Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3494-3502
Epitope Specificity of CD44 for Monoclonal Antibody-Dependent
Facilitation of Marrow Engraftment in a Canine Model
By
Brenda M. Sandmaier,
Rainer Storb,
Kelly L. Bennett,
Frederick R. Appelbaum, and
Erlinda B. Santos
From the Clinical Research Division, Fred Hutchinson Cancer Research
Center, Seattle; the Department of Medicine, University of Washington,
Seattle; and Bristol-Myers Squibb Pharmaceutical Research Institute,
Seattle, WA.
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ABSTRACT |
Primary graft rejection after marrow transplantation occurs more
frequently in patients receiving HLA-haploidentical compared with
HLA-identical sibling transplants. Both human and experimental animal
data suggest that the cells responsible for this phenomenon are either
host natural killer (NK) cells, T cells, or both. To investigate the mechanisms of graft rejection, we have developed a
canine model of marrow transplantation, which uses DLA-nonidentical unrelated donors in the absence of postgrafting immunosuppression. In
this model most animals rejected their marrow grafts after a
preparative regimen of 9.2 Gy total body irradiation (TBI). However,
engraftment of DLA-nonidentical marrow can be facilitated when the
recipients are pretreated with monoclonal antibody (MoAb) S5, which
recognizes CD44. In this report, we extended these observations by
first cloning the canine CD44 and, next, mapping the epitope recognized
by S5, which was located in a region conserved among human and canine
CD44 and was distinct from the hyaluronan binding domain. However, in
vitro binding of S5 caused a conformational change in CD44, which
allowed increased hyaluronan binding. Then, we reexamined the in vivo
model of marrow transplantation and compared results with MoAb S5 to
those with two other anti-CD44 MoAbs, IM7 and S3. Only MoAb S5
significantly increased the engraftment rate of DLA-nonidentical
unrelated marrow, whereas the two other anti-CD44 MoAbs were
ineffective. The enhanced in vivo effect was not related to differences
in the MoAbs' avidities, since both S5 and IM7 had equivalent binding
to CD44, but most likely related to the specific epitope that S5
recognizes. Thus, this study shows that the effect of the anti-CD44
MoAb S5 in facilitating engraftment is epitope specific and if one is
to use an anti-CD44 to facilitate engraftment of marrow in humans, one
cannot assume that any anti-CD44 would work.
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INTRODUCTION |
THE INCIDENCE OF rejection of
HLA-haploidentical related marrow grafts increased in direct proportion
to the number of mismatched HLA antigens on the nonshared
haplotype.1 At our center, the overall incidence of graft
rejection was 12%, ranging from 7% with marrow grafts from
phenotypically HLA-identical donors to 20% with donors who were
mismatched for two or three HLA loci. Graft rejection has been
associated with a high likelihood of subsequent death.
To better understand the mechanisms involved in graft rejection and to
test new therapies, we developed a preclinical canine model of graft
rejection involving marrow transplants from DLA-nonidentical unrelated
donors after conditioning by 9.2 Gy total body irradiation (TBI). In
this setting, 80% to 90% of the grafts fail.2-4 We previously showed that the risk of rejection could be significantly decreased when recipients were treated with six daily intravenous injections of an anti-CD44 monoclonal antibody (MoAb), S5, at 0.2 mg/kg/d before 9.2 Gy TBI and marrow infusion.5 Sixty-seven percent of dogs administered MoAb S5 showed successful engraftment as
compared with only 8% of control dogs not administered MoAb therapy or
given an irrelevant MoAb. The antigen recognized by S5,
CD44,6 is a cellular adhesion molecule expressed on both hematopoietic and nonhematopoietic tissues. CD44 is an integral membrane glycoprotein and serves as the principle receptor for hyaluronan,7 a glycosaminoglycan that is abundantly
distributed in extracellular spaces. CD44 acts as a modulator of
various immune functions including those involving CD2 and
CD3,8-10 such as T-cell activation trigger via
cross-linking of CD2. MoAbs to CD44 can also induce release of
interleukin (IL)-1, tumor necrosis factor (TNF)
,11 and
macrophage colony-stimulating factor (M-CSF)12 from human
monocytes. In addition, we have shown a CD44-dependent increase of
canine natural killer (NK)-cell activity, which is an indirect effect
mediated both by an increase in effector/target cell conjugate
formation and the elaboration of TNF that, additionally, makes the
cells more sensitive to radiation.13-15
In this report, we extended the previous observations with the
anti-CD44 MoAb S5 by first cloning the canine CD44 and next, mapping
the epitope recognized by S5, which was located in a region conserved
among humans and dogs CD44. Then, we reexamined the in vivo model of
marrow transplantation and compared results with MoAb S5 to those with
two other anti-CD44 MoAbs, IM7 and S3, one directed against an
immunologically overlapping epitope and the other recognizing a
discreet epitope on CD44. The endpoints of the in vivo study, graft
rejection, and graft-versus-host disease (GVHD) are mutually exclusive
events in this model. Dogs that reject their graft do not show clinical
or histologic evidence of GVHD. Conversely, because no postgrafting
immunosuppression for prevention of GVHD was used in the present study
in the interest of a simple and rapid study readout, all dogs with
engraftment uniformly developed severe GVHD.16-18 Only MoAb
S5 significantly increased the engraftment rate of marrow from
DLA-nonidentical unrelated donor dogs, whereas the two other anti-CD44
MoAbs were ineffective, suggesting that the epitope recognized by S5 is
important for the graft enhancing effect.
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MATERIALS AND METHODS |
MoAbs.
The murine anti-CD44 MoAbs S5 (subclass IgG1) and S3 (IgG2b) were
generated against canine marrow cells.5,6 The rat anti-CD44 MoAb IM7 (IgG2b), generated against mouse cells,19 was
cross-reactive with canine cells; MoAb MEM-85 (murine IgG1) recognized
human CD44. Negative control murine MoAbs 6.4 (IgG2b) and 31A
(IgG1)20 (which recognized murine Thy-1) did not
cross-react with canine cells. All MoAbs used in vivo were protein A or
protein G affinity purified and were endotoxin free. Protein
concentrations were determined using the Bradford reaction (BioRad
Labs, Hercules, CA).
Cloning of canine CD44.
Peripheral blood and splenic mononuclear cells were isolated from a
Ficoll Hypaque gradient. The cells were cultured for 48 hours in
RPMI-1640 supplemented with 5% fetal calf serum (FCS) in the presence
of 1% phytohemagglutinin (PHA) and 5% pokeweed mitogen. RNA was
extracted on a cesium chloride gradient.21 A cDNA library
in the Lamda Zap cloning vector (Stratagene, La Jolla, CA) was
constructed and packaged.22 A baboon BamH1 fragment containing 600 bp of the 5
portion of the baboon CD44 cDNA was 32P-labeled by extension random hexamers by DNA polymerase
1 in the presence of 32P deoxy cytidine triphosphate
(dCTP) and was hybridized in 35% formamide/5XSSPE/0.1%
Ficoll/0.1% polyvinyl pyrrolidine/0.1% bovine serum albumin/0.5%
sodium dodecyl sulfate (SDS) at 42°C for 18 hours. Plasmids
(pBluescript SK[-]) containing the canine CD44 inserts were subcloned
from the Lambda Zap phage by an in vivo excision process in the
presence of helper virus.22 Clones were sequenced in
pBluescript SK(-) using the dideoxynucleotide method23 as
described in the Sequenase (US Biochemicals, Cleveland, OH) protocol
for single-stranded and double-stranded DNA. cDNA clones were initially
sequenced from the flanking T3 and T7 primers. Specific primers for the
insert were constructed to enable sequencing the complete clone.
Sequence analysis was performed with GenePro 4.2 software (Riverside
Scientific Enterprises, Bainbridge Island, WA).
Preparation of full-length and truncated extracellular domain of
CD44.
As MoAb S5 recognized human CD44, available constructs of full-length
and truncated mutants of the extracellular domain of human CD44 were
used to map the MoAb recognition sites. Both wild-type human CD44H
receptor globulins (Rg)7 and mutant CD44 Rg24 that were ligated into a HindIII/BamHI-cut CDM7 vector
containing genomic sequence encoding the hinge, CH2 and CH3 domains of
human IgG1,25 were transfected into Cos cells as previously
described.7,24 A similar construct of the extracellular
domain of canine CD44 was produced. The fusion proteins were purified
from the culture supernatants using Protein-A-Sepharose as previously
described,7,24 and protein concentration determined.
Binding of MoAb to CD44 Rgs.
Binding of the anti-CD44 MoAb to the purified CD44 Rgs was assessed
using an enzyme-linked immunosorbent assay (ELISA). Wells of
polystyrene ELISA plates were coated with 0.2 µg/mL of the CD44 Rg.
After blocking with nonfat milk in phosphate-buffered saline (PBS), the
plates were incubated with 50 µL of 10 µg/mL of the MoAb. After
washing, the plates were coated with goat antimouse (or antirat) IgG
horseradish peroxidase (HRP) (1/2,000 dilution), followed
by ABTS color solution (Sigma, St Louis, MO). Plates were read on a
Vmax microtiter plate reader (Molecular Devices, Menlo Park, CA) at 405 nm.
Marrow transplantation.
Beagles and beagle/hound crossbreeds were bred and raised at the Fred
Hutchinson Cancer Research Center (FHCRC) or purchased from commercial
kennels. They were vaccinated against distemper, leptospirosis,
hepatitis, and parvovirus and monitored to be disease free before start
of the experimental protocol. The median age of the recipients was 9 months, and the median weight was 10.5 kg at the time of
transplantation. The experimental protocols and husbandry for all dogs
was approved by the FHCRC Institutional Animal Care and Use Committee
per guidelines stipulated by the National Academy of Sciences/National
Research Council.
Unrelated, DLA-nonidentical recipients and donors were selected on the
basis of mutual reactivity of their cells in mixed leukocyte culture,
serotypic differences for dog lymphocyte antigens (DLA-A and -B), and
phenotyping for DLA-D alleles using homozygous typing cells as
previously described.26-29 When possible, donors and
recipients were sex mismatched to differentiate between donor versus
host reconstitution of hematopoiesis. Marrow recipients were
conditioned for transplantation by 9.2 Gy of TBI at 7 cGy/min delivered
from two opposing 60Co sources.30,31
Immediately after irradiation, they received an intravenous infusion of
4 × 108 nucleated marrow cells per kg body weight.
No postgrafting immunosuppression was administered and, thus, no
attempt was made to prevent rapidly fatal acute GVHD. Postgrafting
immunosuppression was omitted because the drugs used for that purpose
have been shown in other studies to be also capable of suppressing
host-versus-graft reactions and, thereby, to enhance
engraftment.16-18 The day of TBI and marrow infusion was
designated as day 0. Supportive care was given after transplant as
described.32,33 Complete peripheral blood counts were
obtained before transplant and daily thereafter. Autopsies were
performed on all dogs at the end of the study to evaluate marrow
cellularity and the presence or absence of acute GVHD. Marrow
engraftment was defined as rising and sustained white blood count
(>500 per mm/m3) after the postirradiation nadir, marrow
cellularity at autopsy (
5% of normal) as estimated by light
microscopy of marrow sections, cytogenetics in some cases where the
donor/recipient were sex mismatched, and by clinical and histologic
evidence of GVHD in allogeneic recipients.
In vivo infusion of MoAbs.
The recipient dogs were treated with daily intravenous injections of
MoAb (0.2 mg/kg/day) for 6 days before TBI. Recipients of the
irrelevant control MoAb 6.4 received the MoAb on days -5 to 0. For the
recipients of MoAb S5, the initial five animals were administered MoAb
on days -5 to 0 and the subsequent 16 animals from day -7 to -2. Three
of the S5-treated animals were given 131I-labeled S5 (1 mCi/kg) for the first dose on day -7 followed by unlabeled MoAb S5 for
the subsequent five doses. The estimated radiation dose delivered via
the 131I administered was 100 cGy,34 which when
combined with 920 cGy TBI, raised the radiation dose to the marrow to
1,020 cGy. Given the previous observation that raising the TBI dose to
1,000 cGy failed to decrease graft rejection in this
model,35 we combined the results in these three dogs with
those in the 18 dogs not administered radiolabeled MoAb for the
purposes of analysis.
The animals that were administered MoAb IM7 and S3 received the MoAb on
the same dose and schedule as the majority of the MoAb S5-treated dogs
(0.2 mg/kg/d, day -7 to -2).
Flow cytometry.
To detect the presence of antibody on the surface of the peripheral
blood mononuclear cells (PBMC) from animals infused with MoAb, cells
were isolated from blood over a Ficoll-Hypaque density gradient (d = 1.074), washed, and incubated with an fluorescein isothiocyanate
(FITC)-conjugated goat antimouse antibody (Biochemica, Indianapolis, IN). To evaluate whether the CD44 expressed on the cell
surface of PBMC was saturated with MoAb from the in vivo infusion, a
one-step procedure was used where the cells were incubated with a
direct FITC conjugate of the same anti-CD44 MoAb that was infused into
the animal to detect unbound CD44.
Serum level of antibody.
A four-step ELISA procedure was used to detect the level of antibody in
the serum of MoAb-infused dogs. Briefly, the method consisted of the
following: (1) polyvinyl 96-well plates (VWR, Seattle, WA) were coated
with 50 µL of 10 µg/mL goat antimouse (or rat) IgG (Biosource
International, Camarillo, CA); (2) after blocking with 5% nonfat milk
in PBS, plates were incubated with 50 µL of sera from the infused
dogs; (3) goat antimouse (or rat) IgG HRP (Tago Inc) was added (1/2,000
dilution); and (4) 100 µL of ABTS color solution was added to each
well. Plates were read with a Vmax microtiter plate reader at 405 nm.
As controls, sera from the dogs before infusion were used, and standard
curves with known amounts of MoAb were established for each MoAb.
NK cell assay.
NK cell assays were performed as previously described.13 In
brief, PBMC from either S5-infused dogs or control dogs were used as
the effectors against canine thyroid adenocarcinoma cells (CTAC), which
is an NK-sensitive target. Triplicate (100 µL) aliquots of the
effector single cell suspensions were pipetted into round bottom
96-well plates with 100 µL of medium (for the spontaneous release) or
100 µL of 2% Triton-X (for maximum release). The target cells were
suspended in 500 µL of medium with 50 µL of Chromium-51 (5 mCi/mL),
and incubated for 1 hour at 37°C, 5% CO2, washed three times with 5 mL of cold medium, resuspended in desired concentration (5 × 104 to 105 cells/mL), and added (100 µL) to the plated effector cells. The plates were incubated at
37°C, 5% CO2 for 16 hours. After incubation, the
plates were spun, 100 µL supernatant obtained, and the released radioactivity measured by a gamma scintillation counter. Percent specific lysis was calculated by the formula:
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Iodination and avidity determination.
Iodination was performed in 20-mL glass scintillation vials coated with
100 µg Iodogen (Pierce Chemical, Rockford, IL).36 Antibody was diluted in PBS to a 1-mL volume in the Iodogen-coated vial, and radioiodine (125I-labeled Na; ICN Biomedicals,
Irvine, CA) was added. The vial was incubated at room temperature with
intermittent agitation for 10 minutes. Unbound iodine was removed by
passage over a Sephadex PD-10, G-25 column (Pharmacia Fine Chemicals,
Piscataway, NJ).
Avidity was determined from Scatchard plots of the binding of labeled
antibody to viable ML3 canine myelomonocytic leukemia cells.36 Known quantities of antibody were diluted in
tissue culture media (RPMI 1640 and 1% bovine serum albumin) and
incubated with 1 × 106 ML3 or Raji cells (human
leukemia cell line used as a negative control) in microfuge tubes in a
total volume of 1 mL for 1 hour at room temperature on a rotating
stand. The cells were washed three times and bound radioactivity was
counted.
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RESULTS |
Cloning canine CD44.
Approximately 1 × 106 recombinant clones from the
canine PBMC and spleen cDNA library were screened by hybridization with
a 32P-labeled baboon CD44 cDNA fragment. Three positive
clones were identified. The complete DNA sequences of these three
clones were determined. In one clone, only the last 2 bp before the
termination codon at the 3 end were missing, whereas another
clone started approximately 200 bp from the 5 end. A full-length
clone was constructed by excising the truncated 3 end and
inserting the correct fragment from the second clone to make a
full-length clone, including both the 5 and 3
untranslated regions. The amino acid sequences translated from
nucleotide sequences for dog, mouse, and human CD44 are shown in
Fig 1. The transmembrane regions as indicated by underlining, has the highest level of conservation between
the species. The overall homology between canine and human CD44 was
85% at the sequence level and 84% at the amino acid level and the
homology between canine and murine CD44 was similar. The membrane
proximal region extracellularly has the greatest divergence in the
three species. This confirms and completes the partial sequence of
canine CD44 that was previously published.37
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| Fig 1.
Comparison of the protein sequences of CD44. The protein
sequence determined from nucleotides of dog is shown on the top line and amino acid differences found in human and mouse (C3H) are presented
in the next two lines. Dashes ( ) are deletions and the putative
transmembrane segment is underlined. Also indicated are the locations
of the truncation variants used for peptide mapping with the 5
end of all the peptides initiating at * and terminating 3 as
indicated (F1-B, F1-C, F2, F3).
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Epitope mapping.
Available constructs of full-length and truncated mutants of the
extracellular domain of human CD44 were used to map the MoAb recognition sites. Wild-type human CD44 receptor globulins
(Rg)7 along with truncated human CD44 Rg (CD44H-Rg) fusion
proteins were used in ELISA to map for CD44 MoAbs S5, S3, IM7, MEM-85, and a negative control MoAb, 31A. MEM-85 was used as a positive control
for the truncation proteins, as it recognized all the variants, but did
not cross-react with canine CD44. CD44D-Rg contained the canine
extracellular domain and was recognized by the three anti-CD44 MoAbs
used in the in vivo transplant studies (S5, IM7, S3)
(Table 1). CD44H-Rg contained the entire
human extracellular domain of the hematopoietic form of CD44. It was
also recognized by all of the CD44 MoAbs tested. Constructs F1-B Rg and
F1-C Rg, which encoded an NH2 terminal 131 and 145 amino
acids, respectively, were bound only by MoAb MEM-85. F2 Rg and F3 Rg,
composed of the first 186 and 210 amino acids, were bound equivalently
by S5, S3, and IM7, indicating that they bound between amino acids 145 and 186 (Fig 1). This region included the NH2-distal domain
consisting of basic amino acids that were required for CD44/HA
interactions,24 although most of the HA binding domain was
located more proximally.
HA binding.
Because IM7 and S5 mapped to one of the HA binding domains of CD44, we
explored CD44/HA interactions in the presence of the MoAbs to evaluate
for possible functional differences. Zheng et al38 have
shown that IM7 could block CD44 binding to HA in some, but not all cell
lines. Using the CD44H-Rg fusion protein in an in vitro HA binding
assay, both S5 and IM7 augmented CD44/HA interactions, whereas MEM-85
blocked CD44 binding to HA (Fig 2).
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| Fig 2.
The abscissa represents increasing concentrations of
MoAb, and the ordinate represents binding of CD44H-Ig to HA in the
presence of antibodies. MoAb MEM-85 blocked the binding of CD44H to its ligand HA, whereas both S5 and IM7 augmented CD44/HA interactions.
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Marrow transplant.
To evaluate whether the previously observed graft enhancing effect of
MoAb S5 was representative of other anti-CD44 MoAbs, MoAbs IM7 and S3
were tested concurrently with MoAb S5 in the DLA-nonidentical unrelated
marrow transplant model. Results are shown in
Tables 2 and 3.
Of 57 historical and concurrent control dogs administered 9.2 Gy TBI
and not treated with any MoAb, 47 rejected marrow grafts from
DLA-nonidentical unrelated donors. Only 10 dogs (17%) engrafted and,
because no postgrafting immunosuppression was administered, all 10 died
with complications of hyperacute GVHD. Seven dogs were treated with
intravenous injection of an irrelevant MoAb 6.4 for 6 days before
TBI.5 Of the seven, six failed to show sustained
hematopoietic engraftment and died of infectious complications. One dog
engrafted and died of GVHD. This result was not statistically different
from the results in control animals not given MoAb (P = 1.0).
Fourteen of 21 dogs treated with S5 engrafted, as determined by
increases in peripheral blood counts and by marrow cellularity at
autopsy. All of the animals that engrafted developed clinical GVHD,
which was confirmed histologically. Of the five animals that were
treated with MoAb IM7 (Table 2), only one showed sustained
hematopoietic engraftment, whereas the remaining four failed to
engraft. Twelve dogs were treated with MoAb S3 (Table 2), three
engrafted and nine had graft failure. In cases where cytogenetic
studies were performed, clinical assessment of engraftment or rejection
was confirmed by karyotype analysis of marrow or peripheral blood cells
(Table 3). Only MoAb S5 significantly enhanced engraftment of
DLA-nonidentical unrelated marrow grafts as compared with controls
(P < .001) (Table 4). The two
other anti-CD44 MoAbs (IM7 and S3) had no significant effect on
engraftment, and results were not significantly different from controls
(P = 1.0 and .69, respectively).
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Table 2.
Data in Dogs Administered 9.2 Gy TBI and Infusion of
DLA-Nonidentical Unrelated Bone Marrow Grafts After Treatment of Marrow Recipients With Intravenous Injection of MoAbs S5, IM7, or S3
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Table 3.
Cytogenetic Results in Female Dogs Administered 920 cGy
TBI, Pretreatment With MoAb, and Marrow Grafts From DLA-Nonidentical, Unrelated Male Donors
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Table 4.
Results in Dogs Administered 9.2 Gy TBI and
DLA-Nonidentical Marrow Grafts From Unrelated Donors With or
Without MoAbs Before Transplant
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Serum level of MoAb.
Serum trough levels were determined for seven dogs administered MoAb
S5, five dogs administered MoAb S3, and five dogs given IM7.
Figure 3 shows the mean MoAb serum levels,
which peaked on day -1 for MoAbs S5 and S3 and day -2 for MoAb IM7.
Overall, serum levels between S5 and S3 were not significantly
different from each other because the curves were overlapping, although
MoAb IM7 serum levels were lower than those of MoAbs S5 and S3
throughout the period of injections. In this regard, the single
IM7-treated dog that engrafted (D109) had a very low serum MoAb level
at the time of marrow infusion, indirectly implying that failure to
engraft in the four other MoAb IM7-treated dogs was not due to
insufficient MoAb serum levels. PBMC obtained from the S5-treated dogs
on day 0 had detectable antibody on their cell surfaces, as measured by
goat antimouse FITC, although not at saturating levels, as additional
S5-FITC was able to bind to the cells, increasing the fluorescence
intensity as detected by flow cytometry
(Fig 4). Similar results were observed for
the IM7 and S3-treated dogs.
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| Fig 3.
The mean serum trough MoAb levels (± standard
error) for seven dogs that received S5, all five dogs that received
IM7, and five dogs that received S3. All animals received MoAb from day -7 through day -2 where day -7 represents the baseline, day -2 is the
last day of MoAb administration, and day 0 is the day of transplant.
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| Fig 4.
The abscissa represents the mean fluorescence intensity
of the cells analyzed from the animals on day -7 (before MoAb
administration), and on day 0 (day of transplant) of an animal that
received MoAb S5. Cells were stained with either goat antimouse FITC
(to detect surface MoAb) or S5-FITC, which is the direct FITC conjugate
of MoAb S5. Cells removed from the animal on day 0 had surface MoAb, as
shown by staining with goat antimouse FITC, although not saturation levels, as additional MoAb S5 binds to cells (S5-FITC).
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Effect of S5 on NK activity of PBMC retrieved from dogs after in vivo
injection of MoAb.
PBMC were obtained from the animals on day -7 (before any MoAb
treatment) during various timepoints of the MoAb infusion, and on day 0 just before and after TBI. There were mild increases in NK activity
among cells from dogs treated with MoAb S5, consistent with previous in
vitro observations of increase of NK activity by S513 (data
not shown). More remarkable was the NK activity in cells obtained on
day 0 just before and just after TBI in two S5-treated dogs studied.
Cells from both dogs showed, on average, 72% reduction in NK activity
after TBI (Table 5). In four controls that
received TBI but no antibody, the reduction in specific lysis was less pronounced on average (32%) (P = .06 by two sample Wilcoxon's rank-sum test).
Cell binding.
Scatchard analyses of S5, IM7, and S3 binding to ML3 cells are shown in
Fig 5. Based on the molecular weight of the
MoAbs and the number of cells in the incubation, for S5 the association constant (Ka) was 1.5 × 109 (mol-1) and
the number of molecules bound per cell was 8.7 × 105;
for IM7, the association constant was 2.83 × 109
(mol-1) and the number of molecules bound was 9.79 × 105; and for S3, the association constant was 3.86 × 107 (mol-1) and the number of molecules bound
was 2.35 × 106.
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DISCUSSION |
The current study extended previous observations5 and
confirmed that treatment of recipients of DLA-nonidentical marrow grafts with an anti-CD44 MoAb, S5, before TBI significantly decreased the incidence of graft rejection. In an attempt to better understand how MoAb S5 accomplished its in vivo effects in the canine graft rejection model and to ultimately develop an analogous antihuman CD44
MoAb, we cloned the canine CD44 gene, mapped MoAb S5, and tested other
anti-CD44 MoAbs in the in vivo model to judge whether this was a
phenomenon applicable to all CD44 MoAbs.
The high homology between canine and human CD44 at both the sequence
and amino acid levels implied conservation of ligand binding regions
and function. This also allowed us to use the human truncation
constructs of CD44 for protein mapping. Although the initial mapping of
S5 determined that S5 recognized a region highly conserved between
human and canine CD44, both IM7 and S3 also mapped to the same general
region of CD44. This region included the NH2-distal domain
comprised of basic amino acids that were required for CD44/HA
interactions, although most of the HA binding domain was located more
proximally.
We previously showed in competitive binding assays that S5 and IM7
blocked each other's binding to CD44,6 whereas S3 (our unpublished observations, June 1989) did not. This suggested that S5
and IM7 saw overlapping epitopes. The observation that S5 recognized only canine CD44 and cross-reacts only with lower affinity with human
CD44 (and not rodent or nonhuman primate CD44), whereas IM7 recognizes
CD44 on mouse, dog, nonhuman primates, and humans indicated that the
epitope recognized by the two MoAbs was not identical.
In an attempt to further localize the epitope bound by S5, peptides (15 amino acids in length) based on the canine sequence were constructed
spanning amino acids 130-184. Using ELISA, multiple anti-CD44 MoAbs,
including S5 and IM7, were tested on plates coated with the peptides,
and all were negative (data not shown). This suggested that the epitope
recognized by S5 might be conformational and/or involve
noncontiguous regions of the glycoprotein.
In addition to extending the in vivo transplant results with S5, we
examined other anti-CD44 MoAbs in vivo to see if the graft enhancement
effect of S5 was a general phenomenon. We showed that only MoAb S5
facilitated engraftment (P < .0001 compared with control),
whereas two other anti-CD44 MoAbs (IM7 and S3) were ineffective. This
suggested that the specific epitope bound by S5 was relevant for the
graft enhancement effect.
Because IM7 and S5 mapped to one of the HA binding domains of CD44, we
explored CD44/HA interactions in the presence of the MoAbs to evaluate
for possible functional differences. Zhang et al38 have
shown that IM7 could block CD44 binding to HA in some, but not all cell
lines. Using the CD44H-Rg fusion protein in an in vitro HA binding
assay, both S5 and IM7 augmented CD44/HA interactions, whereas MEM-85
blocked CD44 binding to HA. This suggested that MoAb S5 and IM7 were
providing a signal to cause a conformational change in the CD44
molecule, which allowed it to bind to a ligand, similar to what has
been reported with T cells.39 These data confirmed that
both S5 and IM7 mapped to a region that was distinct from the HA
binding region. S5 and IM7 did not block HA binding, but caused the
conformational change in CD44, which allowed HA binding as previously
shown by one other antimurine CD44 MoAb.38 This finding
could potentially play a role in the ability of S5 to enhance
engraftment by improving interaction of hematopoietic cells with the
extracellular matrix of the microenvironment, although it is not
sufficient to explain the in vivo effect of engraftment because IM7,
which also augmented HA/CD44 interactions, failed to affect engraftment
of mismatched marrow.
Both determination of serum MoAb levels and saturation studies
performed on cells from animals receiving MoAb were similar between the
three different anti-CD44 MoAbs, which indicated that the differences
seen between S5 and the other two anti-CD44 MoAbs (IM7 and S3) were not
due to variation in pharmacokinetics of the MoAbs.
We have previously shown that S5-treated PBMC had a higher baseline of
NK activity.13 In addition, the S5-treated PBMC were found
to be more sensitive to radiation kill, as shown by the steeper decline
in specific lysis with increasing radiation doses.14 To
evaluate whether this observation was relevant in the in vivo model,
PBMC from a dog that was infused with MoAb S5 were evaluated before and
after 9.2 Gy TBI. The reduction in NK activity after TBI, as compared
with control, was consistent with the in vitro findings of increased
radiation sensitivity of S5-treated NK cells, suggesting that S5
facilitates engraftment by increasing the radiation sensitivity of
recipient NK cells that may be responsible for marrow graft rejection.
To evaluate whether the in vivo differences observed with the three
anti-CD44 MoAbs were the result of lower affinity, lower avidity, or
overall diminished immunoreactivity, binding assays were performed with
the three MoAbs. Although MoAb S3 had a significantly lower avidity to
CD44, S5 and IM7 were indistinguishable, with IM7 having a slightly
higher avidity than S5. These data suggest that the enhanced in vivo
effect on engraftment of S5 is not on the basis of higher avidity, but
rather is due to the epitope bound by S5.
In the canine system, we have determined that pretreatment of PBMC with
MoAb S5 enhances NK activity.13-15 Both in vivo and in
vitro data indicate that S5-treated NK cells are more sensitive to
radiation than NK cells not treated by MoAb, indicating that S5 may
facilitate marrow engraftment by increasing radiation-induced death of
host cells capable of marrow graft rejection.13 We have
also shown previously that S5 augments in vitro hematopoiesis in
long-term marrow cultures (LTMC)40 and increased CD34 cell production (unpublished data, December 1995). Other anti-CD44 MoAbs
tested did not have this effect. If this occurs in vivo, it is
reasonable to postulate that pretreating transplant recipients with S5
could enhance engraftment in two ways, reducing host resistance and
enhancing donor hematopoiesis via effects on host marrow stroma. Whether both mechanisms are required to facilitate engraftment is not
yet known. There was no evidence in either the autologous setting
(unpublished, February 1986) or in the DLA-identical
setting41 that S5 augmented hematopoiesis. In all cases,
the recoveries of neutrophils and platelets were normal, although the
neutrophil recovery may not be sensitive enough to test the hypothesis
that S5 can have a direct hematopoietic effect. Further studies are directed at developing antihuman CD44 MoAbs that are equivalent to MoAb
S5 to prevent graft rejection in patients. To ensure that the epitope
that S5 recognizes is conserved, we will convert the mouse anticanine
CD44 MoAb S5 into a human antihuman CD44 MoAb using phage display
technology and "chain shuffling." This is a proven method of
converting a low-affinity mouse antibody into a high-affinity human
antibody with identical epitope specificity.42,43
Although the epitope mapping data does not discriminate between the
three MoAbs that were used in vivo, it does provide the information
that the functional MoAb, S5, binds to an area of CD44 highly conserved
between the different species and does not involve the HA binding
domain. The data supports the hypothesis that an antibody that will
recognize the same epitope on human CD44 as S5 will have the same
functional effect in human HLA-haploidentical marrow graft recipients.
| |
FOOTNOTES |
Submitted August 18, 1997;
accepted December 15, 1997.
Supported in part by Grants No. CA01483, CA18221, CA31787, HL36444, and
DK42716 from the National Institutes of Health, Department of Health
and Human Services, Bethesda, MD. Laboratory support was also available
through a prize from the Josef Steiner Krebsstiftung, Bern, Switzerland
(awarded to R.S.).
Address reprint requests to Brenda M. Sandmaier, MD, Fred Hutchinson
Cancer Research Center, 1100 Fairview Ave N, D1-100, PO Box 19024, Seattle, WA 98109.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We would like to acknowledge Dr Dana Matthews for assistance in
analysis of binding assays, Dr Ted Gooley for assistance in statistical
analyses, Dr Eileen Bryant and Robert Raff for cytogenetic studies,
Laura Bolles and Jennifer Smith for technical assistance, and Harriet
Childs and Bonnie Larson for their help in manuscript preparation.
| |
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Abstract
Introduction
Abstract
