Note: Descriptions are shown in the official language in which they were submitted.
-
- 1 337756
MEANS AND METHOD FOR PREVENTING AND TREATING
GRAFT FAILURE IN HUMANS
The invention relates to new means and to a
method for preventing and treating graft failure in
humans.
It more particularly relates to means and a
method for preventing and treating organ graft failure,
especially in human leucocyte antigen (or HLA)- mismat-
ched bone marrow transplantation.
Bone marrow transplantation is limited by the
occurence of graft versus host disease and graft
rejection. T cell purging of donor's bone marrow
prevents efficiently graft versus host disease in HLA
matched and mismatched bone marrow transplantation but
leads to a high incidence of graft rejection. This
- method ablates a delicate balance between donor T cells
and residual host immunity. Severe combined immuno-
deficiency is the only disease that can be so far cured
by T cell depleted HLA- mismatched bone marrow transpla-
ntation because of patients' inherent inability to
reject grafts.
Therefore, T cell depleted HLA- mismatched bone
marrow transplantation needs additional procedure for
the prevention of graft failure.
Several approaches have been proposed to prevent
graft rejection such as total lymphoid irradiation and
increasing the dose of total body irradiation or of
chemotherapy.
- Such therapies do however lead to incrèased
toxicity and might not be therefore feasible, specially
in young patients.
Following another approach, the Inventors have
studied the destruction and the blocking in vivo of the
1 337756
_ 2
function of leucocytes by monoclonal antibodies.
They have then found that anti HLA-1 monoclonal
antibody can safely be used to prevent and treat graft
rejection in HLA-mismatched bone marrow transplantation.
This discovery is essentially based on the fact
that it was shown by the Inventors that patients with
congenital LFA-1 deficiency are the only known group of
patients who accept HLA- mismatched haploidentical bone
marrow from their parents, in absence of a major cellu-
lar immunodeficiency.
It is then an object of the invention to provide
an anti LFA-1 monoclonal antibody useful in vivo for
preventing and treating graft failure without side-
effects.
It is another object to provide a new method for
preventing and treating organ graft failure, particular-
ly in HLA- mismatched bone marrow transplantation.
The anti LFA-1 monoclonal antibody or anti-LFA-1
mab infused according to the invention to patients for
preventing or treating graft failure has the following
in vitro properties :
. it does not fix the complement,
. it blocks the mixed lymphocyte culture (MLR),
and partially the PHA-induced proliferation,
. it blocks T cellular-mediated cytolysis with
respect to the population of CD8+ anti-class I clones,
CD4~ anti-class II clones and NR cytolysis assayed on
K562,
. it alters the adhesivity on glass support of
polynuclears and the phagocytose of monocytes,
. it inhibits the fixation of the C3bi component
of the complement on its CR3 receptor,
. it does not react with the leucocyte membrane
of children with a congenital immunodeficiency in
proteins of the LFA-1-C~ receptor and Gp150-90 complex,
1 337756
. it inhibits antigen-specific T cell helper
activity for antibody production,
. it inhibits partially antigen-induced T cell
proliferation.
According to another aspect, the anti-LFA-1 mab
used according to the invention has the following cha-
racteristics :
- it belongs to IgG1 class and is directed
against the a subunit of LFA-1,
- it immunoprecipitates a dimeric glycoprotein
consisting of two chains a and ~, having a molecular
weight of 180 and 90 Kd respectively,
- it reacts with the a chain of the membrane
glycoprotein,
- its tissular distribution is as follows :
15. it reacts with 60 % of the peripheric
blood cells,
. it reacts with T and B lymphocytes,
monocytes, macrophages and polymorphonuclears
(neutrophils and eosinophils),
20. it reacts with the following T cell
lines : MOLT 4, HPB-ALL, CEM,
. it gives a negative reaction with RPMI
8402 line, and 1301,
; it reacts with 60 ~ of the thymocytes.
25According to still another aspect, the anti-
LFA-1 mab used according to the invention is obtainable
by immunizing Balb/c mice with the Tm20 human cytotoxic
T cell clone, bearing the following phenotype : T3+,
T4+, T8- and of HLA-DRw6 specificity, followed by f-
usion with a murine myeloma.
According to a preferred embodiment of the
invention, the mab is a murine anti-LFA-1 mab selected
from an hybridoma resulting from the fusion of myeloma
- 63-Ag 8-653 with Balb/c spleen cells.
-- 1 337756
Taking into account the therapeutical applica-
tion of the mab produced, it is particularly preferred
to use a subclone having specific qualities, i.e.
particularly devoid of mycoplasms, and of murine virus.
A preferred subclone is constituted by 25.3.2
subclone and is obtained from the hybridoma strain
25.3.2, deposited under n- I-635 on December 3, 1986 at
the Collection Nationale de Culture de Microorganismes
(C.N.C.M.).
Advantageously said anti-LFA-1 mab has been
proven to be safe in humans and to allow organ engraf-
tment of donor cells as compared to control groups.
Said mab is then useful in vivo as active
principle in pharmaceutical compositions.
The pharmaceutical compositions of the invention
comprise said mab as purified from ascitic fluids by
affinity on protein A and are sterile and devoid of
pyrogenic substances, murine virus and mycoplasms.
The purification step on protein A is performed
with an ascite liquid containing said mabs, devoid of
fibrin and lipids. Advantageously the ascite liquid is
equilibrated at a basic pH. Preferably the pH is
adjusted to above 8.5. The ascite liquid is contacted
with protein A, in an affinity column, protein A being
fixed to a support, for example polysaccharides such as
those marketed under the trademarks ~Sepharose" and
"Ultrogel". The mab preparation are recovered by using a
buffer having a lower ionic strength than the one used
for equilibrating the column.
Preferably, the purification of the mab further
comprises an elimination step of the nucleic acids by
dialysis against a buffer as PBS ~phosphate buffer
saline).
The invention relates in particular to solutions
of purified mab in a buffer such as PBS, containing 0,5
-- 1 337756
to 3mg of mab per ml of buffer, preferably about 1
mg/ml.
The concentrated solutions can be diluted by any
usual solute.
The solutions can be constituted from freeze
dried powder of mab.
The invention also relates to a method for
preventing and treating graft failure which comprises
using in vivo said purified mab, advantageously to
complement a conditioning regimen. The treatment will be
made from about day -15 to day +15.
From the clinical results, it appears recomman-
dable to infuse to the patient every day, from days -3
to day +6, about 0.1 to 5 mg of mab/kg, preferably about
0.2 mg/kg.
Conditioning regimen usually include busulfan,
cyclophosphamide and antilymphocyte globulin.
They will however vary depending of the thera-
peutical indications. For example, in the following
indications, the regimen will include :
. hereditary diseases : busulfan and cyclophosphamide
. leukemia : total body irradiation, total lymphoid
irradiation, cyclophosphamide and possibly VP16,
aracytive, CCNU and nelphalan.
. SCID : no treatment
To prevent significant graft versus host disea-
se, it is advantageous to deplete T cells and to give an
- appropriate therapy.
It is recommended to have a quality of T cell
depletion above 2 log. Said quality can be checked by
the T cell proliferation method in the presence of Il2
and cytofluometry analysis controls in the presence of a
standard amount of leads as indicator.
- 1 3377 56
-
Various methods are available in that respect. A
satisfactory depletion of bone marrow T cells is obtai-
ned with sheep erythrocyte rosetting ex vivo.
Significant graft versus host disease is preven-
ted by further giving a therapy such as cyclosporin-A
during several weeks for example, 8 weeks.
Said method has been successful for obtaining
stable and functional engraftment, even in patients at
high risk for graft rejection.
More particularly said method enables engraf-
tment in HLA- mismatched bone marrow transplantation in
patients having a normal or subnormal immunity : acute
or chronic leukemia, lymphomas, solid tumors, medullar
aplasia, and bone marrow hereditary diseases, including
immunodeficiencies and various hereditary disorders.
Rapid hematological recovery has been observed.
The granulocytes reached a level higher than
500/mm in 12,7 + 2 days.
The last platelet infusions were required at 24
+ 1~ days.
The regenerating leucocytes were of donor origin
in all cases and in some cases chimera were observed.
Side effect was limited to fever.
The results obtained suggest that said treatment
may lead to long term maintenance of donor bone marrow
cells.
Said method is also useful for preventing and
treating graft rejection in organ transplantation, par-
ticularly in kidney transplantation.
The invention will be more fully illustrated
with reference to the following examples.
The immunological analysis were carried out as
follows : Lymphocyte populations were determined by
immunofluorescence using specific monoclonal antibodies
to CD3 ~T3), CD4 (T4), CD8 (T8), and CD2 (T11).
1 337756
B lymphocyte populations were enumerated using anti-
antisera to Ig heavy and light chains (1). Mitogen and
antigen (Candida albicans, tetanus toxoid, influenza
virus), allogenic cell-induced lymphocyte proliferations
were performed as previously described (1). Serum immu-
noglobulin levels and specific antibody titers to polio-
virus, tetanus and diphteria toxoids, bordetella
pertusis, influenza virus were measured with usual
serological techniques. Chimerism was assessed by
karyotyping and quinacrine staining of the Y chromosome
in sex mismatched and by HLFA typing in all cases. Ig
allotype studies were performed using indirect hemaglu-
tination with specific antisera.
~XA~PLE 1 : MONOCLONAL ANTIBODY USED IN VIVO FOR PRE-
VENTING AND TREATING GRAFT FAILURE -
Obtention of the ascites
Said mab is obtained by injecting 5x106C of the
hybridoma strain (subclone 25.3.2) which results from
the fusion of myeloma, 63-Ag 8-653 with spleen cells
8alb/c, to mice (8alb/c pretreated with pristan).
Ten days following the injection, the ascite
liquid is recovered, the cells are eliminated by cen-
trifugation (4000 g during 10 min.), the supernatant is
recovered and freezed.
Purification
All the steps are conducted under sterile
conditions. The buffers which are used are prepared with
apyrogen, distillated water, and are filtrated and
sterilized in an autoclave.
The ascite liquid is defreezed at 4- C, the
fibrin is eliminated by centrifugation at 4000 g during
10 min.
The cleared liquid is filtrated on Millipore
AP25 to eliminate traces of lipids. The ascite liquid is
then equilibrated to pH 8.5 with a phosphate buffer
~-- 1 337756
(1M pH 8.5) by adding 1 volume of said buffer to 9
volumes of ascite.
The purification is performed on protein A
(protein A Sepharose CL4B R (Pharmacia) or protein A
Ultrogel (IBF). The column is equilibrated with a
5 phosphate buffer (0.1.M pH 8.5) by washing the gel with
said buffer (two times the volume of the column).
The volume of ascite loaded on the column must
exactly correspond to the dead volume of the column.
After the loading step, the ascite is allowed to
incubate in the column during 45 min. The column is then
rinsed with a phosphate buffer (0.1.M., pH 8.5) up to
total absence of protein in the effluent. The IgG1
25.3.2 are taken down with a citrate buffer (0.1.M,
pH 6) and the fractions rich in proteins are recovered.
The column is then regenerated with 0,58% acetic
acid 0.15M NaCl and equilibrated with said buffer
phosphate.
Biochemical and biological activity control, are
carried on. The IgG1 thus purified are analyzed with
acrylamid gel SDS using dissociating conditions. After
staining, two strips are observed at 50 and 25 kd res-
pectively, corresponding to the heavy and the light
chains of the IgG1.
The preparation is also analyzed by FPLC using a
mono G column. Only one optical density peak correspon-
ding to IgG1 must be observed.
The IgG1 biological activity is checked by
fluorimetry on peripheral blood lymphocytes. The cells
are successively incubated with purified antibodies at
concentration of 20, 10, 5 ~g/ml. The fixation of the
antibodies to the cells is revealed by a fluorescent
probe (goat antimouse FITC). The results are analyzed
with a cytofluorograph.
The material used is a sterile, apyrogenic
preparation of mab 25.3-2l purified as disclosed above,
1 337756
diluted in PBS (flask with 0,5 mg in 0,5 ml of P~S).
~X~MpLE 2 : CLINICAL EXPERIMENTS
a) study group :
Seven patients were transplanted between June
1985 and May 1986 (age : between 2 months and 2 years
and a half). The original diseases were Wiskott Aldricht
syndrome (WAS ; n = 3), combined immunodeficiency (CID ;
n = 2) and osteopetrosis (n = 2). Patients with WAS
required bone marrow transplantation because of the
severity of the clinical course. One patient had sple-
- 10 nectomy, steroids and azathioprine for autoimmune
pancytopenia. The second patient with WAS had severe
thrombocytopenia with <10,000/mm3 platelets which could
not be corrected by splenectomy . The third patient with
WAS had vasculitis. The first patient with CID had
15 multivisceral vasculitis that required the use of
steroids and i.v. infusion of cyclosphosphamide.
The patients' age and degree of HLFA incompati-
bility with the donors are given in table 1 hereinafter
All patients in the study group showed signi-
ficant MLR reactivity against their parent's leucocytes.One patient (n-2) showed HLA-A and HLA-B identity but
exhibited the highest degree of MLR reactivity.
b) control group
Seven patients, consecutively transplanted
between September 1984 and May 1985, were recorded as
historical controls (age : between 3 months and 3 years
and a half). These patients were also transplanted for
WAS (n = 1), CID due to defective synthesis of HLA class
II molecules (n = 2), CID of unknown origin (n = 1) ;
30 Chediak-Higashi syndrome (CHS ; n = 2) and osteopetrosis
(n = 1). Bone marrow transplantation was considered for
the severity of their disease. The patient with WAS has
a protacted diarrhea requiring parenteral nutrition for
months. Patients with CHS were in the acute phase of the
-- 1 3 3 7 7 5 6
disease which was controlled only by chemotherapy
consisting of VP16. Patients wiht CID and osteopetrosis
were in good condition, but these diseases have poor
prognosls .
The pretransplant observations are reported in
table 1 hereinafter.
TABLE I :
PATIENTS
.CONTROL GROUP STUDY GROUP
HLA incompatibility with donor HI.A incompatibi3ity with donor
1 HLA class II (-) CIDA-B-DR 1 WAS A, B, DR
2 WAS A-B-DR 2 Osteopetrosis DR
3 CID A-B-DR 3 WAS - A, DR
4 CHS A-B-DR 4 Osteopetrosis A, B, DR
5 HLA class II (-) CIDA-B-DR 5 CID A, B, DR
6 CHS DR 6 CID B, DR
7 Osteopetrosis DR 7 WAS A, B, DR
CID: Combined ImmunoDeficiency - CHS: Chediak Higashi Syndrome
WAS: t'iskott-Aldrich Syndrome
-- 1 337756
Patients were isolated in a sterile
isolator (La Calhene, Paris, France) and received
absorbable antibiotics and ketoconazole daily and
immunoglobulins (CTS France) weekly for 2 months.
Patients in both study and control groups
according to the protocol used for HLA-matched bone
marrow transplantation, were given :
- busulfan 4 mg/kg daily from day -9 to -6;
- cyclophosphamide 50 mg/kg from day -5 to -2,
and
- antilymphocyte globulins (Merieux, Lyon) 2,5
mg/kg, on days -10, -8, -6, -4,
- cyclosporine 60 days.
In addition, patients from the study group
received 0.1 mg/kg of said monoclonal antibody at days
15 -3, -1, +1, +3, +5.
1 ml of serum is taken at day -3 (before injec-
tion of the mab), then every day up to day + 10 for
titration.
The serum is frozen at -20-C. The titration is
20 carried out according to ELISA method using a rabbit
mice anti-chain K antibody (Immunotech).
The graft take is evaluated by markers.
Bone marrow T cell depletion was performed in
all cases by sheep erythrocyte (E) rosetting method as
previously described (1). T cell depletion of donor's
bone marrow resulted in a mean infusion of 1.46 x 108
cells/kg (range .54 - 2,3 x 108) containing 5.5 x 105/kg
T3 + lymphocytes (range 2.7 - 10 x 105) in the seven
patients of the study group treated with anti-HLFA-1
antibody.
These data did not differ from those of the
control group (mean of total cells 1.3 x 108/kg - range
.4 - 2.7 x 108, mean of T3 + lymphocytes 6 x 105/kg -
range 1.6 - 13 x 105).
-- 1 337756
12
~X~MpLE 3 : RESULTS
a) Study group
- Side effects of antiHLFA-1 mab infusion.
The administration of anti-HLFA-1 antibody
resulted occasionally in fever up to 40 C. Fever was
transient. There has been no other side effects.
- Hematoloqical recoverY.
Granulocytes reached 500/~l at a mean of 12.7
day (range 10-15d). The last platelet transfusion was
performed between days 10 and 40 (mean 24 days). There
has been no secondary blood count abnormalities except
in patient n- 2 in whom a steroid sensitive auto immune
hemolytic anemia occured at day 120.
- ~nqraftment.
As depicted in table 2 hereinafter, engraftment
of donor cells has been proven in all patients of the
study group. HLA typing indicated the presence of donor
cells in the patients. A mixed chimerism has been
observed in two patients either by HLA typing or
karyotyping. In both cases the mixed chimerism appears
stable, after one year and 6 months respectively.
The evidence for the engraftment of donor bone
marrow in patients receiving mab-infusion is given in
table 2 hereinafter.
- Correction of underlYinq disease.
After 60-395 d (mean 225 d), the patients did
exhibit the disappearance of clinical and biological
manifestations of their underlying disease and are doing
well. The specific antigen induced T and B cell
responses that were deficient in two patients (n- 5 and
6) prior to transplant have been shown to develop
following immunizations within 2 to 4 months post
transplant.
-- 1 337756 - 13 -
b) Control group
No donor cells engraftment could be documented in
6 out of 7 patients. On one, a partial lymphocyte
engraftment was proven by karyotyping and HLA typing.
This patient eventually died from a lymphoma while the
proportion of donor lymphocytes was less than 20%. Four
patients died from infections in the absence of
hematological reconstitution. Finally, in two a complete
or partial autologous hematological reconstitution
occurred. These patients are still alive, in poor
condition due to their primary disease progression.
Patients HLAKaryotyping Other clinical
typing evidence of
engraftment ~
l Donor Donor Correction of I.D.
2 Mixed - Bone clearing
lS 3 Donor - Increase in platelet
size
4 Donor - Rising of urine
calcium excretion
Donor Mixed (2/3 Correction of I.D.
donor)
6 Donor Donor Correction of I.D.
7 Donor - Correction of
thrombocytopenia
- = not informative
= Ig allotypes are of donor origin
-_ 1 3 3 7 7 5 6
- 13a -
& = The following abnormalities have been corrected
following BMT:
Patient 1 WAS: Thrombocytopenia with small platelet size,
T lymphocytopenia, Auto antibodies to red cells, PMN and
platelets, Low serum IgM, Low antigen-induced lymphocyte
proliferation.
Patient 2 Osteopetrosis: Anemia, Thrombocytopenia,
Presence of immature myeloid cells, Low levels of calcium
in serum and urine, Hepato and splenomegaly, Abnormal
bone density.
Patient 5 CID: Absence of antigen-induced lymphocyte
proliferation and skin tests, Absence of antibody
production, Eosinophilia, Protracted diarrhea.
Patient 6 CID: Absence of antigen-induced lymphocyte
proliferation and skin tests, Absence of antibody
response to vaccinal and infectious antigens, Vasculitis,
Diarrhea (Cryptosporidia).
Patient 7 WAS: Thrombocytopenia.
~ 1 3 3 7 7 5 6
14
Said results demonstrate the safety of using
anti-HLFA-1 in vivo.
First, it will be observed that in the patients
of the study group hematological recovery was fast, or
even faster, than seen in patients receiving fully
matched bone marrow without anti-HLFA-1 preconditioning.
The second main observation of the study is the
contrasting finding seen in two groups of patients. Both
groups have been given busulfan, cyclophosphamide, ALG
and cyclosporin therapy and the only major difference
between the groups was the infusion of anti-HLFA-1
In the anti-HLFA-1 group rapid bone marrow take
was seen in patients. This group includes, to the
knowledge of the inventors, the first children succes-
sfully receiving haplotype- or HLA-DR mismatched bone
marrow transplantation for treatment of Wiskott-Aldrich
syndrome and osteopetrosis. These good results are not
attributable to a low degree of HLA incompatibility,
since the MLR between all recipients ant their donors
was strong, and since 6/7 donor-recipient pairs differed
for at least 2 HLA antigens. Clinical and biological
manifestations of their previous diseases have disap-
peared in all patients.
By contrast, in the conventionally treated group
of bone marrow transplantation recipients no lasting
dominant bone marrow take was observed and only 2
patients survive without the correction of their
underlying immunodeficiency following their autologous
reconstitution.
In conclusion, these results indicate that the
blocking of the functional HLFA-1 receptor has an
important role in bone marrow transplantation.
