DESCRIPTION
IMMUNOLOGICAL RECONSTITUTION PROMOTER OR PROPHYLACTIC AGENT
FOR INFECTIONS EACH OF WHICH MAINTAINS GRAFT-VERSUS-TUMOR
EFFECT
TECHNICAL FIELD
The present invention relates to an immunological
reconstitution promoter or a prophylactic agent for infections
in allogeneic hematopoietic stem cell transplantation therapy
for tumors.
BACKGROUND ART
Hematopoietic stem cell transplantation is a mode of
treatment which, after a malignant tumor has been destroyed by
a pre-transplant regimen involving a combination of
chemotherapy and radiotherapy, builds a new hematopoietic
system by the transfusion of donor-derived or the patient's
own hematopoietic stem cells. Of these, allogeneic
hematopoietic stem cell transplantation involving the
transplantation of donor-derived hematopoietic stem cells can
be expected to have an anti-tumor effect, i.e., a graft-
versus-tumor effect (also referred to below as the "GVT
effect"), on various tumors of the hematopoietic system and
solid tumors against which other therapeutic methods are
likely to be ineffective. However, at the same time, there is

a possibility that such therapy may be accompanied by graft-
versus-host disease (also referred to below as "GVHD") and by
infections attributable to delayed immune reconstitution
following transplantation. Such concerns have limited the
expansion in the use of this approach as a cancer
immunotherapy (Non-Patent Documents 1 and 2).
GVHD is a syndrome characterized by skin rash, jaundice
and diarrhea, and is understood to arise from the infiltration
of activated donor T-cells into, for example, the skin, liver
and intestinal tract. With the appearance of
immunosuppressants, which were rapidly developed starting in
the late 1980s, the prevention of and treatment outcomes for
GVHD improved significantly. At the same time, as a result of
the decreased mortality from GVHD, lethal infections
associated with immune deficiency following allogeneic
hematopoietic stem cell transplantation emerged as a major
factor affecting the prognosis of such transplantation.
However, the pathogenic mechanism and effective treatments for
delayed immune reconstitution following such transplantation
have yet to be established. The situation is such that no
alternative currently exists but to rely on symptomatic
treatment involving the administration of immunoglobulin
preparations and antibiotics.
The present invention sets out to employ a substance
capable of depleting CD4 positive (also referred to below as

CD4+) cells (which substance is also referred to below as a
"CD4+ cell-depleting substance") so as to promote immunological
reconstitution or prevent infection following allogeneic
hematopoietic stem cell transplantation. Such substances have
not yet been reported in the literature.
Non-Patent Document 1: Shlomchik, W.D., Nature Reviews
Immunology, 7(5), 340-352 (2007).
Non-Patent Document 2: Abrahamsen, I.W., and other 5
researchers., Haematologica, 90(1), 86-93 (2005).
DISCLOSURE OF THE INVENTION
The object of this invention is to provide an
immunological reconstitution promoter or a prophylactic agent
for infections in allogeneic hematopoietic stem cell
transplantation therapy for tumors.
The inventors, noting that GVHD severity and delayed
immune reconstitution exhibit a strong correlation in the
clinical course and also that myelosuppression (cytopenia)
manifests at the time of GVHD onset, have conducted extensive
and repeated investigations. As a result, they have
discovered that:
(1) diffuse bleeding, structural breakdown and hematopoietic
disorders which arise in bone marrow tissue following
allogeneic hematopoietic stem cell transplantation (sometimes
referred to below as "bone narrow GVHD") suppress the

differentiation and proliferation of T and B lymphocyte
precursor cells in the bone narrow, retarding the recovery of
cell-mediated immunity and humoral immunity by lymphocytes;
(2) such disorders are caused by donor CD4+ T lymphocytes;
(3) such disorders are ameliorated by treatment involving the
depletion of CD4+ T cells at an early stage following such
transplantation, promoting T and B lymphocyte reconstitution;
and
(4) such treatment does not impair the GVT effect.
Based on these discoveries, the inventors ultimately arrived
at the present invention.
Accordingly, the invention provides the following.
[1] A prophylactic agent for infection which maintains a
graft-versus-tumor effect of allogeneic hematopoietic stem
cell transplantation, comprising a substance capable of
depleting CD4 positive cells, and being administered to a
tumor patient who has received an allogeneic hematopoietic
stem cell transplantation on the same day as transplantation
or in the interval from day 1 to about day 60 following
transplantation, from once a day to once in about 60 days.
[2] The prophylactic agent for infection of the foregoing
[1], which is administered in the interval from day 5 to day
14 following transplantation, from once a day to once in ten
days.
[3] The prophylactic agent for infection of the foregoing

[1] or [2], wherein the substance capable of depleting CD4
positive cells is a CD4 antibody or an altered and/or modified
form thereof.
[4] The prophylactic agent for infection of the foregoing
[3], wherein the CD4 antibody is a humanized anti-human CD4
antibody or a human anti-human CD4 antibody.
[5] The prophylactic agent for infection of the foregoing
[3], wherein the CD4 antibody is administered in a dose of
from 1 to 30 mg/kg each time.
[6] The prophylactic agent for infection of the foregoing
[1], wherein the tumor is a hematopoietic tumor.
[7] The prophylactic agent for infection of the foregoing
[6], wherein the hematopoietic tumor is acute leukemia,
myeloma or malignant lymphoma.
[8] The prophylactic agent for infection of the foregoing
[1], wherein the allogeneic hematopoietic stem cell
transplantation is bone marrow transplantation, peripheral
blood stem cell transplantation or umbilical cord blood
transplantation.
[9] The prophylactic agent for infection of the foregoing
[1], wherein a donor of the allogeneic hematopoietic stem cell
transplantation is a HLA-matched related donor, HLA-matched
non-related donor, HLA-mismatched related donor or HLA-
mismatched non-related donor.
[10] The prophylactic agent for infection of the

foregoing [1], wherein the allogeneic hematopoietic stem cell
transplantation is non-myeloablative transplantation or
myeloablative transplantation.
[11] The prophylactic agent for infection of the
foregoing [1], wherein pre-transplant treatment in allogeneic
hematopoietic stem cell transplantation comprises anti-cancer
drug administration, exposure to radiation, or a combination
thereof.
[12] The prophylactic agent for infection of the
foregoing [1], wherein the infection is pathogenic viral
infection, pathogenic bacterial infection, pathogenic fungal
infection or pathogenic parasitic infection.
[13] The prophylactic agent for infection of the
foregoing [1], wherein the prophylaxis of infection comprises
amelioration of delayed immune reconstitution due to a graft-
versus-host reaction in bone marrow.
[14] An immunological reconstitution promoter which
maintains the graft-versus-tumor effect of allogeneic
hematopoietic stem cell transplantation, which comprising a
substance capable of depleting CD4 positive cells and being
administered to a tumor patient who has received an allogeneic
hematopoietic stem cell transplantation on the same day as
transplantation or in the interval from day 1 to about day 60
following transplantation, from once a day to once in about 60
days.

The inventive drug makes it possible to promote
immunological reconstitution or prevent infections following
allogeneic hematopoietic stem cell transplantation, while
maintaining the GVT effect of such transplantation.
Specifically, in a tumor patient who has received such a
transplantation, especially a patient in whom immune
reconstitution is delayed, such reconstitution can be promoted
by administering, at an early stage following transplantation,
a substance capable of depleting CD4+ cells. Early completion
of infection management in the patient and improvement in the
survival rate are anticipated as a result. In addition, the
risk of complications associated with allogeneic hematopoietic
stem cell transplantation is reduced, enabling more widespread
use of this therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of the pathogenic mechanism
for bone marrow GVHD.
FIG. 2 shows the change over time in the number of each
of the following cells in the BMT and GVHD groups: total bone
marrow cells (FIG. 2A), total splenic cells (FIG. 2B) and
total thymic cells (FIG. 2C); and in the number of each of the
following cells in bone marrow within the BMT and GVHD groups:
granulocytes (FIG. 2D), monocytes (FIG. 2G), erythroblastic
cells (CD71+ and Terll9+/-) (FIGS. 2F and 21), and splenic CD4+

and CD8+ T cells (FIGS. 2E and 2H) .
FIG. 3 shows the change over time in the number of each
type of donor T cell (FIGS. 3A) and 3B) and donor bone marrow-
derived cell (FIGS. 3C to 3F) in the BMT and GVHD groups (FIGS.
3A, 3B, 3D and 3E are the spleen results, and FIGS. 3C and 3F
are the thymus results).
FIG. 4 shows the change over time in the number of B
cells in the bone marrow (FIGS. 4A and 4C) and the thymus
(FIGS. 4B and 4D) in the BMT and GVHD groups.
FIG. 5 shows the mRNA expression of factors (CXCL12, Pax5,
E2a, Ebf1, IFNgamma, IL-7R, Irf1, GATA3) which take part in
hemocyte differentiation within the total bone marrow cells
(FIGS. 5A and 5C) and within the c-Kit(+) Sca-1(+) fractions
thereof (FIGS. 5B and 5D).
FIG. 6 shows immunohistological staining patterns (x200)
in the intestinal tract and fecal IgA concentrations (using
ELISA) on day 14 after transplantation in the BMT and GVHD
groups.
FIG. 7 shows hematoxylin-eosin stained images of bone
marrow sections on day 21 after transplantation in murine
models of GVHD.
FIG. 8 shows SDF-1 (CXCL12) expression in the bone marrow
within the BMT and GVHD groups.
FIG. 9 shows B cell production and body weight change
over time in experiments wherein mutant FasL-bearing gld

mouse-derived splenic T cells and mutant Fas-bearing lpr
mouse-derived bone marrow cells were used in various
combinations (in the figures, combinations are labeled as
"bone marrow cells/splenic cells" [BM/SPL]).
FIG. 10 shows the influence of administering CD4 antibody
or CD8 antibody on the number of B cells.
FIG. 11 shows the influence of administering CD4 antibody
or CD8 antibody on the number of naive (TN) or effector (TE)
CD4+ T cells.
FIG. 12 shows the influence of CD4 antibody
administration upon the serum immunoglobulin concentration on
day 100 after transplantation.
FIG. 13 shows the influence of CD4 antibody or CD8
antibody administration on the percent survival of murine
models of GVDH (FIG. 13A: GVT effect with concurrent
transfusion of P815 cells), the GVHD score (FIG. 13B), and the
body weight change over time (FIG. 13C).
BEST MODE FOR CARRYING OUT THE INVENTION
The invention is described more fully below.
Graft-versus-host reaction (sometimes abbreviated below
as "GVH reaction") refers herein to the reaction that arises
when transplanted hematopoietic cells from the donor, owing to
the immune response by the cells, attack recipient organs.
The GVH reaction is provoked by the infiltration of donor T

cells in, for example, the skin, liver or intestinal tract,
and is characterized by causing, as the main symptoms, skin
rashes, jaundice and/or diarrhea. The present invention is
based on the discovery that delayed immune reconstitution or
lowered immune function in a patient who has received a bone
marrow transplant is caused by the GVH reaction to bone marrow
(referred to below as the "bone marrow GVH reaction" or "bone
marrow GVHD"). This mechanism is shown in FIG. 1. Following
allogeneic hematopoietic stem cell transplantation, the
transfused donor hematopoietic stem cells colonize blood
marrow hematopoietic niches present in blood marrow
microenvironments and proliferate, producing various leukocyte
subsets or precursor cells. The mechanisms of blood marrow
GVHD are thought to be the indirect suppression of
hematopoiesis via impairment of bone marrow hematopoietic
niches by donor T cells and the direct suppression of
hematopoiesis in which inflammatory factors such as IFN and
TNF suppress hemocyte proliferation and differentiation. As
shown in the examples, the Fas-FasL pathway participates to
some degree in bone marrow GVHD via donor T cells; moreover
the donor T-cell subset which is the main cause of bone marrow
GVHD is CD4+ cells. Such bone marrow GVHD stops the
production and differentiation of B cells in the recipient and
at the same time also suppresses the production of
granulocytic cells and T cells, thus playing a large role in

delayed immune reconstitution and increasing the opportunities
for post-transplantation infection.
The "graft-versus-tumor effect" refers to the cancer or
tumor growth suppressing, shrinking and eliminating effects
that can be observed as a result of T cells present in the
transplanted donor-derived hematopoietic stem cells
recognizing and attacking the patient's cancer or tumor cells
as foreign matter. This is therefore an effect which can be
observed only in allogeneic transplantation involving the
transfusion of cells collected from a donor having a different
HLA type. This effect, with respect to leukemia in particular,
is called the graft-versus-leukemia effect (GVL effect).
Similarly, this effect is called the graft-versus-lymphoma
effect with respect to lymphoma, and the graft-versus-myeloma
effect with respect to multiple myeloma.
In the use of the drug of the present invention, the
tumor patient who has received an allogeneic hematopoietic
stem cell transplantation refers primarily to malignant tumor
patients, examples of which include patients having one or
more of the following carcinomas: hematopoietic system tumors,
cancer of the large intestine, kidney cancer (e.g., clear cell
carcinoma), melanoma (e.g., metastatic malignant melanoma),
prostate cancer (e.g., hormone-refractory prostate
adenocarcinoma), breast cancer, colon cancer, lung cancer
(e.g., non-small-cell lung cancer), bone cancer, pancreatic

cancer, skin cancer, head and neck cancer, skin or orbital
malignant melanoma, ovarian cancer, rectal cancer, anal cancer,
stomach cancer, testicular cancer, uterine cancer, fallopian
tube carcinoma, endometrial carcinoma, cervical carcinoma,
vaginal carcinoma, vulvar carcinoma, esophageal cancer, cancer
of the small intestine, endocrine system cancer, thyroid
cancer, parathyroid cancer, adrenal cancer, soft tissue
sarcoma, urethral cancer, cancer of the penis, pediatric solid
cancer, bladder cancer, kidney or ureteral cancer, renal
pelvic carcinoma, tumors of the central nervous system (CNS),
primary CNS lymphoma, tumor angiogenesis, vertebral tumors,
brain stem glioma, pituitary adenoma, Kaposi sarcoma,
epidermoid cancer, squamous cell cancer, and environmentally
induced cancers, including asbestos-induced cancers. Here,
tumor patients in whom the use of the drug of the present
invention is preferred are hematopoietic system tumor patients.
Hematopoietic system tumors are exemplified by leukemia
and malignant lymphoma. Examples of leukemia include
lymphatic leukemia (e.g., hairy cell leukemia, acute lymphatic
leukemia, prolymphocytic leukemia, chronic lymphatic leukemia
(e.g., B-cell chronic lymphatic leukemia), adult T-cell
leukemia (adult T-cell lymphoma, adult T-cell leukemia bone
marrow infiltration), lymphatic leukemia bone marrow
infiltration), myeloma (e.g., plasma cell leukemia, solitary
myeloma, multiple myeloma (e.g., Bence Jones multiple myeloma,

multiple myelomatous joint disease, nonsecretory multiple
myeloma, myeloma kidney, multiple myeloma bone marrow
infiltration), myelodysplastic syndrome (e.g., RAEB, RAEB-t,
refractory anemia, RARS (primary sideroblastic anemia)),
myeloid leukemia (e.g., acute myeloid leukemia, acute
myelomonocytic leukemia, acute promyelocytic leukemia,
basophilic leukemia, eosinophilic leukemia, neutrophilic
leukiemia, myelomonocytic leukemia, chronic myeloid leukemia
(e.g., malignant changes in chronic myeloid leukemia, chronic
phase of chronic myeloid leukemia, transitional stage of
chronic myeloid leukemia, atypical chronic myeloid leukemia),
chronic myelomonocytic leukemia (e.g., juvenile myelomonocytic
leukemia), myeloid leukemia bone marrow infiltration), acute
leukemia, chronic leukemia, monocytic leukemia (e.g., acute
monocytic leukemia, chronic monocytic leukemia), smoldering
leukemia, Letterer-Siwe disease, acute histiocytosis, acute
mastocytoma, acute megakaryoblastic leukemia, plasmacytoma,
myelofibrosis (e.g., acute myelofibrosis, primary
myelofibrosis, secondary myelofibrosis, idiopathic
myelofibrosis), myeloproliferative diseases, mixed cell
leukemia, meningeal leukemia, erythroid leukemia, monoclonal
immunoglobulinemia, hypoplastic leukemia, secondary leukemia,
leukemic joint disease, atypical leukemia, mast cell leukemia,
mast cell tumor disorders, and Crow-Fukase syndrome. Examples
of malignant lymphoma include B cell lymphoma, diffuse

lymphoma (e.g., diffuse mixed lymphoma, diffuse small cell
lymphoma, diffuse small cleaved cell lymphoma, diffuse large
cell lymphoma, diffuse undifferentiated lymphoma,
lymphoblastocytic lymphoma, immunoblastocytic lymphadenopathy,
reticulosarcoma), Hodgkin's disease (e.g., lymphocyte depleted
Hodgkin's disease, lymphocyte predominant Hodgkin's disease,
nodular sclerosing Hodgkin's disease, mixed cell Hodgkin's
disease), lymphoma, malignant lymphoma of the stomach, orbital
malignant lymphoma, malignant lymphoma of the neck, malignant
lymphoma of the thyroid gland, malignant lymphoma of the bones,
malignant lymphoma of the duodenum, malignant mediastinal
lymphoma, malignant lymphoma of the small intestine, malignant
lymphoma of the large intestine, malignant lymphoma of the
brain, non-Hodgkin's lymphoma, peripheral T-cell lymphoma
(e.g., T zone lymphoma, Sezary syndrome, Lennert lymphoma,
mycosis fungoides), malignant tonsillar lymphoma, malignant
lymphoma of the spleen, follicular cell lymphoma (e.g.,
medium-sized cell type follicular cell lymphoma, mixed cell
type follicular cell lymphoma, large cell type follicular cell
lymphoma), MALT lymphoma, malignant lymphoma of the heart,
malignant lymphoma of the colon, malignant lymphoma of the
rectum, malignant lymphoma bone marrow infiltration, malignant
immunoproliferative diseases (e.g., alpha-H chain disease,
gamma-H chain disease, primary macroglobulinemia,
immunoproliferative small intestinal disease), and malignant

lymphoma of the nose and throat. Here, hematopoietic system
tumors in which the use of the drug of the present invention
is preferred are acute leukemias.
"Allogeneic hematopoietic stem cell transplantation" in
the use of the drug of the present invention refers herein to
a method of reconstituting hematopoiesis by transfusing
hematopoietic stem cells from a related donor or an unrelated
donor having an HLA type that is identical or similar. In
order to collect or acquire the hematopoietic stem cells, it
is necessary first to screen the HLA types of relatives or to
search bone marrow banks (e.g., the Japan Marrow Donor
Program) or umbilical cord blood banks (e.g., the Japanese
Cord Blood Bank Network) for a donor having a HLA type which
is identical or similar to that of the patient. Allogeneic
hematopoietic stem cell transplantation can be expected to
have a GVT effect, but there is a risk of GVHD onset. It is
for this reason that the drug of the present invention is
effective. Another type of hematopoietic stem cell
transplantation is autologous hematopoietic stem cell
transplantation which is a method of reconstituting
hematopoiesis by transfusing one's own hematopoietic stem
cells. However, the need for the drug of the present
invention is low in such cases because there is no concern
over GVHD and there is little GVT effect.
Hematopoietic stem cell transplantation falls into three

categories, depending on the type of cell used in
transplantation: bone marrow transplantation, peripheral
blood stem cell transplantation and umbilical cord blood
transplantation, each to which the drug of the present
invention can apply. Bone marrow transplantation is a method
of transplanting hematopoietic stem cells by transplanting
bone marrow fluid. Bone marrow fluid can be obtained by
placing the donor under general anesthesia and using a bone
marrow needle to collect about 15 to 20 mL of fluid per body
weight from three to five places on the left and right sides
of the dorsum of the pelvis. Peripheral blood stem cell
transplantation is a method wherein peripheral blood stem
cells which have been mobilized in a large quantity from the
bone marrow into the blood by G-CSF administration is
transplanted. Peripheral blood stem cells can be obtained by
subcutaneously injecting about 10 µg/kg/day of G-CSF for 4 to
6 days, and using a blood component collection system to
collect the cells on days 4 to 6 following injection. The
timing of cell collection can be set by measuring the number
of cells positive for the CD34 antigen, which is a
hematopoietic stem cell marker present in the blood.
Umbilical cord blood transplantation is a method of
transplanting hematopoietic stem cells present in umbilical
cord blood. Cord blood which matches the patient can be
sought from a cord blood bank by means of a HLA type test. In

cord blood transplantation, although the number of stem cells
that can be collected from umbilical cord blood is limited,
compared with the other types of transplantation, GVHD does
not readily arise. As a result, even if two out of six HLA
type are incompatible, transplantation is possible. Each of
the above methods of transplantation and methods of collecting,
preparing or screening for bone marrow, peripheral blood stem
cells or umbilical cord blood can be carried out based on
Manual of hematopoietic stem cell transplantation and
diagnosis, first edition (published by Nihon Igakukan), or
Manual of hematopoietic cell transplantation, revised third
edition (published by Nihon Igakukan).
The donor for the allogeneic hematopoietic stem cell
transplantation may be selected based on the HLA (human
leukocyte antigen) type. Given that three HLA types (HLA-A, -
B and -DR) are inherited from each parent, in principle, the
number of HLA types which should be considered in allogeneic
hematopoietic stem cell transplantation is six. Because HLA
type incompatibility can cause severe GVHD after
transplantation, it is desirable for the HLA type to be
matched. However, a certain degree of incompatibility may
have the opposite effect of leading to a strengthened GVT
effect. Hence, it is preferable to select a suitable donor
according to the type of tumor, the age and health status of
the patient, and the type of hematopoietic stem cell to be

transplanted. Donor selection is carried out based on, in
principle, the following classification.
(1) HLA-matched related donors. Because the HLA type is
inherited, there is a 1/4 probability of compatibility among
siblings. For this reason, the frequency of GVHD and
transfusion-related complications is generally low. In
allogeneic hematopoietic cell transplantation, it is
desirable first to seek a compatible donor from among
relatives.
(2) HLA-matched unrelated donors. In cases where an HLA-
matched relative for all the HLA types has not been found, an
HLA-matched person (HLA-matched unrelated donor) can be
sought from a bone marrow bank.
(3) HLA-mismatched related donors. Given that the success
rate for allogeneic hematopoietic stem cell transplantation
from related donors in which five of the six HLA types match
is comparable to that from HLA-matched non-related donors,
even in cases where a relative that matches for all HLA types
has not been found, a related donor with a partial mismatch
may be selected.
(4) HLA-mismatched unrelated donors. In cases where a
suitable donor cannot be found from among HLA-matched
individuals or HLA-mismatched relatives, an HLA-mismatched
unrelated person may be selected as the donor. However, in
such cases, there is an increased risk of GVHD.

In addition, in selecting the donor, it is preferable to
make a judgment which is also based on, for example,
respiratory function, circulatory function, liver function,
medical history for various diseases, and the presence or
absence of infections and allergies. For more detailed
selection criteria, reference may be made to Manual of
hematopoietic stem cell transplantation and diagnosis, first
edition (published by Nihon Igakukan), or Manual of
hematopoietic cell transplantation, revised third edition
(published by Nihon Igakukan).
In connection with the use of the drug of the present
invention, allogeneic hematopoietic stem cell transplantation
includes also pre-transplant preparation. Here, "pre-
transplant preparation" refers to treatment which is carried
out prior to transplantation and involves anticancer drug
administration, irradiation or a combination thereof, and also,
where necessary, the administration of immunosuppressants, in
order to eradicate cancer cells or lower the immunity of the
patient so as to facilitate the engrafting of the donor's
hematopoietic stem cells. Such pre-transplant preparation may
be carried out from about 7 to 10 days prior to hematopoietic
stem cell transplantation.
Examples of anticancer drugs which can be employed in
pre-transplant treatment using the drug of the present
invention include alkylating agents (e.g., cyclophosphamide,

busulfan, melfalan, hydroxyurea, nimustine hydrochloride,
carmustine, lomustine, ranimustine, nitramine, iphosphamide,
melphalan thiotepa, carboquone, busulfan, dacarbazine,
temozolomide, procarbazine hydrochloride, nitrogen mustard-N-
oxide hydrochloride), antimetabolites (e.g., enocitabine,
capecitabine, carmofur, cladribine, gemcitabine, cytarabine,
cytarabine ocfosfate, methotrexate, mercaptopurine,
fludarabine, fluorouracil, tegafur, tegafur uracil, tegafur-
gimeracil-oteracil potassium, doxifluridine, nelarabine,
hydroxycarbamide, pemetrexed, pentostatin, mercaptopurine),
anticancer antibodies (e.g., daunorubicin hydrochloride,
doxorubicin hydrochloride, pirarubicin, mitoxantrone,
idarubicin hydrochloride, bleomycin, actinomysin D,
aclarubicin, amrubicin, epirubicin, zinostatin stimalamer,
peplomycin, mitomycin C, mitoxantrone), alkaloids (e.g.,
vincristine, vindesine, etoposide, irinotecan, etoposide,
sobuzoxane, docetaxel, nogitecan, paclitaxel, vinorelbine,
vinblastine), molecular markers (e.g., ibritumomab tiuxetan,
imatinib, erlotinib, gefitinib, gemtuzumab ozogamicin,
sunitinib, cetuximab, sorafenib, tamibarotene, trastuzumab,
tretinoin, panitumumab, bevacizumab, bortezomib, rituximab),
and platinum-containing drugs (e.g., oxaliplatin, carboplatin,
cisplatin, nedaplatin). Anticancer drug administration
protocols may be carried out according to commonly known
methods in this field.

The irradiation which can be employed in pre-transplant
treatment using the drug of the present invention may be
carried out according to a commonly known protocol in this
field. For example, it is preferable for irradiation to be
carried out by total-body irradiation for acute leukemia,
malignant lymphoma and some solid carcinomas, and by local
irradiation for ordinary solid carcinomas. The dose required
will vary depending on such factors as the method of
irradiation (single-dose or fractionated exposure), the type
of tumor, and the susceptibility of the tumor to radiation.
For example, in total-body irradiation, 10 to 12 Gy is
regarded as a standard dose. On the other hand, to reduce
organ damage from radiation, frequent use is being made
recently of fractionated irradiation. For example,
irradiation may entail one or two exposures daily at a dose
level of about 1.8 to 2 Gy per exposure for a period of about
4 to 7 days. The fractionated irradiation may be equally
fractionated exposure or unequally fractionated exposure, and
may be suitably modified according to the burden on the
patient, side effects and the therapeutic effects. Radiation
therapy may be carried out in parallel with anticancer drug
therapy.
The types and dosages of anticancer drugs or the dose of
radiation when using the drug of the present invention may be
selected in accordance with the type of tumor or hematopoietic

stem cell or in accordance with the age or health status of
the patient. The drug of the present invention may be
employed either in "allogeneic hematopoietic stem cell
transplantation with a myeloablative pre-transplant regimen
(myeloablative transplantation)" involving the use of a
conventional powerful pre-transplant regimen, or in
"allogeneic hematopoietic stem cell transplantation with a
non-myeloablative pre-transplant regimen (non-myeloablative
transplantation)" which is able to reduce the toxicity by
weakening the strength of the pre-transplant regimen (see
Manual of hematopoietic stem cell transplantation and
diagnosis, first edition (published by Nihon Igakukan), or
Manual of hematopoietic cell transplantation, revised third
edition (published by Nihon Igakukan)).
In addition, an immunosuppressant (e.g., cyclosporine,
tacrolimus) may be optionally administered to keep acute GVHD
from occurring.
In the present invention, "CD4+ cell-depleting substance'
refers to, for example, a substance which eradicates donor-
derived CD4+ cells or a substance which suppresses the
proliferation or function of donor-derived CD4+ cells.
Examples include CD4 antibodies having a complement-dependent
cytotoxicity (abbreviated below as "CDC") and/or an antibody-
dependent cytotoxicity (abbreviated below as "ADC"), or such
CD4 antibodies to which a cytotoxin or a cytotoxic drug has

been added.
Here, "CD4+ cells" refers to immune cells which express
CD4 at the cell surface. Examples include CD4+ T cells, CD4+
dendritic cells, CD4+ macrophages and CD4+ NKT cells. The
contribution of CD4+ T cells to the onset of bone marrow GVHD
in the present invention is especially large.
Here, the CD4 antibodies may be any which bond to human
CD4 and, by destroying CD4+ cells or suppressing their
proliferation or function, reduce or eliminate such cells from
the patient's blood or various tissues. However, humanized
anti-human CD4 antibodies and human anti-human CD4 antibodies
are preferred.
Here, "humanized anti-human CD4 antibody" refers to an
antibody obtained by grafting the complementarity determining
region (also referred to as "CDR") of an anti-human CD4
antibody derived from another mammal such as a mouse onto the
framework (also referred to as "FR") sequence of a human
antibody. Such an antibody may be produced based on the
methods described in, for example, U.S. Patent Nos. 4,816,567,
5,225,539, 5,530,101, 5,585,089 and 6,180,370. Anti-human CD4
antibodies derived from other mammals may be produced by, for
example, the hybridoma technique (Kohler, G. et al., Nature,
256(5517), 495-497 (1975)). Amino acids on the FR in the
variable region of the antibody may be substituted so that the
CDR of the humanized anti-human CD4 antibodies forms a

suitable antigen-bonding site (Sato, K. et al., Cancer
Research, 53, 851-856 (1993)).
"Human anti-human CD4 antibody" is an anti-human CD4
antibody in which the entire structure of the CDR and FR, etc.
is derived from humans. Such antibodies may be produced using
an HuMAb mouse (registered trademark) (see, for example, U.S.
Patent Nos. 5,545,806, 5,569,825, 5,625,126 and 5,633,425), a
KM mouse (registered trademark) (see WO 02/43478), a XenoMouse
(registered trademark) (see U.S. Patent Nos. 5,939,598,
6,075,181, 6,114,598, 6,150,584 and 6,162,963), a TC mouse
(registered trademark) (see Tomizuka et al., Proc. Natl. Acad.
Sci. USA 97(2), 722-727 (2000), or a human immune cell-
reconstituted SCID mouse (see U.S. Patent No. 5,476,996 and
5,698,767). The human anti-human CD4 antibody may also be
prepared by a phage display method for human immunoglobulin
gene library screening (see U.S. Patent Nos. 5,223,409,
5.403,484 and 5,571,698).
The CD4 antibody in the present invention also includes
such antibody fragments as Fab, F(ab)'2 and ScFv of the above
antibody, and low-molecular antibodies such as Sc(Fv)2 and
diabodies.
Isotypes of the CD4 antibody include IgG (IgG1, IgG2, IgG3
and IgG4), IgA (IgA1 and IgA2), IgM, IgD and IgE. IgG is
preferred, and IgG1 or IgG3, in which the ADCC or CDC is
stronger, are even more preferred.

In addition, the latent immunogenicity of the CD4
antibody in the present invention can be lowered by changing
at least one residue within the FR or at least one residue
within at least one CDR and removing a T-cell epitope (see U.S.
Patent Publication No. 20030153043).
Also, by altering or modifying the variable region or
constant region of the CD4 antibody in the present invention,
it is possible to change the antigen-binding activity,
stability, biological half-life, complement-fixing activity,
CDC, Fc receptor-binding activity and/or ADCC.
The antigen compatibility of the CD4 antibody in the
present invention can be increased by eliminating the
glycosylating site on the variable region FR via an amino acid
substitution (see U.S. Patent Nos. 5,714,350 and 6,350,861).
Also, by altering the number of cysteine residues in the hinge
region of CH1, the assembly of heavy chains and light chains
can be promoted or the antibody stability can be enhanced (U.S.
Patent No. 5,677,425). In addition, the biological half-life
can be lengthened by the amino acid substitutions mentioned in
U.S. Patent No. 6,277,375 or 5,869,046 and U.S. Patent No.
6,121,022, or by PEG conversion using a method known in this
technical field.
Moreover, with regard to the CD4 antibody in the present
invention, the effector function can be altered by an amino
acid substitution in the Fc region (see U.S. Patent Nos.

5,624,821 and 5,648,260), the CDC can be enhanced by the
method described in U.S. Patent No. 6,194,551, and the
complement-fixing activity can be enhanced by the method
described in WO 94/29351.
Also, the affinity of the CD4 antibody in the present
invention to ADCC and/or Fcγ receptors can be enhanced by the
method described in WO 00/42072. Similarly, the antibody ADCC
can be increased by using the methods or cells described in
U.S. Patent Publication No. 20040110704, EP Patent No.
1,176,195, WO 03/035835 or WO 99/54342 to alter glycosylation
or reduce the fucose residues.
Here, preferred examples of the CD4 antibody in the
present invention include MTRX-1011A, TRX-1 (see WO
2002/102853), Ibalizumab (see WO 92/09305), BT-061, huB-F5 and
Zanolimumab (see WO 97/13852), 4162W94, Clenoliximab or
Keliximab (see WO 93/02108), AD-519 or PRO-542 (see WO
92/13947), and Cedelizumab (see WO 96/36359).
To increase the CD4+ cell-depleting ability,.the CD4
antibody in the present invention may optionally bind a
cytotoxic molecule such as a cytotoxin or a cytotoxic drug.
Illustrative examples of cytotoxins include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin, etoposide, teniposide, vincristine, vinbastine,
colchicine, doxorubicin, daunorubicin,
dihydroxyanthracenedione, mitoxantrone, mitramycin,

actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine,
tetracaine, lidocaine, propranolol, puromycin, duocarmycin,
calicheamicin, maytansine, auristatin, and derivatives thereof.
More preferred examples include duocarmycin, calicheamicin,
maytansine, auristatin, and derivatives thereof. Meanwhile,
illustrative examples of cytotoxic drugs include
antimetabolites (e.g., metotrexate, 6-mercaptopurine, 6-
thioguanine, cytarabine, 5-fluorouracil dacarbazine),
alkylating agents (e.g., mechlorethamine, thioepa chlorambucil,
melfalan, carmustine (BSNU), lomustine (CCNU),
cyclophosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, cis-dichlorodiamineplatinum (II)), anthracyclines
(e.g., daunorubicin and doxorubicin), antibiotics (e.g.,
dactinomycin, bleomycin, mithramycin, anthramycin (AMC)), and
antimitotic agents (e.g., vincristine, vinblastine). The
binding of cytotoxins or cytotoxic drugs to CD4 antibodies may
be carried out by a known method in this technical field.
The drug of the present invention may be used for the
prevention of post-transplant infection, such as pathogenic
viral infection, pathogenic bacterial infection, pathogenic
fungal infection and pathogenic parasitic infection. Here,
illustrative examples of pathogenic viruses include HIV,
hepatitis viruses (e.g., HCV, HBV, HAV), herpesviruses (e.g.,
VZV, HSV-1, HAV-6, HSV-II, CMV, Epstein-Barr virus),
adenoviruses, influenzaviruses, flaviviruses, echoviruses,

rhinoviruses, coxsackieviruses, coronaviruses, respiratory
syncytial viruses, mumps viruses, rotaviruses, measles viruses,
rubella viruses, parvoviruses, vaccinia viruses, adult T-cell
leukemia viruses (HTLV), dengue viruses, papillomaviruses, the
molluscum contagiosum virus, polioviruses, rabies viruses, JC
viruses, arboviruses, and encephalitis viruses. Illustrative
examples of pathogenic bacteria include chlamydia, rickettsia
bacteria, mycobacteria, pneumococci, staphylococci,
streptococci, pneumococci, meningococci, gonococci,
Escherichia coli, enterococci, conococcus, Klebsiella, Proteus,
Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, and
the bacteria responsible for cholera, tetanus, botulism,
anthrax, plague, leptospirosis and Lyme disease. Illustrative
examples of pathogenic fungi include Candida (e.g., albicans,
krusei, glabrata, tropicalis), Cryptococcus neoformans,
Aspergillus (e.g., fumigatus, niger), Mucorales (e.g., Mucor,
Absidia, Rhizopus), Sporothrix schenckii, Blastomyces
dermatitidis, Paracoccidioides brasiliensis, Coccidioides
immitis, and Histoplasma capsulatum. Illustrative examples of
pathogenic parasites include Entamoeba hisolytica, Balantidium
coli, Naegleria fowleri, Acanthamoeba spp., Giardia lamblia,
Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax,
Babesia microti, Trypanosoma brucei, Trypanosoma cruzi,
Leishmania donovani, Toxoplasma gondii, and Ancylostoma
braziliense.

In the present invention, the CD4+ cell-depleting
substance as an active ingredient may be administered by a
parenteral pathway, such as intravenously, intramuscularly,
intracutaneously, peritoneally, subcutaneously or spinally, as
an injection or infusion of a composition prepared, together
with a pharmaceutically acceptable carrier.
The dose of CD4+ cell-depleting substance is typically,
for example, from about 0.1 to about 100 mg/kg, preferably
from about 0.1 to about 50 mg/kg, and more preferably from
about 1 to about 30 mg/kg.
The period of administration for the inventive drug is
the same day as allogeneic hematopoietic stem cell
transplantation or from 1 to about 60 days following
transplantation, preferably from 1 to about 30 days after
transplantation, more preferably from day 5 to day 14 after
transplantation, and still more preferably from day 5 to day 7
after transplantation. In cases where myelosuppression
associated with chronic GVHD is observed, additional
administration may be carried out even more than 60 days after
transplantation.
The inventive drug may be administered anywhere from once
daily to once in about 60 days, although administration is
preferably once daily, more preferably once in 3 days, and
even more preferably once in 10 days.

The injection or infusion containing the CD4+ cell-
depleting substance in the present invention may be used as a
solution, a suspension or an emulsion. The solution may use,
for example, distilled water for injection, physiological
saline, a glucose solution and an isotonic solution (e.g., a
solution of sodium chloride, potassium chloride, glycerol,
mannitol, sorbitol, boric acid, sodium borate, propylene
glycol). In addition, the injection may also include, for
example, stabilizers, solubilizing agents, suspending agents,
emulsifying agents, soothing agents, buffers, preservatives,
antiseptics and pH adjustors. Examples of stabilizers that
may be used include albumin, globulin, gelatin, mannitol,
glucose, dextran, ethylene glycol, propylene glycol,
diethylene sufite, ascorbic acid, sodium bisulfite, sodium
thiosulfate, sodium edetate, sodium citrate and
dibutylhydroxytoluene. Examples of solubilizing agents that
may be used include alcohols (e.g., ethanol), polyalcohols
(e.g., propylene glycol, polyethylene glycol), and nonionic
surfactants (e.g., Polysorbate 80 (registered trademark), HCO-
50). Examples of suspending agents that may be used include
glycerol monostearate, aluminum monostearate, methylcellulose,
carboxymethylcellulose, hydroxymethylcellulose and sodium
lauryl sulfate. Examples of emulsifying agents that may be
used include gum arable, sodium alginate and gum tragacanth.
Examples of soothing agents that may be used include benzyl

alcohol, chlorobutanol and sorbitol. Examples of buffers that
may be used include phosphoric acid buffers, acetic acid
buffers, boric acid buffers, carbonic acid buffers, citric
acid buffers, Tris buffers, glutamic acid buffers and e-
aminocaproic acid buffers. Examples of preservatives that may
be used include methyl p-oxybenzoate, ethyl p-oxybenzoate,
propyl p-oxybenzoate, butyl p-oxybenzoate, chlorobutanol,
benzyl alcohol, benzalkonium chloride, sodium dehydroacetate,
sodium edentate, boric acid and sodium borate. Examples of
antiseptics that may be used include benzalkonium chloride, p-
oxybenzoic acid and chlorobutanol. Examples of pH adjustors
that may be used include hydrochloric acid, sodium hydroxide,
phosphoric acid and acetic acid.
The injection or infusion may be produced by
sterilization in the final step or by sterilization involving
aseptic manipulation, such as filtration with a filter or the
like, followed by filling into an aseptic container.
Injections may be preserved by freezing or may be preserved
after first removing water by freeze-drying. In the latter
case, at the time of use, distilled water for injection or the
like is added to the preserved injection so as to re-dissolved
it for use.
The entire contents of all patents and reference
documents explicitly cited in this specification are
incorporated herein as part of this specification.

EXAMPLES
The present invention is described in detail below by way
of examples, although the invention is not limited by these
examples.
Example 1: Production of Animal Model of GVHD after
Allogeneic Hematopoietic Stem Cell Transplantation
On the day prior to hematopoietic stem cell
transplantation, the recipient mice (6-week-old female
C57BL/6xDBA2 Fl (BDFl, H2d/b)) were lethally irradiated (11 Gy)
in two split doses given 3 hours apart. Some of the
irradiated mice received both C57BL/6 (B6, H2b)-derived T cell-
depleted bone marrow cells (5xl06 cells) and splenic T-cells
(5xl06 cells negatively enriched against CDllb, B220, Terll9
and NK1.1). These mice are referred to below as the GVHD
group. Others of the irradiated mice received only the
C57BL/6 (B6, H2b)-derived T cell-depleted bone marrow cells
(5xl06 cells). The latter are referred to below as the BMT
group.
Example 2: Flow Cytometric Analysis of Donor Hematopoietic
Stem Cell-Derived Bone Marrow Hematopoiesis after
Transplantation
Bone marrow, spleen and thymus were harvested from each
animal in each group of mice prepared in Example 1, and the
donor hematopoietic stem cell-derived bone marrow
hematopoiesis from day 7 to day 28 following transplantation

was analyzed over time by flow cytometry. Flow cytometry was
carried out by a method known to persons of ordinary skill in
the art.
FIGS. 2(A) to (I) show the change over time in the number
of each of the following cells in BMT and GVHD groups: total
bone marrow cells, total splenic cells and total thymic cells;
and in the number of each of the following cells in bone
marrow within the BMT and GVHD groups: granulocytes,
monocytes, erythroblastic cells (CD71+ and Terll9+/"), and
splenic CD4+ and CD8+ T cells. Decreases in the total number
of bone marrow, splenic and thymic cells in the GVHD group, a
recovery in splenic CD4+ T-cells in the BMT group, and a
decrease in erythroblastic cells in the GVHD group were
observed.
Example 3: Production of Animal Model of GVHD after
Allogeneic Hematopoietic Stem Cell Transplantation (2)
On the day prior to hematopoietic stem cell
transplantation, CD45.2+ recipient mice (6-week-old female
C57BL/6xDBA2 Fl (BDFl, H2d/b)) were lethally irradiated (11 Gy)
in two split doses given 3 hours apart. Some of the
irradiated mice received both CD45.1+CD45.2+ congenic mouse-
derived T cell-depleted bone marrow cells (5xl06 cells) and
CD45.1+ congenic mouse-derived splenic T-cells (5xl06 cells
negatively enriched against CD11b, B220, Terll9 and NK1.1).
These mice are referred to below as the GVHD group. Others of

the irradiated mice received only the CD45.1+CD45.2+ congenic
mouse-derived T cell-depleted bone marrow cells (5xl06 cells).
The latter are referred to below as the BMT group.
Example 4: Flow Cytometric Analysis of Donor Hematopoietic
Stem Cell-Derived Bone Marrow Hematopoiesis after
Transplantation (2)
Bone marrow, spleen and thymus were harvested from each
animal in the groups of mice prepared in Example 3, and the
donor hematopoietic stem cell-derived bone marrow
hematopoiesis from day 7 to day 28 following transplantation
was analyzed over time by flow cytometry. Flow cytometry was
carried out by a method known to persons of ordinary skill in
the art.
FIGS. 3(A) to (F) show the change over time in the number
of each type of donor T cell and donor bone marrow-derived
cell in the BMT and GVHD groups. Recovery by the donor bone
marrow-derived T-cells is delayed in the GVHD group compared
with the BMT group. Moreover, although the donor bone marrow-
derived T-cells in the GVHD group is suppressed, they are
gradually producing.
FIGS. 4(A) to (D) show the change over time in the number
of B cells in the bone marrow and the thymus in the BMT and
GVHD groups. In the GVHD group, excessive impairment of B-
cell differentiation and production persists throughout.
Hence, in the GVHD group, declines in the myelocytic and

erythroblastic cells, and in particular a delayed recovery of
systemic T-cells and B-cells due to delayed recovery of
lymphatic progenitor cells, are observed. These results
indicate that immunosuppression by GVHD has occurred.
Example 5: Analysis of Hemocyte Differentiation by Real-Time
RT-PCR
To determine which stage of hemocyte differentiation the
bone marrow GVHD impairs, bone marrow was harvested from the
GVHD murine models produced in Example 1, the total RNA was
prepared by a method known to persons of ordinary skill in the
art, and the mRNA expression of factors which participate in
hemocyte differentiation (CXCL12, Pax5, E2a, Ebfl, IFNgamma,
IL-7R, Irfl, GATA3) was analyzed by real-time RT-PCR. The
real-time RT-PCR was conducted by a method known to persons of
ordinary skill in the art.
FIGS. 5(A) to (D) shows the results obtained by
concentrating and analyzing the total bone marrow cells and
the c-Kit(+)Sca-l(+) fraction thereof. In the GVHD group,
extreme declines are apparent in the expression of Pax5, E2a
and Ebfl, which are transcription factors essential for B-cell
differentiation and proliferation. A decrease in IL-7R, which
is required for the differentiation of lymphocyte precursors,
was also observed. These results indicate that, due to the
onset of GVHD, the production and differentiation of B-cells
is specifically or continuously impaired from a very early

stage.
Example 6: Immunohistological Staining of Intestinal Tract
and ELISA Analysis of Fecal IgA
To understand the influence of GVHD on intestinal tract
immunity, the intestinal tracts and feces of the GVHD murine
models prepared in Example 1 were collected, and
immunohistological staining of the intestinal tract and ELISA
measurement of fecal IgA were carried out. Immunohistological
staining and ELISA analysis were carried out by methods known
to persons of ordinary skill in the art.
FIG. 6(A) and (B) shows intestinal tract
immunohistological staining patterns (x200) and fecal IgA
concentrations on day 14 after transplantation in the BMT and
GVHD groups, respectively. IgA production was clearly
depressed (disappearance of light areas indicated by arrows in
immunohistological staining patterns) in the GVHD group
compared with the BMT group. The same was also true of the
ELISA measurements of fecal IgA. These results show that IgA
production is impaired in GVHD.
Example 7: Pathological Analysis in Bone Marrow at Time of
GVHD Onset
Bone marrow sections on day 21 after transplantation were
prepared for the GVHD murine models produced in Example 1, and
hematoxylin-eosin staining was carried out. Hematoxylin-eosin
staining was carried out by a method known to persons of

ordinary skill in the art.
As shown in FIG. 7, a distinct decline in the number of
nucleated cells (the number of cells represented on the image
by black shadows) and spotted clusters of erythrocytes
(arrows)
in the GVHD group were observed. These were characteristic
bone marrow findings associated with GVHD. Although not shown,
in the GVHD group on day 28 after transplantation, bleeding
and blood clots within the bone marrow were conspicuous,
indicating severe breakdown of the normal structure.
At the same time, although not shown, the distinct
decline in cellularity and the hemorrhagic picture observed in
the GVHD group on day 21 after transplantation improved in the
group given CD4 antibodies. Specifically, a recovery in the
number of nucleated bone marrow was observed and the spotted
clusters of erythrocytes decreased. It should be noted that
200 µg of the CD4 antibodies (GK1.5, Medical & Biological
Laboratories) was administered intraperitoneally once each on
days 4 and 6 after transplantation in the above GVHD murine
models.
Example 8: Analysis of GVHD Sites of Action in Bone Marrow
Tissue
To identify the GVHD sites of action in the bone marrow
tissue, bone marrow was harvested from the GVHD murine models
produced in Example 1, total RNA was prepared by a method

known to persons of ordinary skill in the art, and the
expression of SDF-1 (CSCL12) was measured over time by real-
time RT-PCR. SDF-1 is one hematopoiesis-associated molecule
which is essential during interactions with bone marrow stroma
at sites of proliferation and differentiation by hematopoietic
stem cells and hemocytic precursor cells at various stages of
differentiation. Real-time RT-PCR was carried out by a method
known to persons of ordinary skill in the art.
As shown in FIGS. 8(A) to (D), the expression decreased
markedly in the GVHD group. This result indicates that the
bone marrow stroma is a target for GVHD. This is also
supported by the absence of a significant difference between
the GVHD group and the BMT group in the expression of SDF-1
receptor CXCR4 on B cells and is moreover corroborated by the
fact that, even in transplantation experiments using mutant
Fas-containing 1pr mouse-derived bone marrow cells, B cell
production was unable to recover in the GVHD group. By
contrast, in the group given CD4 antibodies, the decrease in
SDF-1 expression showed an improving trend with the passage of
time (FIGS. 8C and 8D).
Example 9: Identification of Effector Molecules which Induce
Bone Marrow GVHD
On the day prior to hematopoietic stem cell
transplantation, the recipient mice (6-week-old female
C57BL/6xDBA2 Fl (BDFl, H2d/b)) were lethally irradiated (11 Gy)

in two split doses given 3 hours apart. The irradiated mice
received as the transplanted bone marrow either C57BL/6 (B6,
H2b)-derived T cell-depleted bone marrow cells (5xl06 cells) or
mutant Fas-containing lpr mouse-derived T cell-depleted bone
marrow cells (5xl06 cells). In cases where GVHD was to be
induced, the mice received ordinary wild-type (also referred
to as WT) B6 splenic T cells (5xl06 cells negatively enriched
against CDllb, B220, Terll9 and NKl.l) or mutant FasL-
containing gld mouse-derived splenic T cells (5xl05 cells
negatively enriched against CDllb, B220, Terll9 and NKl.l) in
various combinations (also referred to as GVHD groups). The
mutant FasL-containing gld mice and the mutant Fas-containing
lpr mice were obtained from Japan SLC, Inc.
[0066]
In FIG. 9, (WT/-), (lpr/-), (WT/WT), (lpr/WT) and
(WT/gld) indicate the combination of bone marrow cells and
splenic cells (bone marrow/spleen) at the time of
transplantation.
Bone marrow was harvested from each animal in the
respective groups of mice, and hemocyte differentiation from
day 7 to day 28 after transplantation was analyzed over time
by flow cytometry. Flow cytometry was carried out by a method
known to persons of ordinary skill in the art.
As shown in FIGS. 9(A) to (C), partial recovery of the B
cells was observed in the WT/gld group. This result shows

that the FasL of the donor T cells plays a limited role in the
onset of bone marrow GVHD. Also, because the recovery of B
cells is not observed in the lpr/WT group in which mutant Fas-
containing lpr mouse-derived bone marrow cells were used as
the transplanted bone marrow cells, there is a possibility
that the target of FasL is stromal cells rather than hemocytic
cells. Also, the fact that hematopoiesis does not recover
even though bone marrow which does not express Fas was
transfused suggests the possibility that, instead of the bone
marrow cells being directly impaired by the Fas-FasL pathway
of the donor T cells, the bone marrow stromal cells which are
the hematopoietic micro-environments that directly interfere
with the bone marrow cells and play the essential role of
"fields" for differentiation of the bone marrow cells are
impaired.
Example 10: Identification of T-Cell Subset which Induces
Bone Marrow GVHD
The CD4 antibody (200 µg) or the CD8 antibody (53-6.7,
Medical and Biological Laboratories) mentioned in Example 7
were intraperitoneally administered to the GVHD murine models
produced in Example 1, once each on days 4 and day 6 after
transplantation, and, using improvement in GVHD as the
indicator, the causative T cell subset was identified.
As shown in FIG. 10, a marked recovery in B cells due to
CD4 antibody administration can be observed, suggesting that

CD4 is the main effector. On the other hand, no effects due
to CD8 antibody administration were observed. An analysis of
thymic T cell differentiation by flow cytometry demonstrated
that, as shown in FIGS. 11(A) to (D), the recovery of naive T
cells in the thymus was promoted by CD4 antibody
administration. Also, as shown in FIG. 12, with the
administration of CD4 antibody, a recovery in various types of
immunoglobulins was observed on day 100 after transplantation.
Example 11: Action of CD4 Antibody on Bone Marrow GVHD and
GVT Effect
To confirm the action of CD4 antibody administration on
GVHD and on the GVT effect, the GVHD murine models produced in
Example 1 were intravenously injected with 1x104 cells of P815
(a DBA2 mouse-derived mast cell tumor (ATCC: TIB-64)) 2 hours
before bone marrow transplantation, and were intraperitoneally
administered 200 ng of anti-CD4 antibody once each on days 4
and day 6 after transplantation. The survival of the mice,
the GVHD scores and the changes in body weight were analyzed.
As shown in FIG. 13A to 13C, in the CD4 antibody group,
the GVHD was suppressed without a loss in the GVT effect (13B),
and 100% survival was maintained up to nearly day 40 following
transplantation (13A). In the BMT group, tumor deaths due to
the metastasis of tumor cells to the liver and spinal cord
occurred on days 15 to 20 after transplantation.
Example 12: CD4 Antibody Therapy in Myeloablative

Transplantation
Following various forms of chemotherapy (in the case of
various types of acute or chronic leukemia, a single
administration of a suitable amount of endoxan; in the case of
malignant lymphoma, treatment involving any combination of
suitable amounts of melfalan, endoxan, lastet and
dexamethasone), total-body irradiation (TBI) at 12 Gy
(fractionated exposure: 4 Gy per day for 3 days) is carried
out from 1 to 3 days prior to transplantation, after which
hematopoietic stem cell transplantation is carried out.
Preferably, CD4 antibodies are administered once daily a total
of three times from day 5 to day 7 after transplantation.
Example 13: CD4 Antibody Therapy in Non-Myeloablative
Transplantation
Following chemotherapy (a combination of fludarabine and
busulfan), TBI (2 to 4 Gy) is carried out as fractionated
exposure for 1 to 2 days before transplantation, then
hematopoietic stem cell transplantation is carried out. Next,
CD4 antibodies are preferably administered once daily a total
of three times from day 5 to day 7 after transplantation.
INDUSTRIAL APPLICABILITY
The present invention is useful in that it can prevent
the risk of complications, particularly infections, associated
with allogeneic hematopoietic stem cell transplantation.

CLAIMS:
1. A prophylactic agent for infection which maintains a
graft-versus-tumor effect of allogeneic hematopoietic stem
cell transplantation, comprising a substance capable of
depleting CD4 positive cells, and being administered to a
tumor patient who has received an allogeneic hematopoietic
stem cell transplantation on the same day as transplantation
or in the interval from day 1 to about day 60 following
transplantation, from once a day to once in about 60 days.
2. The prophylactic agent for infection according to
claim 1, which is administered in the interval from day 5 to
day 14 following transplantation, from once a day to once in
ten days.
3. The prophylactic agent for infection according to
claim 1 or 2, wherein the substance capable of depleting CD4
positive cells is a CD4 antibody or an altered and/or modified
form thereof.
4. The prophylactic agent for infection according to
claim 3, wherein the CD4 antibody is a humanized anti-human
CD4 antibody or a human anti-human CD4 antibody.
5. The prophylactic agent for infection according to
claim 3, wherein the CD4 antibody is administered in a dose of
from 1 to 30 mg/kg each time.
6. The prophylactic agent for infection according to
claim 1, wherein the tumor is a hematopoietic tumor.

7. The prophylactic agent for infections according to
claim 6, wherein the hematopoietic tumor is acute leukemia,
myeloma or malignant lymphoma.
8. The prophylactic agent for infection according to
claim 1, wherein the allogeneic hematopoietic stem cell
transplantation is bone marrow transplantation, peripheral
blood stem cell transplantation or umbilical cord blood
transplantation.
9. The prophylactic agent for infections according to
claim 1, wherein a donor of the allogeneic hematopoietic stem
cell transplantation is a HLA-matched related donor, HLA-
matched non-related donor, HLA-mismatched related donor or
HLA-mismatched non-related donor.
10. The prophylactic agent for infection according to
claim 1, wherein the allogeneic hematopoietic stem cell
transplantation is non-myeloablative transplantation or
myeloablative transplantation.
11. The prophylactic agent for infection according to
claim 1, wherein pre-transplant treatment in allogeneic
hematopoietic stem cell transplantation comprises anti-cancer
drug administration, exposure to radiation, or a combination
thereof.
12. The prophylactic agent for infection according to
claim 1, wherein the infection is pathogenic viral infection,
pathogenic bacterial infection, pathogenic fungal infection or

pathogenic parasitic infection.
13. The prophylactic agent for infection according to
claim 1, wherein the prophylaxis of infections comprises
amelioration of delayed immune reconstitution due to a graft-
versus-host reaction in bone marrow.
14. An immunological reconstitution promoter which
maintains a graft-versus-tumor effect of allogeneic
hematopoietic stem cell transplantation, comprising a
substance capable of depleting CD4 positive cells, and being
administered to a tumor patient who has received an allogeneic
hematopoietic stem cell transplantation on the same day as
transplantation or in the interval from day 1 to about day 60
following transplantation, from once a day to once in about 60
days.

The object of the invention is to provide an
immunological reconstitution promoter or a prophylactic agent
for infections for use in allogeneic hematopoietic stem cell
transplantation therapy for tumors. The promoter or
prophylactic agent enables the amelioration of delayed immune
reconstitution or the prevention of infection following
transplantation, while maintaining the GVT effect of
allogeneic hematopoietic stem cell transplantation.
Specifically, in a transplant patient in whom immune
reconstitution is delayed, such reconstitution can be promoted
by administering, at an early stage following transplantation,
a substance capable of depleting CD4+ cells. Early completion
of infection management in the patient and improvement in the
survival rate are anticipated as a result. In addition, the
risk of complications associated with allogeneic hematopoietic
stem cell transplantation is reduced, enabling more widespread
use of this therapy.