U.S. patent application number 11/575024 was filed with the patent office on 2008-07-03 for adsorbent for lymphocyte proliferation inhibitor and treating method.
This patent application is currently assigned to KANEKA CORPORATION. Invention is credited to Akira Kobayashi, Katsuo Noguchi, Shinya Yoshida.
Application Number | 20080159990 11/575024 |
Document ID | / |
Family ID | 36036483 |
Filed Date | 2008-07-03 |
United States Patent
Application |
20080159990 |
Kind Code |
A1 |
Kobayashi; Akira ; et
al. |
July 3, 2008 |
Adsorbent For Lymphocyte Proliferation Inhibitor and Treating
Method
Abstract
The invention has for its object to provide a porous material
capable of relieving the lymphocyte proliferation inhibition in
lymphocyte culture and improving the proliferative nature of
lymphocytes as well as a method for proliferating lymphocytes and a
method for producing lymphocytes each of which utilizes such porous
material. The invention relates to a porous material for body fluid
treatment for promoting lymphocyte proliferation in lymphocyte
culture which contains a high-molecular compound having an angle of
contact with water within the range of 40.degree. to 98 .degree.;
and a porous material for body fluid treatment which comprises
activated carbon. It also relates to a treatment device wherein the
porous material is used; a method for proliferating lymphocytes; a
method for producing mammalian lymphocytes; a method for producing
a pharmaceutical composition; an additive body fluid to be added to
a culture medium on the occasion of lymphocyte culture; a method
for treating a disease against which a therapeutic effect is
produced by returning extracorporeally activated mammalian
lymphocytes into the body; and the use of activated carbon or a
high-molecular compound having an angle of contact with water
within the range of 40.degree. to 98.degree. in manufacturing
porous materials for body fluid treatment for promoting the
lymphocyte proliferation in lymphocyte culture.
Inventors: |
Kobayashi; Akira; (Hyogo,
JP) ; Yoshida; Shinya; (Hyogo, JP) ; Noguchi;
Katsuo; (Tokyo, JP) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ LLP
1875 EYE STREET, N.W., SUITE 1100
WASHINGTON
DC
20036
US
|
Assignee: |
KANEKA CORPORATION
Osaka
JP
|
Family ID: |
36036483 |
Appl. No.: |
11/575024 |
Filed: |
September 9, 2005 |
PCT Filed: |
September 9, 2005 |
PCT NO: |
PCT/JP05/16601 |
371 Date: |
October 19, 2007 |
Current U.S.
Class: |
424/93.7 ;
435/372; 604/358 |
Current CPC
Class: |
C12N 5/0636 20130101;
C12N 2533/00 20130101; C12N 2533/30 20130101; A61M 1/3679 20130101;
A61K 35/17 20130101 |
Class at
Publication: |
424/93.7 ;
435/372; 604/358 |
International
Class: |
A01N 63/00 20060101
A01N063/00; C12N 5/00 20060101 C12N005/00; A61F 13/15 20060101
A61F013/15 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 2004 |
JP |
2004-264044 |
Claims
1. A porous material for body fluid treatment for promoting
lymphocyte proliferation in lymphocyte culture which contains a
high-molecular compound having an angle of contact with water
within the range of 40.degree. to 98.degree..
2. The porous material according to claim 1 which has a
molecular-weight exclusion limit of not higher than
1.5.times.10.sup.5.
3. The porous material according to claim 1, wherein the
high-molecular compound is an aromatic high-molecular compound.
4. The porous material according to claim 3, wherein the aromatic
high-molecular compound is polystyrene or a styrene-divinylbenzene
copolymer.
5. The porous material according to claim 1, wherein the
high-molecular compound contains no amine residue bound
thereto.
6. The porous material according to claim 1, wherein the
high-molecular compound has no other compound immobilized
thereon.
7. A porous material for body fluid treatment for promoting
lymphocyte proliferation in lymphocyte culture which comprises
activated carbon.
8. A treatment device for promoting lymphocyte proliferation in
lymphocyte culture which comprises the porous material according to
claim 1 and a container therefor.
9. The device according to claim 8, wherein said container has a
liquid inlet and a liquid outlet.
10. A method for proliferating lymphocytes which comprises bringing
a body fluid into contact with the porous material according to
claim 1 and cultivating lymphocytes using the body fluid after
contacting with said porous material.
11. The method for proliferating lymphocytes according to claim 10,
wherein said body fluid is an autologous body fluid of a mammal and
said mammal is in a condition of poor lymphocyte proliferation.
12. A method for producing mammalian lymphocytes which comprises
bringing a mammalian body fluid into contact with the porous
material according to claim 1, cultivating lymphocytes using the
body fluid after contacting with the porous material and recovering
the thus-produced lymphocytes.
13. A method for producing a pharmaceutical composition which
comprises producing lymphocytes according to the method according
to claim 12 and blending the lymphocytes with a pharmaceutically
acceptable additive.
14. An additive body fluid to be added to the culture medium on the
occasion of lymphocyte culture which is obtained by bringing a
mammalian body fluid into contact with the porous material
according to claim 1.
15. A method for treating a disease against which a therapeutic
effect is obtained by returning extracorporeally activated
mammalian lymphocytes into the body which comprises bringing a body
fluid derived from a mammal requiring or not requiring treatment
into contact with the porous material according to claim 1,
cultivating lymphocytes using the body fluid after contacting with
the porous material, and administering the thus-obtained
lymphocytes to said mammal.
16. The use of activated carbon or a high-molecular compound having
an angle of contact with water within the range of 40.degree. to
98.degree. in producing a porous material for body fluid treatment
to promote the lymphocyte proliferation in lymphocyte culture.
17. The porous material according to claim 2, wherein the
high-molecular compound is an aromatic high-molecular compound.
18. The porous material according to claim 2 wherein the
high-molecular compound contains no amine residue bound
thereto.
19. The porous material according to claim 3 wherein the
high-molecular compound contains no amine residue bound
thereto.
20. The porous material according to claim 4 wherein the
high-molecular compound contains no amine residue bound thereto.
Description
TECHNICAL FIELD
[0001] The present invention relates to a porous material for
overcoming the lymphocyte proliferation inhibition in lymphocyte
culture in activated autologous lymphocyte therapy, among others,
which comprises taking immunocompetent cells (lymphocytes in
particular) in blood out of the body, culturing them for
stimulation/activation and for proliferation and again returning
them into the body to thereby preventing the advance of cancer, an
infectious disease or an immune disease, and to a device for
lymphocyte proliferation wherein the porous material mentioned
above is utilized. Furthermore, it relates to a method for
preparing a body fluid with a reduced lymphocyte proliferation
inhibitor concentration as well as to a method for proliferating
lymphocytes using a medium with such body fluid added thereto.
BACKGROUND ART
[0002] In recent years, attention has been focused on activated
autologous lymphocyte therapy, which comprises taking
immunocompetent cells (lymphocytes in particular) in blood out of
the body, culturing them for stimulation/activation and for
proliferation and again returning them into the body to thereby
prevent the advance of cancer. This technique produces little side
effects and makes it possible to maintain the quality of life (QOL)
at high levels even during treatment and, therefore, is becoming
more and more popular in the field of cancer treatment as a fourth
choice of cancer therapy next to the three major cancer treatment
methods, namely surgical therapy, radiotherapy and chemotherapy.
The technique is already in actual use as one of tailor-made highly
advanced medical treatment methods in university hospitals, cancer
centers and specialized clinics and, expectedly, it will be used
still more widely. This technique generally comprises collecting a
portion of the blood of a patient, separating a lymphocyte fraction
by density gradient centrifugation, adding the autologous plasma to
a medium for exclusive use and cultivating the lymphocytes.
Generally, the number of lymphocytes arrives at about 100 times the
number of lymphocytes in the primary culture in a week. It has
become known, however, that there are some such cancer patients
that lymphocytes derived therefrom can hardly proliferate in the
presence of autologous plasma but can proliferate in the presence
of the plasma derived from another person (resulting from blood
donation). Cancer cells produce cellular immunity inhibiting
factors, for example such cytokines as transforming growth factor
beta (hereinafter abbreviated as TGF-.beta.), interleukin 4
(hereinafter abbreviated as IL4), interleukin 6 (hereinafter
abbreviated as IL6) and interleukin 10 (hereinafter abbreviated as
IL10) as well as prostaglandin E2 (hereinafter abbreviated as
PGE2). Thus, the possibility is suggested that such factors might
inhibit the proliferation of lymphocytes. However, the
concentrations of these factors in the blood of cancer patients are
almost the same as those in persons in normal health, although
their local concentrations in cancer foci are high. Further, when
such factors commercially available as reagents are dissolved in
plasma at high concentrations and the influences thereof on
lymphocyte proliferation are examined, little inhibition is
observed. Such and other findings suggest that there is an unknown
mechanism other than the involvement of such factors.
[0003] As a matter of fact, an adsorbent for adsorptively removing
immunosuppressive acidic proteins (IAPs) (Patent Document 1), an
adsorbent for adsorptively removing interleukins in body fluids
(Patent Document 2) and an adsorbent capable of adsorbing
TGF-.beta. in body fluids (Patent Document 3), among others, have
so far been disclosed. However, all of them are limited in scope to
the adsorptive removal of cytokines and there is no report about an
adsorbent capable of improving the proliferative activity of
lymphocytes. In recent years, the number of patients having
activated lymphocyte therapy has been increasing year by year with
the marked advance of such therapy and, on the other hand, the
number of patients relying on blood donation because of poor
lymphocyte proliferation has also been increasing. In the case of
blood donation, it is necessary to secure non-autologous plasma in
conformity with patient's therapeutic cycle. Further, there are a
number of problems to be taken up from the safety viewpoint, for
example the risk of infection; therefore, it is desired that a
method for overcoming the lymphocyte proliferation inhibition in
lymphocyte culture by a simple procedure without losing other
useful substances be developed. Furthermore, in cancer patients as
well whose lymphocyte can proliferate in the presence of autologous
body fluids, it is expected that further improvements in
proliferation rate and in cytokine producing activity, for
instance, be achieved when the inhibition of lymphocyte
proliferation in lymphocyte culture is broken down. Currently,
however, neither adsorbent, nor porous body, nor device nor
treatment method is available for such purposes. Further,
adsorbents prepared by causing a material capable of adsorbing
cytokines and like immunosuppressive proteins to bind to a
water-insoluble carrier have been disclosed (Patent Document 4 to
6). However, the effect of those adsorbents depends on an affinity
between an amine residue and such immunosuppressive proteins as
cytokines. Therefore, those adsorbents require the presence of an
amine residue.
[0004] At present, a treatment is eagerly anticipated which will
promote the proliferation of those lymphocytes which are in a
poorly proliferative condition in lymphocyte culture for the
treatment of a disease against which a therapy comprising returning
activated lymphocytes again into the body, typically activated
autologous lymphocyte therapy, is therapeutically effective.
[0005] Patent Document 1: Japanese Kokai Publication
Sho-56-092824
[0006] Patent Document 2: Japanese Kokai Publication
Hei-08-257398
[0007] Patent Document 3: Japanese Kokai Publication
2001-218840
[0008] Patent Document 4: Japanese Kokai Publication
2003-310751
[0009] Patent Document 5: Japanese Kokai Publication
2003-339854
[0010] Patent Document 6: Japanese Kokai Publication 2004-73618
SUMMARY OF THE INVENTION
[0011] Accordingly, it is an object of the present invention to
provide an adsorbent useful in such treatment for promoting
lymphocyte proliferation, a method for such treatment, and
lymphocytes proliferated by treatment with the porous material.
[0012] The present inventors made intensive investigations in
search of a porous material capable of breaking down the inhibition
of lymphocyte proliferation while minimizing the loss of useful
substances necessary for the proliferation of lymphocytes, etc.
and, as a result, found that when a subject-derived body fluid is
treated in advance with a water-insoluble porous material
containing a high-molecular compound having an angle of contact
with water within the range of about 40.degree. to 98.degree. or a
porous material containing activated carbon, the lymphocyte
proliferation rate in lymphocyte culture is markedly improved. Such
and other findings have now led to completion of the present
invention.
[0013] Thus, the present invention relates to
[0014] a porous material for body fluid treatment for promoting
lymphocyte proliferation in lymphocyte culture
[0015] which contains a high-molecular compound having an angle of
contact with water within the range of 40.degree. to 98.degree.;
and to
[0016] a porous material for body fluid treatment for promoting
lymphocyte proliferation in lymphocyte culture
[0017] which comprises activated carbon.
[0018] The invention also relates to
[0019] a treatment device for promoting the lymphocyte
proliferation in lymphocyte culture
[0020] which comprises the above-mentioned porous material and a
container therefor.
[0021] Further, the invention relates to
[0022] a method for proliferating lymphocytes;
[0023] a method for producing mammalian lymphocytes;
[0024] a method for producing a pharmaceutical composition;
[0025] an additive body fluid to be added to the culture medium on
the occasion of lymphocyte culture;
[0026] a method for treating a disease against which returning
extracorporeally activated mammalian lymphocytes into the body is
therapeutically effective; and
[0027] a use of activated carbon or a high-molecular compound
having an angle of contact with water within the range of
40.degree. to 98.degree. in producing a porous material for body
fluid treatment to promote the lymphocyte proliferation in
lymphocyte culture.
DETAILED DESCRIPTION OF THE INVENTION
[0028] First, the porous material of the invention is
described.
[0029] The porous material of the invention is a porous material
for use in body fluid treatment for promoting the lymphocyte
proliferation in lymphocyte culture which contains a high-molecular
compound having an angle of contact with water within the range of
40.degree. to 98.degree..
[0030] In the practice of the invention, the angle of contact with
water can be determined by preparing a smooth film specimen
constituted of the high-molecular compound, which is the main
constituent of the porous material, forming a liquid drop on the
film in a horizontal state using a microinjector and measuring the
contact angle at room temperature. When the porous material is
soluble in an organic solvent, the contact angle can also be
measured after dissolving the porous material and preparing a cast
film specimen on a flat sheet using the resulting solution. For
details of the measurement method, reference can be made, for
example, to "Shin-Jikken Kagaku Koza (Lectures in Experimental
Chemistry, New Series) 18: Kaimen to Koroido (Interface and
Colloid)" (First Edition, published October 20, Showa 52 (1977) by
Maruzen Co., Ltd.). Thus, a flat sheet specimen having a
mirror-finish level of smoothness is placed horizontally so that
the atmosphere surrounding the same may be filled with the
saturated vapor of the liquid to be subjected to measurement, and a
liquid drop is formed thereon using a microinjector. The size of
the liquid drop is such that the contact diameter is about 3 mm or
smaller. The contact angle can be determined by measuring the angle
formed upon allowing the liquid drop to advance toward the solid
surface (at the time when after the liquid is allowed to develop
and spread on the specimen, the liquid drop becomes stable at a
certain size) using a reading microscope (having a magnification of
about 20) equipped with a goniometer. The visibility of the image
becomes very good when the lens barrel is inclined by 1 to 2
degrees downward from the horizontal. The drop is illuminated from
the front with light transmitted through an opalescent glass or
with parallel beams of light transmitted through a heat
ray-absorbing glass.
[0031] The contact angle data reported herein were measured by the
method described later in the example section.
[0032] The high-molecular compound having an angle of contact with
water within the range of 40.degree. to 98.degree. includes as
typical examples, but is not limited to, synthetic high-molecular
compounds such as nylon 6, nylon 6,6, nylon 11, polyethylene,
poly(vinylidene chloride), poly(vinyl chloride), poly(vinyl
acetate), polystyrene, styrene-divinylbenzene copolymers,
poly(trifluoroethylene), poly(chlorotrifluoroethylene),
poly(ethylene terephthalate), polypropylene, polyacrylic esters
(e.g. poly(methyl acrylate)), polymethacrylic esters (e.g.
poly(methyl methacrylate)), crosslinked polyacrylates and
crosslinked polyamides as well as cellulose and like
water-insoluble ones. Among them, polymers or copolymers produced
by polymerizing an aromatic monomer or monomers (e.g. monomers
selected from among alkylstyrenes which may optionally be
substituted, for example methylstyrene and ethylstyrene;
divinylbenzene and benzo-condensed cyclic compounds which may
optionally be substituted, for example divinylnaphthalene and
divinylanthracene) are preferred from the lymphocyte proliferation
rate viewpoint. In particular, polystyrene and
styrene-divinylbenzene copolymers are preferred.
[0033] Any conventional polystyrene species can be used as the
polystyrene. The polystyrene is an arbitrary styrene polymer or
styrene-based polymer.
[0034] The styrene-divinylbenzene copolymers can be obtained by
crosslinking the above-mentioned styrene compound with m-, o- or
p-divinylbenzene, which may optionally be substituted.
[0035] The high-molecular compound mentioned above may optionally
be substituted by a halogen, alkyl, alkenyl, alkynyl, aralkyl,
aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, alkylcarbonyl,
alkoxycarbonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfonyl,
etc. The substituents mentioned above may further be substituted by
a substituent(s) other than amines, and such substituents on the
above-mentioned substituents are preferably other than amide, urea,
ester and ether groups.
[0036] The high-molecular compound mentioned above is preferably
one having no amine residue bound thereto. As the amine residue,
there may be mentioned those residues resulting from chemical
bonding of ammonia, primary to tertiary amine or the like to the
high-molecular compound.
[0037] Further, the high-molecular compound mentioned above is
preferably one comprising no other compound immobilized thereon.
The other compound is not particularly restricted but includes, for
example, amines, alcohols, glycidyl ethers, carboxylic acids and
derivatives thereof, acid halides, halides, halogenated silanes,
thiols, aldehydes and antibodies.
[0038] For the purpose of the present invention, namely from the
lymphocyte proliferation promotion viewpoint, the above-mentioned
angle of contact with water is preferably greater than 60.degree.
but not greater than 96.degree., more preferably about 70.degree.
to 94.degree., still more preferably about 75.degree. to
91.degree..
[0039] In the practice of the invention, the porous material is
preferably insoluble in water.
[0040] The porous material of the invention contains not less than
about 50% by weight, preferably not less than about 60% by weight,
more preferably not less than about 70% by weight, still more
preferably not less than 80% by weight, of the high-molecular
compound mentioned above.
[0041] As the component which can be contained in the porous
material in addition to the above-mentioned high-molecular compound
having an angle of contact with water within the range of
40.degree. to 98.degree., there may be mentioned, for example,
polyvinyl alcohol (contact angle 36.degree.), poly(hydroxyethyl
methacrylate) (contact angle 13.degree.) and paraffin (contact
angle 105 to 106.degree.), among others.
[0042] By using two or more of the high-molecular compounds
mentioned above having an angle of contact with water within the
range of 40.degree. to 98.degree. in combination and/or by using
any of the above-mentioned high-molecular compounds having an angle
of contact with water of 40.degree. to 98.degree. as the main
component and incorporating a component other than the
high-molecular compound as the auxiliary material, among others, it
is possible to adjust the angle of contact with water of the porous
material to be obtained.
[0043] It is also preferred that the porous material have an angle
of contact with water within the range of the angle of contact with
water of the high-molecular compound mentioned above. Thus, the
angle of contact with water of the porous material is preferably
40.degree. to 98.degree., more preferably greater than 60.degree.
but not greater than 96.degree., still more preferably about
70.degree. to 94.degree., particularly preferably about
75.degree.to 91.degree..
[0044] The porous material of the invention occurs as a solid at
ordinary temperature and ordinary pressure and has pores
appropriate in size, namely has a porous structure.
[0045] As for the size of pores in the porous material, the
molecular-weight exclusion limit of the water-insoluble porous
material as determined using polystyrene beads is preferably not
higher than 1.5.times.10.sup.5, more preferably not higher than
1.4.times.10.sup.5. When the molecular-weight exclusion limit is
higher than 1.5.times.10.sup.5, the level of unspecific adsorption
tends to increase, the loss of useful proteins in the body fluid
tends to occur and/or the ability of lymphocytes to proliferate
tends to decrease with ease. For lessening the influence of
unspecific adsorption while maintaining the lymphocyte
proliferation rate at high levels, the molecular-weight exclusion
limit is more preferably not higher than 1.3.times.10.sup.5,
particularly preferably not higher than 1.2.times.10.sup.5, most
preferably not higher than 1.0.times.10.sup.5.
[0046] The molecular-weight exclusion limit can be easily
controlled, for example, by adjusting the content of the
above-mentioned high-molecular compound, which is the main
constituent of the porous material, in the step of porous material
production. Thus, as the content of the high-molecular compound
increases, the molecular-weight exclusion limit lowers and, as the
content of the high-molecular compound decreases, the
molecular-weight exclusion limit rises.
[0047] The molecular-weight exclusion limit is the molecular weight
of those molecules which cannot enter the pores (are excluded by
the pores) in chromatography but are smallest in molecular weight
among molecules incapable of enter the pores.
[0048] The molecular-weight exclusion limit can be measured in the
following manner. Polystyrene beads differing in particle diameter
are passed through a column packed with the porous material, and
the pore size is determined on the basis of an excluded polystyrene
beads-based exclusion curve. Then, the above-mentioned pore size is
extrapolated to a spherical protein (e.g. dextran) the diameter and
molecular weight of which are known, to thereby determine the
molecular weight on the spherical protein equivalent basis; this is
regarded as the molecular-weight exclusion limit.
[0049] The shape of the porous material may effectively be
spherical, granular, flat membrane-like, fibrous, or hollow
fiber-like, for instance. From the adsorption performance
viewpoint, however, the spherical or granular form is more
preferably used.
[0050] When the porous material is in a spherical or granular form,
the average particle size thereof is preferably about 5 .mu.m to
1,000 .mu.m, more preferably about 20 to 800 .mu.m, still more
preferably about 30 to 600 .mu.m.
[0051] The average particle diameter can be determined in the
following manner. The porous material in a wet state is developed
on a dish and scores of particles are photographed using a CCD
camera. Then, the average particle diameter is calculated from the
image captured using the particle diameter measurement software
"Image-Pro plus" (product of Medical Cybernetics, Inc.).
[0052] The porous material of the invention can be produced, for
example, in the following manner. One or more raw material monomer
compounds are dispersed/suspended in a solvent having an
appropriate viscosity (e.g. water). Suspension polymerization is
carried out in the conventional manner while stirring the
suspension to give the desired porous material.
[0053] Further, the porous material of the invention may also
comprise activated carbon.
[0054] Usable as the activated carbon are, for example, fibrous
activated carbon derived from phenolic fibers; coconut
shell-derived activated carbon, petroleum pitch-derived activated
carbon, peat-derived activated carbon, charcoal-based activated
carbon and like granular activated carbon species.
[0055] The average particle diameter of the activated carbon is not
particularly restricted but is preferably about 5 .mu.m to 1,000
.mu.m, more preferably about 20 to 800 .mu.m, still more preferably
about 30 to 600 82 m.
[0056] Among the porous material components each comprising a
high-molecular compound or activated carbon, polystyrene,
styrene-divinylbenzene copolymers and activated carbon are
preferred and, in particular, styrene-divinylbenzene copolymers are
preferred, from the lymphocyte proliferation rate viewpoint.
[0057] As described hereinabove, activated carbon or a
high-molecular compound having an angle of contact with water
within the range of 40.degree. to 98.degree. can be used in
producing the porous material for body fluid treatment for
promoting the lymphocyte proliferation in lymphocyte culture.
[0058] Now, the treatment device of the invention is a treatment
device for promoting lymphocyte proliferation in lymphocyte culture
which comprises the porous material mentioned above as contained in
a container.
[0059] The container to be used in the treatment device is not
particularly restricted in shape, size or material.
[0060] The shape may be an arbitrary one, for example a sphere,
container, bag, tube or column. As a preferred typical example,
there may be mentioned, for example, a transparent or
semitransparent cylindrical container with a capacity of about 0.1
to 400 ml and a diameter of about 0.1 to 10 cm.
[0061] The container can be manufactured using an arbitrary
structure material. More specifically, as the structure material,
mention may be made of unreactive polymers, biocompatible metals,
alloys and glass, for instance.
[0062] As the unreactive polymers, there may be mentioned
acrylonitrile-based polymers such as
acrylonitrile-butadiene-styrene terpolymers; halogenated polymers
such as polytetrafluoroethylene, polychlorotrifluoroethylene,
tetrafluoroethylene-hexafluoropropylene copolymers and polyvinyl
chloride; polyamides, polysulfones, polycarbonates, polyethylene,
polypropylene, polyvinyl chloride-acrylic copolymers,
polycarbonate-acrylonitrile-butadiene-styrene, polystyrene and
polymethylpentene, among others.
[0063] As metallic materials useful as the container material,
there may be mentioned stainless steel, titanium, platinum,
tantalum, gold, and alloys thereof, as well as gold-plated alloy
iron, platinum-plated alloy iron, cobalt-chromium alloys and
titanium nitride-coated stainless steel, among others.
[0064] Materials resistant to autoclaving are particularly
preferred and, specifically, silicone-coated glass, polypropylene,
polyvinyl chloride, polycarbonates, polysulfones, polymethylpentene
and the like may be mentioned as such materials.
[0065] The treatment device is preferably one comprising a
container having a liquid inlet and a liquid outlet and equipped
with means for preventing porous material leakage and the
water-insoluble porous material packed in the container, though the
device is not limited to such one.
[0066] The means for preventing porous material leakage may be a
mesh, nonwoven fabric, cotton plug or like filter.
[0067] The method for proliferating lymphocytes according to the
invention is now described.
[0068] The method for proliferating lymphocytes of the invention is
a method which comprises bringing the above-mentioned porous
material into contact with a body fluid and cultivating lymphocytes
using the body fluid after contacting with the porous material.
[0069] As the method for proliferating lymphocytes of the
invention, namely the treating method for promoting lymphocyte
proliferation, there may specifically be mentioned such methods as
mentioned below. (1) The method which comprises packing the
treatment device (container having a body fluid inlet and a body
fluid outlet and equipped, at the outlet, with a filter allowing
the body fluid passage but allowing no porous material passage)
with the porous material, bringing a body fluid into contact
therewith and then using the same in lymphocyte cultivation, (2)
the method which comprises collecting a body fluid in a bag already
containing the porous material and, after a predetermined period of
contacting, filtering off the porous material and using the
filtrate in lymphocyte cultivation, and (3) the method which
comprises causing the porous material to coexist in the lymphocyte
culture system and filtering off the porous material from
lymphocytes during cultivation or after completion of
cultivation.
[0070] Referring to the method (1), the method for contacting
consists, for example, in circulating the body fluid using a feed
pump for a certain period of contacting or in allowing a certain
period of contacting without circulation. As for the contacting
time, 1 minute or a longer period of contacting is preferred and,
from the adsorption performance viewpoint, about 15 minutes to 6
hours of contacting is more preferred. From the sufficient
adsorption performance and cell treatment efficiency viewpoint,
about 20 minutes to 4.5 hours of contacting is more preferred and
about 30 minutes to 3 hours of contacting is still more
preferred.
[0071] As the method (2), there are available, among others, the
method which comprises collecting a body fluid directly in a bag
already containing the porous material and effecting a certain
period of contacting; and the method which comprises preparing a
plasma or serum fraction from blood by centrifugation, for
instance, placing the fraction in such a bag and effecting a
certain period of contacting with the porous material. As for the
contacting time, about 1 minute or a longer period of contacting is
preferred and, from the adsorption performance viewpoint, about 10
minutes to 10 hours of contacting is more preferred and about 15
minutes to 6 hours of contacting is still more preferred. From the
sufficient adsorption performance and cell treatment efficiency
viewpoint, about 30 minutes to 3 hours of contacting is
particularly preferred. As another method, it is also possible to
add the porous material to a system wherein lymphocyte cultivation
is in progress with patient's plasma added, for causing the porous
material coexist in the system and, after the lapse of a certain
period of time, the porous material is separated from lymphocytes
by filtration using a filter allowing no passage of the porous
material.
[0072] The temperature during contacting of the porous material
with the body fluid in the above-mentioned method 1) or (2) can be
arbitrarily selected but is preferably about 4.degree. C. to
50.degree. C., more preferably about 10.degree. C. to 45.degree.
C.
[0073] Referring to the method (3), the porous material is caused
to coexist with lymphocytes in a lymphocyte culture vessel and the
porous material is separated from lymphocytes by filtration on the
occasion of medium exchange or after completion of the cultivation.
The contacting time is such that the contacting lasts until
completion of lymphocyte cultivation at the longest, and the amount
of the porous material to be added is preferably such that a
sufficient space for lymphocyte proliferation can be secured
without physical suppression thereof.
[0074] While the invention is not restricted to those mentioned
above, the above method (1) is procedurally simple and most
preferred as the method for overcoming lymphocyte proliferation
inhibition in lymphocyte culture.
[0075] There may be some unknown mechanism of inhibiting the
lymphocyte proliferation, as referred to hereinabove. An unknown
"lymphocyte proliferation inhibiting factor" may be involved in
that mechanism. Therefore, the porous material of the invention can
function as an adsorbent for lymphocyte proliferation inhibiting
factors.
[0076] When the treatment device mentioned above is used in
carrying out the method for proliferating lymphocytes of the
invention, an anticoagulant may be used.
[0077] As the anticoagulant, use may be made of any of heparin,
low-molecular heparin, nafamostat mesilate, gebexate mesilate,
argatroban, acid-citrate-dextrose (ACD) solution,
citrate-phosphate-dextrose (CPD) solution and like
citrate-containing anticoagulants, among others. Among them,
heparin may generally be mentioned as the most preferred
anticoagulant.
[0078] In the case of serum preparation, for instance, the
above-mentioned anticoagulant may not be contained in the
serum.
[0079] The body fluid so referred to herein includes blood, plasma
and serum. In addition, the body fluid includes other body fluids,
such as ascetic fluid, lymph and intraarticular fluid and fractions
derived from these as well as other living body-derived fluid
components.
[0080] For the purpose of the invention, it is a simple and easy
way to collect blood from the subject and, if desired, prepare
plasma by separation from blood corpuscle fractions by such means
as centrifugation, for the subsequent use. Further, serum may be
prepared for the subsequent use.
[0081] Also usable as the body fluid other than blood, plasma and
serum are dilutions of these or supernatants obtained from them by
pretreatment by specific gravity gradient centrifugation using
Ficoll, Percoll, Vacutainer tube, Lymphoprep or the like.
[0082] While the body fluid may be used immediately after blood
collection, refrigerated or lyophilized blood and preparations may
also be used. Further, it is also possible to treat the body fluid
using the adsorbent and then refrigerate or lyophilize the same,
followed by thawing for use when required. The invention is not
restricted to such modes, however.
[0083] The body fluid released from lymphocyte proliferation
inhibition in lymphocyte culture, after lymphocyte proliferation
promoting treatment according to the invention, can be obtained
from the outlet side filter allowing no passage of the porous
material as a result of filtering off of the porous material. When
the target is blood, the desired plasma can be obtained by further
carrying out a centrifugation procedure. It is also possible to
obtain lymphocytes and the desired plasma simultaneously from the
blood after treatment with the porous material by using Ficoll,
Percoll, Vacutainer tube, Lymphoprep or the like.
[0084] The term "lymphocytes" as used herein refers to T cells and
B cells, among others, occurring in mammalian peripheral blood,
lymph vessels and bone marrow. The lymphocytes also include cells
which are neither T cells nor B cells, for example natural killer
cells. The T cells are not particularly restricted but include
helper T cells, cytotoxic T cells and killer T cells.
[0085] In the practice of the invention, the lymphocyte culture can
be carried out, for example, in the following manner. A mammalian
body fluid is brought into contact with the porous material
mentioned above and then sowed in a lymphocyte culture medium. The
lymphocyte culture medium is incubated at an arbitrary temperature
(preferably about 20.degree. C. to 45.degree. C., more preferably
about 30.degree. C. to 40.degree. C., still more preferably about
37.degree. C.) for an arbitrary period of time (for example about 3
days to 30 days, preferably about 7 days to 21 days, more
preferably about 10 days to 18 days, most preferably about 14
days). As a result, lymphocytes proliferate.
[0086] The lymphocyte culture medium may be a conventional culture
medium. Such medium includes, but is not limited to, such
lymphocyte culture media as PB-MAX medium, AIM V medium, CHANG
medium, LGM-3 medium, KBM400, GIT, Ham F-12, Dulbecco MEM,
.alpha.-MEM, MEM, IMEM, RPMI-1640 and McCoy's 5A medium, among
others.
[0087] On the occasion of lymphocyte culture, an antibody or the
like can also be used to activate lymphocytes. As the antibody,
there may be mentioned, for example, anti-CD3 antibody (OKT3) and
so forth.
[0088] When the body fluid is a mammalian autologous body fluid and
the mammal is in a condition of poor lymphocyte proliferation, the
above-mentioned method for proliferating lymphocytes is more
effective.
[0089] The term "poor lymphocyte proliferation" indicates that when
patient's body fluid is added to a lymphocyte culture system, the
lymphocyte proliferation rate is lower than the lymphocyte
proliferation rate determined using a normal human-derived body
fluid.
[0090] The lymphocyte proliferation rate is the proliferation rate
(number of cells after 7 days/number of cells sowed) found after 1
week of lymphocytes cultivation at 37.degree. C. using a culture
medium supplemented with 0.1% (v/v) to 20% (v/v) of a
patient-derived body fluid treated with the porous material or a
normal human-derived body fluid not yet treated with the porous
material.
[0091] In the case of packing a column with the porous material for
using the same, it is important that clogging will never occur on
the occasion of fluid passage, among others. For that reason, the
porous material is required to have sufficient mechanical strength.
Therefore, the porous material to be used in the practice of the
invention is more preferably a hard one. In the case of a granular
gel material, the term "hard one" as used herein refers to a gel
which, when a cylindrical column is uniformly packed with the gel
and an aqueous fluid is passed therethrough, shows a linear
relationship between pressure loss .DELTA.P and flow rate until
about 0.3 kg/cm.sup.2. In the case of using the porous material by
placing the same in a bag, however, it may be a soft one.
[0092] Now, the method for producing mammalian lymphocytes
according to the invention is a method which comprises bringing a
mammalian body fluid into contact with the porous material
mentioned above, cultivating lymphocytes using the body fluid after
contacting with the porous material, and recovering lymphocytes
produced.
[0093] The lymphocyte recovery can be carried out by any arbitrary
method, for example by centrifugation, membrane filtration or
chromatography.
[0094] Now, the method for manufacturing a pharmaceutical
composition according to the invention is a method which comprises
producing lymphocyte by the above-mentioned lymphocyte production
method and blending the lymphocytes with a pharmaceutically
acceptable additive.
[0095] As the pharmaceutically acceptable additive, there may be
mentioned, for example, anticoagulants, vitamins and other nutrient
sources, and antibiotics.
[0096] The pharmaceutical composition manufacture can be carried
out using an acceptable pharmaceutical technique to give an
appropriate galenic form composition (e.g. for transfusion, drip
infusion or injection).
[0097] The additive body fluid of the invention, which is to be
added to a culture medium on the occasion of lymphocyte culture, is
one obtained by bringing a mammalian body fluid into contact with
the porous material mentioned above.
[0098] The additive body fluid can be prepared, for example, by
bringing a body fluid derived from a mammal with or without a
disease against which a therapeutic effect is produced by
extracorporeally activating lymphocytes and returning them into the
body into contact with the porous material mentioned above.
[0099] Further, the method of the invention for treating a disease
against which a therapeutic effect is produced by extracorporeally
activating lymphocytes and returning them into the body is a method
which comprises bringing a body fluid derived from a mammal
requiring or not requiring treatment into contact with the porous
material mentioned above, cultivating lymphocytes using the body
fluid after contacting with the porous material, and administering
the thus-obtained lymphocytes to the mammal.
[0100] The disease against which a therapeutic effect is produced
by extracorporeally activating lymphocytes and returning them into
the body includes, but is not limited to, cancer, infectious
diseases and immune diseases, among others.
[0101] The activation includes, within the meaning thereof, an
increase in the number of lymphocytes, a change in lymphocyte
population as a result of proliferation and/or an improvement in
the functions intrinsic in lymphocytes, and the like.
(Effects of the invention)
[0102] The present invention has made it possible to overcome the
lymphocyte proliferation inhibition in lymphocyte culture and thus
markedly increase the lymphocyte proliferation rate by adsorbing
lymphocyte proliferation inhibiting factors from a body fluid
derived from a subject with a disease against which a therapeutic
effect is produced by extracorporeally activating lymphocytes and
returning them into the body, for example a cancer subject with
poor lymphocyte proliferation, without adsorbing factors necessary
for lymphocyte proliferation from that body fluid. The invention is
useful in that it provides a porous material for relieving the
lymphocyte proliferation inhibition in lymphocyte culture using a
target body fluid in activated autologous lymphocyte therapy, for
instance, according to which a disease is treated or the progress
of a disease is inhibited by taking immunocompetent cells
(lymphocytes in particular) in blood out of the body, culturing
them for stimulation/activation and for proliferation and again
returning them into the body, as well as a method for proliferating
lymphocytes which utilizes the above-mentioned porous material.
BEST MODES FOR CARRYING OUT THE INVENTION
[0103] The following examples specifically illustrate the present
invention. These examples are, however, by no means limitative of
the scope of the invention.
[0104] In the following examples etc., the angle of contact with
water, molecular-weight exclusion limit and average particle
diameter measurements were made in the following manner.
(1) Angle of Contact with Water
[0105] A flat and smooth film was prepared by compressing the
high-molecular compound sample at a high pressure. The
thus-obtained flat and smooth sheet specimen was placed
horizontally, and a liquid drop was formed thereon using a
microinjector. The size of the liquid drop was such that the
contact diameter was about 1 to 2 mm. The contact angle was
determined by measuring the angle formed upon allowing the liquid
drop to advance toward the solid surface, at room temperature
(20.degree. C.), using a reading microscope (having a magnification
of about 20) equipped with a goniometer.
(2) Molecular-weight Exclusion Limit
[0106] Polystyrene beads differing in particle diameter were passed
through a column packed with the porous material, and the pore size
was determined on the basis of an excluded polystyrene beads-based
exclusion curve. Then, the above-mentioned pore size was
extrapolated to the spherical protein dextran the diameter and
molecular weight of which were known, to thereby determine the
molecular weight on the dextran equivalent basis; this was reported
as the molecular-weight exclusion limit.
(3) Average Particle Diameter
[0107] The porous material in a wet state was developed on a dish
and scores of particles were photographed using a CCD camera. Then,
the average particle diameter was calculated from the image
captured using the particle diameter measurement software
"Image-Pro plus" (product of Medical Cybernetics, Inc.).
EXAMPLE 1
(1) Lymphocyte Preparation
[0108] A winged needle for intravenous injection was connected to
an adaptor and a holder was connected to the other end of the
adaptor. The injection needle was stabbed into the brachial region
of a normal subject and about 7.5 ml of blood was collected in a
tube for lymphocyte separation (Vacutainer tube (product of Becton
Dickinson and Company)). After blood collection, the Vacutainer
tube was subjected to 20 minutes of centrifugation at 3,000 rpm at
room temperature. The lymphocyte layer was recovered and
supplemented with 40 ml of physiological saline, and the resulting
mixture was centrifuged at 1,500 rpm at 4.degree. C. for 5 minutes.
This procedure was repeated several times for washing lymphocytes,
and a lymphocyte suspension with a predetermined concentration was
prepared by resuspending the lymphocytes in KBM 400 medium (product
of Kohjin Bio Co., Ltd.).
(2) Preparation of a Plate with OKT3 Immobilized Thereon
[0109] OKT3(product of Dainippon Pharmaceutical) was diluted to a
concentration of 5 .mu.g/ml with physiological saline, and the
dilution was distributed in 500-.mu.l portions into the wells of a
24-well polystyrene microplate (product of Sumitomo Bakelite Co.,
Ltd.). After 2 hours of standing still at room temperature, the
OKT3 solution was removed, and the plate was washed with two equal
portions of physiological saline; a plate with OKT3 immobilized
thereon was thus prepared.
(3) Preparation of a Porous Material
[0110] A monomer mixture composed of 100 parts by weight of
divinylbenzene for industrial use (divinylbenzene content 57%), 100
parts by weight of toluene, 60 parts by weight of isoamyl alcohol
and 1 part by weight of benzoyl peroxide (content 75%) was added to
an aqueous solution composed of 572 parts by weight of water, 23
parts by weight of sodium chloride, 1 part by weight of polyvinyl
alcohol and 0.03 part by weight of sodium nitrite, and the
polymerization was carried out at 80.degree. C. in a nitrogen
atmosphere for 5 hours with stirring so that droplets of the
monomer mixture might be dispersed and suspended. The polymer
particles formed were filtered off, washed with water and then
deprived of such residual components as the solvent, monomer and
initiator by extraction with acetone and again thoroughly washed
with water and hot water. Porous styrene-divinylbenzene copolymer
beads with a volume average particle diameter of about 400 .mu.m
were obtained (molecular-weight exclusion limit about
8.times.10.sup.4, angle of contact with water about
85.degree.).
(4) Plasma Treatment
[0111] The porous styrene-divinylbenzene copolymer beads were
thoroughly washed with physiological saline and then 0.17 ml
thereof were measured and placed in a cryotube. The physiological
saline was thoroughly removed from the porous beads, 1 ml of cancer
patient's serum was added thereto, followed by 2 hours of
incubation at 37.degree. C. with stirring (40 rpm) on a MIX
rotor.
(5) Lymphocyte Cultivation
[0112] The plasma treated in the above manner was added to the
lymphocyte culture medium KBM 400 (product of Kohjin Bio Co., Ltd.)
to a concentration of 9% (v/v). Using the resulting culture medium,
a lymphocyte suspension with a lymphocyte number of
1.0.times.10.sup.5 cells/ml was prepared and sowed onto the
previously prepared plate with OKT3 immobilized thereon in an
amount of 444 .mu.l/well (n=3 wells).
[0113] After 7 days of cultivation, lymphocytes were recovered and
the number of cells was counted using a hemocytometer, and the
lymphocyte proliferation rate was calculated according to the
following formula (1).
Proliferation rate (times)=number of lymphocytes after 7 days of
cultivation/number of lymphocytes sowed (1)
[0114] As a result, in the case of treatment of cancer patient's
plasma with the adsorbent, the number of lymphocytes increased to
16.7 times (proliferation rate) the number of cells sowed.
EXAMPLE 2
[0115] The lymphocyte proliferation rate was determined in the same
manner as in Example 1 except that petroleum pitch-derived
activated carbon with a particle diameter of about 500 .mu.m was
used as the adsorbent in lieu of the porous styrene-divinylbenzene
copolymer beads. As a result, the number of lymphocytes increased
to 10.6 times (proliferation rate) the number of cells sowed.
EXAMPLE 3
[0116] The lymphocyte proliferation rate was determined in the same
manner as in Example 1 except that porous polystyrene (angle of
contact with water about 85.degree. C.) beads with a molecular
weight exclusion limit of not higher than 1.times.10.sup.4 and a
particle diameter of about 400 .mu.m were used. As a result, the
number of lymphocytes increased to 11.5 times (proliferation rate)
the number of cells sowed.
EXAMPLE 4
[0117] Porous material preparation: A cellulose solution with a
viscosity of about 1,000 cP was jetted, in the form of uniform
droplets, into a gaseous phase under direct application of
vibrations at a frequency of about 20,000 Hz to the solution. After
causing the droplets to make a sufficient flight to take a
spherical form, they were captured in a coagulation bath, deprived
of the solvent and washed to give porous cellulose particles with a
particle diameter of about 400 .mu.m (molecular-weight exclusion
limit not higher than 3.times.10.sup.4, angle of contact with water
about 50.degree.).
[0118] The lymphocyte proliferation rate was determined in the same
manner as in Example 1 except that the porous particles were used.
As a result, the number of lymphocytes increased to 6.9 times
(proliferation rate) the number of cells sowed.
EXAMPLE 5
[0119] The lymphocyte proliferation rate was determined in the same
manner as in Example 1 except that porous cellulose beads with a
molecular-weight exclusion limit of not higher than
6.times.10.sup.4 and a particle diameter of about 400 .mu.m (angle
of contact with water about 40.degree.) were used. As a result, the
number of lymphocytes increased to 5.7 times (proliferation rate)
the number of cells sowed.
COMPARATIVE EXAMPLE 1
[0120] The lymphocyte proliferation rate was determined in the same
manner as in Example 1 except that the patient's plasma was used
without contacting with any adsorbent. As a result, the number of
lymphocytes increases to 3.9 times (proliferation rate) the number
of cells sowed.
COMPARATIVE EXAMPLE 2
[0121] The lymphocyte proliferation rate was determined in the same
manner as in Example 1except that the porous particles used in
Example 4 were used after binding dextran sulfate thereto via
epichlorohydrin (molecular-weight exclusion limit not higher than
3.times.10.sup.4, angle of contact with water about 35.degree.). As
a result, the number of lymphocytes increased to 3.6 times
(proliferation rate) the number of cells sowed.
COMPARATIVE EXAMPLE 3
[0122] The lymphocyte proliferation rate was determined in the same
manner as in Example 1 except that the porous particles used in
Example 5 were used after binding dextran sulfate thereto via
epichlorohydrin (molecular-weight exclusion limit not higher than
3.times.10.sup.4, angle of contact with water about 30.degree.). As
a result, the number of lymphocytes increased to 3.2 times
(proliferation rate) the number of cells sowed.
[0123] The results of the lymphocyte proliferation rate evaluation
in Examples 1 to 5 and Comparative Examples 1 to 3 are shown in
Table 1. From these results, it is evident that the lymphocyte
proliferation rate could be markedly increased by cultivating
lymphocytes using the specific porous materials of the invention
after contacting with a body fluid as compared with the case of
non-use of the porous material (adsorbent) or the cases where
dextran sulfate was immobilized on the high-molecular compounds to
render them hydrophilic.
TABLE-US-00001 TABLE 1 Influence of use of patient's plasma on
lymphocyte proliferation Lymphocyte proliferation rate after 7 days
of cultivation (calculated according to equation 1)) Lymphocyte
proliferation rate (times) Example 1 16.7 .+-. 3.2 Example 2 10.6
.+-. 2.7 Example 3 11.5 .+-. 3.0 Example 4 6.9 .+-. 1.5 Example 5
5.7 .+-. 1.6 Comp. Ex. 1 3.9 .+-. 1.4 Comp. Ex. 2 3.6 .+-. 0.8
Comp. Ex. 3 3.2 .+-. 0.5 n = 3 .+-. S.D
INDUSTRIAL APPLICABILITY
[0124] The present invention has made it possible to overcome the
lymphocyte proliferation inhibition in lymphocyte culture and thus
markedly increase the lymphocyte proliferation rate by adsorbing
lymphocyte proliferation inhibiting factors from a body fluid
derived from a subject with a disease against which a therapeutic
effect is produced by extracorporeally activating lymphocytes and
returning them into the body, for example a cancer subject with
poor lymphocyte proliferation, without adsorbing factors necessary
for lymphocyte proliferation from that body fluid. The invention is
useful in that it provides a porous material for relieving the
lymphocyte proliferation inhibition in lymphocyte culture using a
target body fluid in activated autologous lymphocyte therapy, for
instance, according to which a disease is treated or the progress
of a disease is inhibited by taking immunocompetent cells
(lymphocytes in particular) in blood out of the body, culturing
them for stimulation/activation and for proliferation and again
returning them into the body, as well as a method for proliferating
lymphocytes which utilizes the above-mentioned porous material.
* * * * *