-CD3F(ab’),RTA could inhibit established GVHD across the H-2 barrier,
B1O.BR mice were given 8 Cy total body irradiation (TBI)
and then transplanted with 25 X lo6 C57BL/6 BM cells and
25 X IO6 splenocytes. In situ studies showed that at day 9
after BMT, there is a dramatic infiltration of CD3-expressing
lymphocytes observed in the colon of these mice (data not
shown). No such cells were observed in controls given BM
grafts without GVHD-causing splenocytes. Treatment with
anti-CD3F(ab’),-RTA was initiated a week after GVHD
induction to permit a graft-versus-host reaction before treatment. Mice were givendaily IP injections of 30 pg IT beginning on day 7 and continuing through day 11. Figure 1A
shows that anti-CD3F(ab’),-RTA-treated mice had a median survival time (MST) of greater than 80 days. At 3
months posttransplant, 22% of the animals were still surviving. In contrast, control PBS-treated animals had an MST
Table 2. Depletionary Effect of Anti-CDBF(ab’),-RTA on Mice
Undergoing Syngeneic BMT
CD3’ Cells by Flow Cytometty
Treatment
96 Positive
PBS
0.1
400 pg anti-CDJF(ab’),
150 pg anti-CD3F(ab’)?-RTA
anti-Thyl.2
0.18 + C’ (in vitro)
36
27
9
25
Absolute Cell Count IXlO-’)
1
0.05
0.02
~~~
____
C57BU6 mice were given 9 GyTB1 on day -1 and 1 day later were
given a syngeneic BMT with 15 x 10’ C57BU6 BM cells and 15 x 10’
splenocytes. Different groups (n = 3 per group) were given IP injections of PBS, 400 pg anti-CD3F(ab‘)2once on day 0, or 150 pg antiCD3F(ab’),-RTA once on day 0. One group wasgiven BM mixed with
T-cell-depleted splenocytes. Splenocytes were harvested on day 2
and examined for CD3’ cells by flow cytometry.
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4370
VALLERA ET AL
I
anti-Thyl.2+C’
l !
T
Ap~yi-CD3F(:”-RTA
p
,001 compared to PES
anti-CD&anti-CDB
0
B
3
22
Fig1. The in vivo effect ofanti-CDBF(ab’),-RTA
injections on establishad GVHD induced across the
MHC barrier in B10.M recipients of C57Bt.16 grafts.
Irradiated (8 Gyl recipientswere given bone marrow
andspleencells (25 x 10’ ofeach).Onegroup
of
mice received donorBM that was depletedof Tcells
using anti-Thyl.2
C‘ before BMT. Groups were
injectedon days7 through 11with PES, 50 p g l d antiCDBF(ab’), fragments, or 30 pgld anti-CD3F(ab’)2RTA. The remaining groups were given a single injection of 400 p g of intact anti-CD3 or 400 p g of antiC M (clone GK1.5) plus anti-CD8 (clone 2.43) on day
7. (A) Survival is plolted in an actuarial manner (n =
8 per group). (B) Mean weight curves are shown for
the same mice. The course of injection of anti-CD3
F(ab’),-RTA is indicated by the bar.
+
Days Post Transplant
of only 20 days, and none survived beyond 45 days postBMT. Treatment with 50 pg ar1ti-CD3F(ab’)~X 5 days on
days 7 through 11 (250 pg total) did not result in significant
protection against GVHD. Treatment with 400 pg of intact
CD3 on day 7 resulted in a characteristic high percentage
of early mortality (MST, 8 days) due to the activation of T
cells and toxic side effects of anti-CD3 treatment, aspreviously reported.” Transient and nonsignificant protection
from GVHD was noted when a combination of 400 pg antiCD4 and anti-CD8 MoAbs was administered on day 7. As
a positive control, T cells were eliminated from the donor
grafts before BMT using anti-Thy 1.2 plus complement in
one group of animals. Eighty-eight percent of these mice
were survivors on day 90, indicating that elimination of all
donor T cells can prevent GVHD mortality in this model.
We also studied the effects of an irrelevant control hamster
F(ab’)2-RTA in a separate experiment using the exact dose
and schedule for GVHD therapy as with anti-CD3F(ab’)zRTA (data not shown). No extension of MST was observed.
Collectively, these findings indicate that anti-CD3F(ab‘)2RTA is potent and specific in its ability to inhibit an ongoing
GVHD response across a full MHC disparity.
To select the dayof GVHD treatment initiation, we identified the onset of GVHD in the mice shown in Fig 1A by
monitoring weight loss over time. Our data in Fig 1B show
that the first detectable GVHD occurred on days 7- 11. However, after an initial decline, weights of mice treated with
anti-CDSF(ab’)*-RTA stabilized and remained around 18 g
for the duration of the experiment. More precipitous drops
in weight were noted in mice with higher GVHD mortality.
These groups included mice treated with control PBS, antiCD3F(ab’)2 alone, or a combination of anti-CD4 plus antiCD8 MoAbs. Only the group of mice in which T cells were
eliminated from the donor graft before BMT with antiThyl.2 plus complement underwent a steady increase in
weight over the 76-day interval. These findings indicated
that although anti-CD3F(ab’),-RTA inhibited GVHD, it
was still present in these treated mice.
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437 1
ANTI-CD3F(ab‘)z IMMUNOTOXIN FOR GVHDTHERAPY
Table 3. Effect of Various Dosages of Anti-CD3F(ab)2-RTA on
Established GVHD
Treatment
PBS
Anti-CD3F(ab’)z-RTA
Anti-CD3F(ab’)?-RTA
Anti-CMF(ab’)z-RTA
Anti-CDJF(ab’)z-RTA
Anti-CDBF(ab’),-RTA
Anti-CD3
C‘
Anti-Thyl.2
+
Dose
(pg/d)
-
Schedule
d 7-11
10
30
50
75
d 7-11
12’
d 7-11
d 7-11
d 7-11
400 + 25 d 7-11
400
d 7 only
vitro
In
-
MST
(d)
26
36
42*
34
22
18
9
>76*
% Actuarial
Survival Rate
at Day 60
0
0
0
0
0
0
100’
B1O.BR recipients were given lethal TB1 (n = 8 per group) on day
-1, and on day 0 were transplanted with C57BU6 BM and spleen cells
(25 x 10‘ of each). On day 7, mice were given a 5-day course of
various concentrations of anti-CDBF(ab’),-RTA or unconjugated antiCDBF(ab’), fragments, or a mixture of unconjugated anti-CD3F(ab’)z
fragments plus unconjugated RTA.One group was given a single
and spleen
injection of 400 pg anti-CD3. Another was given donor BM
cells that were treated with anti-Thyl.2
C’ before BMT. Survival
was monitored.
* P < .05 compared with PES control.
+
Tissues from mice in the same experiment that were
treated with unconjugated anti-CD3F(abr)2, anti-CD4 plus
anti-CD8, or intact anti-CD3 and that were morbid between
day 19 and day 39 were collected immediately before death
(n = l or 2). Tissues were embedded in paraffin, blocked,
sectioned, and stained with hematoxylin and eosin for histopathologic examination. All mice showed evidence of colonic GVHD including infiltrates and tissue necrosis. Two
mice treated with anti-CD3F(abr),-RTA that survived were
killed on day 90 post-BMT. These mice showed positive
signs of GVHD in the colon, liver, and lung. Infiltrates and
tissue necrosis were the most prominent features.
Effect of varying dosages of anti-CD3F(ab’)2-RTA on
ongoing GVHD across the H-2 barrier. In a different experiment, B 1O.BR recipients given C57BL/6 BM and spleen
were treated with increasing concentrations of antiCD3F(ab’),-RTA (Table 3). Mice given control PBS had
an MST of 26 days, similar to the previous experiment, and
died of GVHD. MST was increased to 36 days in mice given
a 5-day (days 7 through 11) course of 10 pg per injection
anti-CD3F(abf),-RTA and to 42 days in mice given a course
of 30 pg per injection anti-CD3F(abr),-RTA. However,
MST decreased when the dosage was increased to 50 or 75
pg per injection. A group of control mice given a 5-day
course of 400 pg unconjugated anti-CD3F(ab‘), plus 25 pg
unconjugated RTA showed no protective effect from GVHD
onset. A group of mice given anti-CD3 had an MST of only
9 days. Positive controls in which T cells were eliminated
before transplant with anti-Thyl.2 plus complement in vitro
had an MST of greater than 76 days. Using these data, we
selected the dose of 30 pg per injection anti-CD3F(ab‘),RTA for subsequent studies. Our findings on the effectiveness of the IT were reproducible, and we established a working dose and schedule.
Effect of anti-CD3F(abt),-RTA in protecting against es-
tablished GVHD across non-H-2, minor histocompatibiliry
barriers in two Afferent mouse models. To determine
whether anti-CD3F(ab’),-RTA could be used to treat
GVHD generated across non-H-2 minor histocompatibility
antigen disparities, CBA mice were given 8 Gy T B 1 and
then 20 X lo6 T cell-depleted, H-2-matched B1O.BRBM
cells and 50 X lo6splenocytes (twice the splenocyte number
used in the C57B46 into BlO.BR model). Table 4 shows
that anti-CD3F(abr),-RTA treatment significantly extended
the MST from 18 days in PBS controls to 46 days in ITtreated mice. Daily weight measurements showed that antiCD3F(ab’),-RTA enhanced the anti-GVHD effect compared with untreated controls, but that GVHD-related weight
loss still occurred later (data not shown). Whereas GVHD
generation in the mismatched C57BL/6-into-B10.BR model
is CD4+ T cell-dependent, GVHD in this minor model is
CD8+ T cell-dependent, indicating that this agent was active
against both types of T cells.34
In a separate experiment ina different modelinwhich
minor antigens are mismatched, DBN2 recipients given 8
Gy TB1 and 20 X lo6T cell-depleted B10.D2 BM cells and
50 X lo6 splenocytes developed GVHD. The MST was 21
days in a group of mice given control PBS injections. AntiCD3F(ab’),-RTA significantly extended the MST to 34
days in this minor mismatched model. Weight loss data indicated that the mice that died did undergo GVHD-related
weight decline (data not shown). In both of these models,
in vitro depletion of T cells from donor grafts before BMT
resulted in an MST of greater than 83 days, with 100% of
the animals surviving at day 60. Collectively, these findings
show that anti-CD3F(abr),-RTA is notonly
effective
against lethal GVHD induced across the major histocompatibility barrier, but is also effective against GVHD in aggressive models in which minor antigens, and not major antigen
differences, serve as targets for lethal GVHD. The data also
Table 4. In Vivo Effect of Anti-CD3Flab)z-RTA Injections on
Established GVHD Induced Acrossthe Minor Histocompatibility
Barrier in Two Ditferent Strain Combinations
Treatment
E1O.BR into CBA
PBS
Anti-CD3F(ab‘)z-RTA
Anti-Thyl.2 + C’
B10.D2 into DBA-2
PBS
Anti-CDJF(ab‘)p-RTA
30
Anti-Thyl.2
C’
+
Dose
Idd)
MST
Id)
% Survival
at Dav 60
d 7-11
d 7-11
- vitroIn
18
46’
>85*
0
20s
100’
-
21
34*
>83*
Schedule
-
30
d 7-11
d 7-11
- vitroIn
0
0
loo*
BMTs were performed with two strain combinations. First, CBA
recipients (n = 10 per group) were given 8 Gy lethal TB1 on day -1,
and on day 0 were transplanted with 20 x lo6 B1O.BR BM cells and
50 x 10‘ spleen cells. On day 7, mice were given a 5-day course of
anti-CDfF(ab’),-RTA or control PES. Another group was given donor
BM and spleen cells that were treated with anti-Thyl.2 + C‘ before
BMT. Survival was monitored. A second experiment was performed
identically, except B10.D2 BM and splenocytes were transplanted into
irradiated DBA-2 recipients (n = 10 per group).
P < .05 compared with PBS control.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
4372
VALLERA ET AL
Table 5. Effect of Anti-Lyl-F(ab’),-RTA on Established GVHD in
the Mismatched C57BL/G-lnto-BlO.BR Model
Dose
MST
(d)
Treatment
(pg/dl
PBS
- 7-11d
21
Anti-Lyl-F(ab’kRTA
Anti-human CD5-RTA
30
30
30
20
25
Anti-CD3F(abf),-RTA
Schedule
7-11d
d 7-11
7-11d
77”
% Actuarial
Survival Rate
at Day 60
0
0
0
62*
B1O.BR recipients (n = 8 per group) were given lethal TB1 on day
-1, and on day0 were transplanted withC57BU6 BM andspleen cells
(25 x I O 6 of each). On day7, mice were given a 5-day course
of PBS,
anti-Lyl-F(ab’)2-RTA, anti-CD3F(abr),-RTA, or a control anti-human
CD5-RTA. Survival was monitored.
* P < .01 compared with PBS control.
show that anti-CD3F(abr),-RTAisactive
in models in
which CD8+ T cells play a major role.
EfSect of anti-Lyt-lF(ab’),-RTA on ongoing GVHD induced across the H-2 MHC barrier. As
anti-LytlF(ab’),-RTA is the murine equivalentof anti-human CD5RTA currently used in clinical trials, we directly compared
anti-Lyt-lF(ab’),-RTA
and
anti-CD3F(abr),-RTA
for
their efficacies in treating ongoing GVHDin the same experiment. Table 5 shows that anti-Lyt-lF(ab’),-RTA given in
a5-day course did not result in asignificant anti-GVHD
effect in B 1O.BR recipients of C57BL/6 BWspleen.In contrast, a 5-day course of anti-CD3F(ab’),-RTA significantly
increased MST to day 77, as compared with day 21 in controls. Anti-human CD5-RTA, a noncrossreactive control IT,
did not inhibit GVHD.
To determine whether the failure of anti-Lyt-lF(ab’),RTA as opposedto
anti-CD3F(ab‘),-RTA
to protect
against GVHD was related to IT potency or perhaps to a
difference in the clearance of the two proteins in vivo, we
performed additional studies. Pharmacokinetic
studies
showed that the serum
half-lives of anti-Lyt-lF(ab’),-RTA
and anti-CD3F(ab’),-RTA were similar (8.9 and 9.0 hours,
respectively) in normal B10.BR mice. Both ITS accumulated
in a similar manner in the spleen and thymus. In contrast,
the abilities of these two ITS to inhibit T-cell proliferation
in PHA assays were different, because the 50% inhibition
concentration (I(&) of anti-Lyt-lF(ab’),-RTAwas10.2
pg/mL and the ICs0 of anti-CD3F(abf),-RTA was 2.1 pg/
mL when compared in the same assay without the addition
of any potentiators. Collectively, these findings suggest that
the failure of anti-Lyt-IF(ab’),-RTA to inhibit GVHD
is
more likely related to an issue of potency of the IT, rather
than clearance
and
localization.
second
A anti-Lyt1F(ab’)2-RTA
was
prepared
using
another
MoAb,
SK69.6.38 generously provided by Dr Edward Boyse (Memorial Sloan-Kettering Cancer Center, New York, NY) and
findings were identical.
DISCUSSION
Ongoing GVHD is acknowledged asa significant clinical
problem and few murine studies have been
directed at studying ongoing GVHD. The majorfinding of thisstudy was that
antiLCD3F(ab’),-RTA extended MST in major and minor
antigen-mismatched BMT recipients with established
GVHD longer than any other IT thus far reported. MST was
also extended in two additionalmodels of lethal GVHD
where donors and recipients were disparate at minor antigen
loci only. The effects of anti-CD3F(ab’),-RTA were specific, because flow cytometry studies on mice given syngeneictransplants [to studytheeffects
of anti-CD3F(ab’),RTA in the absence of the immunosuppressive effects of
allogeneic transplants] revealed that anti-CD3F(abr),-RTA
was selectively depletionary in its effect on CD3-expressing
T cells, butnot on cells expressing the
unrelated NK 1.1
marker. An irrelevant IT control, hamster CD3F(abf)?-RTA,
did not extend MST.
Previous studies usingmiceindicatedthat
effectiveIT
could be synthesized using anti-CD3 MoAb totarget T cells,
but only if we inhibited the ability of the MoAb to trigger
T-cell activation through the removal of the Fc region.” ITS
made with activating, intact anti-CD3 caused a high mortality rate and a precipitous release of inflammatory cytokines.
In fact, therapy with anti-CD3 alone has shown potential for
preventingorgangraftrejection,
but has been limited by
toxic side effects due to in vivo activation of T cells induced
by ligation of the TCR and theFc region of anti-CD3.3”.35~”X
ITS made with nonactivating anti-CD3F(ab’), did not cause
cytokine release, were less toxic to nontarget organ systems,
deleted CD3-expressingT cells, and prevented BM rejection
in vivo. Evaluation of anti-CD3F(ab’)>-RTAand
antiCD3-RTA in the same invitro PHA experiment showed
that anti-CD3F(ab’)2-RTA is more potent.” Previous studies have demonstrated that ITS made with F(abO2 are more
potent than ITS made with intact antib~dy.’”~” However,
this
is not always the~ a s e . ~ Collectively,
”~’
these studies indicate
that certain F(ab’),-ITS such as anti-CD3F(ab’),-RTA may
have advantages.
We believed that itwas important to addressthe activation
issue in these studies. CD5 interacts with costimulatory moleculeCD72
onantigen-presentingB
cells.” Anti-CD5
MoAb triggersactivation
of CD5-expressing humanT
cells.” Likewise anti-Lyt-1 triggers murine T cells toproliferate.“
Thus,
we
prepared
nonactivating
anti-LytI.2F(abf),-RTA and compared
it with anti-CD3F(ab’ )?RTA in the same experiment. The same RTA was used to
prepare both reagents to ensure consistency of the toxic moiety of the ITS. Although the clearance andbiodistribution of
thetwo agentsweresimilar,
anti-CD3F(abf),-RTA was
more effective in treatingestablished GVHD.Onefactor
that correlatedwith thesuperior in vivoactivity of antiCD3F(abf),-RTA was its superior fivefold in vitro potency
measured by PHA assay. We concluded that preparation of
anti-Lyt-IF(ab‘)*-RTA provided no advantage.
Then why was CD3 more effective than CD5 as a target
molecule?Thereare severalpossibleexplanations.
(1) In
addition to the previously discussed studies in which antiCD3-RTA was morecompletelyand rapidlyinternalized
than anti-Lyt- 1-RTA,”.” other subcellular compartmentalization studies inwhich anti-CD5-RTA wasradiolabeled
andthendirectlymeasured
in organelle fractions isolated
from Percol gradients show that anti-CD5-RTA IT is rapidly
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
4373
ANTI-CD3F(ab')2 IMMUNOTOXIN FOR GVHDTHERAPY
transported to the lysosome, where unwanted degradation
takes place.44 ( 2 ) Anti-CD5 IT permits the expansion of
GVHD-causing T cells, which do not express CD5. Studies
show that a large proportion of T lymphocytes lack CD5
expression after BMT?5346When human BM cells from
GVHD patients were treated with an antiCD5 IT and then
cultured in vitro for 16 days, CD5-negative T cells mostly
expressing CD3 were detected. CD5-CD3+ cells that escape
IT treatment retain an alloreactive cytotoxic repertoire capable of GVHD reaction?'
These results with anti-CD3F(abf),-RTA are the best that
have been attained in several published studies dealing with
the treatment of ongoing GVHD in the same mismatched
mouse model of allogeneic BMT.'0,30,48
Anti-Lyt-l antibody
linked to RTA" or labeled with r a d i o n ~ c l i d ewas
~ ~ only
partially protective. Attempts to treat established GVHD using the potent anti-T cell drug rapamycin?' a fungal macrolide, also produced transient protection.
Allof these murine studies and the clinical studies resulted in significant but transient anti-GVHD effects. The
lack of complete inhibition of GVHD during the efferent
phase rather than the afferent phase indicates that it is far
more difficult to inhibit GVHD once an alloresponse is initiated in vivo. This could be attributed to the difficulty of
effectively delivering therapy to a relatively small GVHDcausing population of cells in a complex biologic environment. Alternatively, a network of cells may be involved in
GVHD once the process has begun, thus making specific
targeting difficult. Protection was more limited when mice
were transplanted with BM with minor antigenic disparities
because minor histocompatibility antigen differences sometimes can be even more potent at eliciting GVHD responses
than fully mismatched MHC differences, because different
mechanisms of GVHD may be operative in the two different
systems34or because we gave higher numbers of donor T
cells in the minor model. This is a topic of future investigation.
The difficulty in inhibiting ongoing GVHD is evidenced
by our results with anti-CD3F(abf)2 fragments alone. In past
studies, we showed that unconjugated anti-CD3F(abr), fragments can be used prophylactically to prevent GVHD. In
these current studies, anti-CD3F(abf), fragments were much
less effective when compared with anti-CD3F(ab'),-RTA
in treating established GVHD. Fragments given on day 7
post-BMT did not inhibit established GVHD in this model.
This could be explained by the flow cytometry studies, which
showed that anti-CD3F(ab'), fragments caused the modulation of CD3 from the cell surface of the T cells and only
partial T-cell depletion. In contrast, the anti-CD3F(ab'),RTA, which was much more effective against ongoing
GVHD in the present study, caused the deletion of CD3+
T cells. The internalization of anti-CD3F(ab'),-RTA eliminated the GVHD-causing cells. Whereas modulation may be
an effective means of GVHD prophylaxis, it is better to
delete T cells to treat GVHD once the response has been
initiated.
Although we have achieved a high degree of protection
against ongoing GVHD with IT, we have not cured the mice,
as evidenced by late onset of the disease. To further extend
MST with ~ I I ~ ~ - C D ~ F ( ~ ~ ' ) ~it -may
R T Abe, necessary to
combine itwith other ITS or approaches such as d r ~ g s . 4 ~
Some reasons that anti-CD3F(ab'),-RTA is not fully protective are as follows. (1) Inadequate dosing and schedule may
have resulted in incomplete elimination. This has been a
serious limitation to the clinical use of IT, as clinical studies
to date have mostly been limited by vascular leak syndrome
and hepatotoxicity. However, we hope that a more thorough
understanding of IT toxicity and agents that might diminish
their toxicity will be of future help. ( 2 ) Immunohistology
studies show that other donor cells, such as macrophages
and natural killer cells, may play some role in established
GVHD in this model (unpublished data, January 1995). Perhaps even greater anti-GVHD effects might be achieved by
targeting these cells as well. (3) Antibody responses may
develop against the antibody or toxin component of IT. Although several immunoglobulin responses have been noted
in clinical trials:'
it is likely that optimal doses of anti-T
cell IT will not only target GVHD-causing cells, but also
might inhibit the development of immunoglobulin responses,
as these responses are also T cell-dependent.
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