Note: Descriptions are shown in the official language in which they were submitted.
Field of the Inventio~ ~
This invention relates generally to new hybrid cell
lines, and specifically to those hybrid cell llnes which
produce complement-fixlng monoclonal antibodies against
an antigen found in normal human and mouse B lymphoid
cells,macrophages, and activated T lymphoid cells, to the
antibodies so produced and to the antigen components re-
acting with these antibodies.
BACX~;ROI~ND OF THE INVENTION
Descrip'tion''o'f' th'e' Pri'or Art
. _ _
Kohler and Milstein showed that the fusion between
immunized mouse spleen cell and mouse myeloma cell gives
rise to cell lines that produce homogenous single-clone
(so~called monoclonal) antibodies [Nature 256, 495-~97
(1975)]. Since the publication of this basic technique
major efforts have been made to produce various hybrid
cell lines (called hybridomas) and to apply monoclonal
antibodies so produced to the research in basic and ap-
plied sciences. Reference should be made to some recentreports, for example, D. E. Yelton and M.D. Scharff. Annu.
Rev. Biochem. 50, 657-680 (1981) and E.D. Servier et al~
Clin. Chem. 27, 1797-1806 (1981) and to the literature
cited therein. These references indicate many advantages
of monoclonal antibodies produced by hybridomas as well
as technical complications involved. The general techni-
que is well understood conceptually but for specific
cases difficulties are encountered that require some al-
terations. In fact, there is no assurance, prior to the
attempt to prepare given hybridomas, whether or not the
desired hybridomas are obtained, those hybridomas if ob-
tained would produce antibodies, and antibodies so pro-
duced would possess desired specificities. The success
mainly depends on the types of antigens used, and the im-
munization methods and selection techniques to isolate
the desired hybridomas.
8~
The major histocompatibility complex (MHC) plays a
primary role in the interaction of mouse immune cells
Mouse MHC is present in chromosomal H-2 locus. Major cell-
cell interactions in immunological reactions are under the
regulation by genes located in the I-region of MHC. The
I-region is divided into subregions I-Ar I-B, I-I, I-E
and I-C that express Ia antiyens (I-region-associated anti-
gens). These Ia antigens play important roles in the regu-
lation of immune response. Mouse monoclonal antibody HAK-75
descr~ here (vide infra~ reacts with an immune-cell antigen
that controls the aforementioned interactions among immune
cells.
Human Ia antigens are called HLA-DR antigens (human
leukocyte an~igen-D-related antigens). The analogy among
mouse Ia and human HLA-DR antigens is supported by their
linkage with respective MHCs, similarity in tissue distri-
bution and immune reactions. Fur-ther chemical nature of the
antigen structure is quite similar. The antigen of both
origins consists of two subunits of molecular weight
33,000 - 35,000 (~ chain) and 27,000 - 29,000 (~ chain)
and con~ins the same amino acid sequences at the N-termini.
Human HL~-DR antigen molecule is currently considered
to be analogous to the gene product of mouse I-E subregion.
However, it became clear recently that like mouse I region
human HLA-DR region containsseveral genetic loci. Further
anal~sis of these loci is now in progress.
Attempts to produce monoclonal antibodies against human
HLA-DR antigens have been initiated only recently and few re-
ports are found in the literature. The present inventors
after several attempts, used two congenic mouse strains,A.TL
~H-2 1) and A.TH (H-2 ), which possess identical K- and D-
regions of the H-2 locus but differ in immune responsiveness
(namely, the I-region differs) and succeeded in enhancing the
~X~
antigen specificity by imnunizing mouse A.TH (H-2 2) `with
spleen and thymus cells of mouse A.TL (H~2 ) as antigens.
The spleen was dissected out o~ the mouse so immunized and
spleen cells were fused with mouse myeloma cells to produce
hybridomas. Supernatants of these hybridoma cultures re-
acted with human peripheral monocytes and human lymphoid B
cell strain (EBV-Wa) and analyzed by a double fluorescent
staining method for positive hybridoma clones.
In human and animal immune systems there exist two
principal classes uf lymphocytes. The first class of lym-
phocytes (the bone marrow-derived cells, or B cells) are
those which secrete antibodies. They develop from hemopoie-
tic stem cells, but their differentiation does not depend
on the thymus. In birds, they are differentiated in an or-
gan analogous to -the thymus, called the Bursa of Fabricius.
In mammals, however, no equivalent organ has yet been dis-
covered, and it is considered that B cells are differenti-
ated in the bone marrow.
The second class of lymphocytes (the thymus-derived
cells, or T cells) also develop from hemopoietic stem cells
in the thymus. These differentiating cells are called thy-
mocytes when they are within the thymus. Mature T cells
emerge from the thymus and circulate among tissues, lym-
phatics and blood streams. These T cells possess various
immunological specificities and are directly involved, as
effectors, in cell-mediated immune responses, such as in
graft rejection. Unlike B cells, T cells do not secrete
humoral antibodies.
T cells are divided into at least 3 subclasses called
"helper", "suppressor" and "killer" T cells. Helper T cells
stimulate antibody production in B cells and suppressor T
cells suppress antibody production in B cells, whereas killer
T cells kill (lyse) foreign cells. Agammaglobulinemia and
autoimmune diseases are related to the excess or shortage of
these T cells.
Incidentally, macrophages also develop from hemopoie-
tic stem cells, phagocytize foreign objec-ts and present anti-
--3--
:~2~
.
gens to lymphoc~tes. Generally, macrophages are involvedin non-specific immune responses.
~ he role of mouse I-region in the regulation of immune
response is well documented in immunobiochemical terms, but
with regard to human counterpart HLA-DR region, its subregions
and their expressed antigens and functions have been described
only recently.
Monoclonal antibody HAK-75 to which the present inven-
tion is related is extremely useful not only in further de-
tailed analysis of mouse I-region, differential fractionation
of B and T cells and macrophage analysis but also in the
analyses of human lymphocyte subclasses, human HLA-DR region,
Ia antigens of activated T cells and their immunobiological
significance. In addition, since human HLA-DR region is
related to autoimmune diseases such as Takayasu disease,
myasthenia gravis and juvenile diabetes mellitus, it is
quite likely that monoclonal antibodies against human HLA-DR
antigens can be used for diagnosis and for therapy of auto-
immune diseases. These monoclonal antibodies may also have a
positive effect on the success of tissue grafts.
The preparation of so-called conventional polyclonal
antibodies enco~nters difficulties in the process of absorp-
tion and purification to enhance their specificities. Ab-
sorbed and purified antisera contain, in many cases, contam-
inants other than the desired antibodies. These antisera
also contain antibodies against all antigenic determinants
of the same antigen. Thus the concentration of a speci~ic
antibody against each antigen determinant is so low that
it is difficult to use polyclonal antibodies for the analysis
of human HLA-DR antigens which exhlbit high cross-reactivity.
In ~ybridoma cultures, monoclonal antibodies of high specifi-
city and purity can be produced reproductively in a large
quantity. The reference by E.D. Servier et al~ LClin. Chem.
27, 1797-1806 (1981)] further describes disadvantages of the
use of polyclonal antibodies produced by conventional meth-
ods and the advantages of monoclonal antibodies.
~2~
Si~M~lAR~ OF TH~ IN~JENTION
.,
In accordance with the present invention there has
now been discovered a novel hybridoma (designated clone HAX-
75) which is capable of producing a monoclonal antibody
characterized by a) specific cytotoxicity to H-2 haplotype
mouse spleen cells, b) specific reaction with an Ia antigen
expressed in mouse B cells, namely, the gene product of the
I-A subregion, c) specific reaction with an antigen that
is nonpolymorphic and expressed in human B cells, macroph-
ages and activated T cells but not in non-specific cells
and d) killing less than 5% of T cells. Monoclonal anti-
body HAK-75 fixes complement.
The monoclonal an~ibody thus produced exhibits dif-
ferent cross-reactivity from that of previous monoclonal
antibodies reacting with the gene product of mouse I-E sub-
region as well as with human HLA-DR antigens. The antibody
of the present invention is specific to the gene product of
the I-A subregion of II-2 haplotype in the mouse, an in hum-
ans, to the analogous antigen expressed in B cells, macro-
phages and activated T cells, and does not contain other im-
munoglobulins.
Accordingly, one of the purposes of the present in-
vention is to offer hybridomas producing antibodies against
the antigen which is expressed in lymphoid B cells, macro-
phages and activated T cells~ and corresponds to the gene
product of mouse I-A subregion~
Another purpose of the present invention is to offer
a method for the preparation of these hybridomas.
Still another purpose of the present invention is
to offer essentially homogeneous antibodies against the an-
tigen which is expressed in lymphoid B cells, macrophages
and activated T cells, and corresponds to the gene product
of mouse I-A subregion~
Other purposes and advantages of the present inven-
tion will be clarified in the following description.
For the aforementioned purposes and advantages, there
have been offered, in the present invention, novel hybri-
domas and novel antibodies produced by these hybridomas
~2~
,
specifically against the antigen ~hich is expressed in lym-
phoid B cells, macrophages and activated T cells, and cor-
responds to the gene product of mouse I-A subregion. The
said hybridomas themselves are produced by the general
method of Kohler and Milstein. Spleen and thymus cells of
mouse A.TL (H-2 ) immunized congenic mouse A.TH (H-2t2)
of different immune responsiveness. Spleen cells of the
immunized mouse were fused with cells of mouse myeloma line
and hybridomas thus produced were cul-tured. Culture super-
natants reacted with human peripheral monocytes and humanlymphoid B cell strain (EBV-Wa) and were analyzed by a double
fluorescent staining method. Reaction-positive hybrid cells
were further purified by a limiting dilution method for the
final establishment and characterization of the strains pro-
ducing monoclonal antibodies. Consequently, there has been
obtained hybridoma clone HAK-75 which produces antibody HAK-
75 a~ainst the antigen corresponding to the gene product of
mouse I-A subre~ion. This antibody reacts with lymphoid B
cells, macrophages and activated T cells, but not with T
cells and non-specific cells. The analysis by the double
fluorescent staining method further revealed that this anti-
body HAK-75 reacts with spleen cells from human, monkey, goat,
dog, rabbit, guinea pig, rat, and mouse but not with
thymocytes from these animal species. This positive reaction
with so wide a range of animal species is surprising and
suggests some of the merits of monoclonal antibody HAK-75.-
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is the graph to show complement--dependent
cytotoxicity patterns of clone HAK-75 culture supernatant
to spleen cells from various H-2 haplotype and H-2 recombi-
nant mice.
Figure 2 is the graph to show the time course of com-
plement-dependent cytotoxicities of clone HAK-75 culture
supernatant to human T cell fibroblasts (activated T cells)
cultured in the one-way mixed lymphocyte reaction. For the
negative control, immunoglobulins were used.
Figure 3 shows sequential precipitation analysis of
human EB virus-transformed B cell strain (EBV-Wa). 125I-
labeled soluble cell antigen from human B cell strain EBV-Wa
was first incubated with the sera indicated on the right
side of the panels. The resulting il~muno-complexes were
adsorbed to SEPHAROSE ~B (trademark, Pllarmacia AB, Uppsala,
Sweden) beads conju~ated with a rabbit anti-mouse immuno-
globulin and removed by centrifugation. The remaining super-
natants were divided and the aliquots were subjected to the
second precipitation with the sera indicated on ~he top of
the figure. The precipitates were dissolved, reduced, and
analyzed in SDS-12.5% polyacrylamide gel electrophoresis.
The gels were sliced into 1 mm sections and counted for
radioactivity.
DETAILED DESCRIPTION OF THE INVENTION
The procedure for the production of hybridomas in
the present invention followed the general steps as de-
scribed below.
A. Two congenic mice with the same H-2 K and D regions
but with different immune responsiveness, namely, mouse
A.TL (H-2 ) and mouse A.TH (~- ) were used. Mouse A.TH
(H-2t2) was immunized with spleen and thymus cells of mouse
A~TL (H-2 ). The immunization schedule and concentration of
spleen and thymus cells were so chosen as to produce suffi-
cient number of appropriately immunized spleen cells. In
the first immunization, mixed cells 2 x 107 from mouse A.TL
(H-2tl) spleen and thymus were injected intraperitoneally
with 1 x 1~ Bordetella pert ssis organisms (pertussis vac~
cine). Subsequent immunization was carried out every 7 days
for 13 times with similar injection of the mixed cells 2 x 107
per mouse without pertussis vaccine. The above immunization
schedule proved to be effective.
7-
B. The spleen was taken ou-t of t~e immunized mouse'and sus-
pended in an appropriate solution. One ml of solution is
usually enough for the spleen cell suspension from one mouse.
These experimental techniques are well known among investi-
gators.
C. Suspended spleen cells were fused with cells of an ap-
propriate mouse myeloma line in the presence of a suitable
fusion-promoting agent. The preferable ratio of spleen and
myeloma cells was approximately 5 to 1. About 108 spleen
cells were suspended in 0.5 to 1.0 ml of the fusion solution.
Many mouse myeloma cell lines are known and usually obtained
from various institutions such as The Salk Institute Cell
Distribution Center, ~a ~olla, California. Myeloma cells
to be used should preferably be of a "drug-resistant" type
and a culture medium was so chosen as to allow the growth of
hyhrid cells whereas myeloma cells did not grow. Myeloma
cells most commonly used are 8-azaguanine-resistant, which
are devoid of an enzyme, hypoxanthine-guanine phosphoribosyl
transferase, and cannot grow in the HAT (hypoxanthine, aminop-
terin, and thymidine) medium. The myeloma cellline to heused should preferably of a "non-secreting type" where anti-
bodies are not secreted from the cel~ but a secreting type
may also be used. A suitable fusion-promoting agent is poly-
ethylene glycol of the average molecular weight 1,000 to
4,000 ~purchased with such brand names as PEG 100~) but other
fusion-promoting agents known in this field may be used alone
or in various combinations.
D. The mixture of unfused spleen cells, unfused myeloma cells
and fused cells was diluted in a selectivemedium, partitioned
into wells of a 96-well plate to have statistically 1 to 4
cells per well in this limiting dilution and grown for about
a week until the unfused cells were dead. The medium to be
used (for instance HAT medium as used here) should not sup-
port the growth of drug-resistant (for instance 8-azaguanine-
resistant) and unfused myeloma cells. Unfused myeloma cells
usually die in the medium. Unfused spleen cells do not grow
in the medium either since they are not malignant. Fused cells
however, possess both malignancy of the parent myeloma cell
and properties of the parent spleen cell so as to grow in the
_~_
selecti~e medium.
E. Supernatant fluids were obtained from the wells contain-
ing hybridomas, reacted with human peripheral monocytes and
human B cell strain (EBV-Wa~ and analyzed by a double fluore-
scent staining method for antibody production.
F. Reaction-positive hybridomas producing the desired anti-
bodies were selected and further cloned (for instance by
limiting dilution method).
Once the desired hybridomas were selected and cloned,
the desired antibodies could be produced by one of two methods.
For the production of monoclonal antibodies of High purity, the
desired hybridomas were grown in vitro in an appropriate med-
ium and the antibodies were recovered in culture supernatants.
The appropriate medium and growth time could be determined
easily. This ln vitro technique offers specific monoclonal
antibodies essentially without inclusion of other specific
immunoglobulins. Since the medium usually contains exogene-
ously added serum tfor instance fetal calf serum), it is pos-
sible that trace amounts of other immunoglobulins are present
in the monoclonal antibody preparation. This in vitro method
gave rise to monoclonal antibodies at about 50 ~g/ml of cul-
ture supernatants.
For the production of monoclonal antibodies of a very
high concentration, the desired hybridomas were injected
into mice. ~fter a certain period, -tumors arose which pro-
duced antibodies in tHe abdominal ascitic fluid and blood
streams of the host mice at about 5 to 20 mg per ml. The
host mice also possess normal antibodies in the blood streams
and abdominal ascitic fluid but concentrations of these nor-
mal antibodies are only about 5% of the concentration of mono-
clonal antibodies. The titers of monoclonal antibodies thus
produced were higher (activity remaining up to more than
1:50,000 dilution) and the ratio of specific to non-specific
immunoglobulins was high (about 1/20). Therefore in many
cases these monoclonal antibodies could simply be diluted for
use without loss of specificity.
EX~MPLE I ,
.. .. . .... .... ... .... .... . .
Produc't'ioh o'f' mon'oc'lbn'al''antibody
A) Immuniz'at'ion and som'atic'cell fusion
Mouse A.TH (H-2 ~) (8 week-old) was immunized intra-
peritoneally with mixed cells 2 x 10 of mouse A.TL (H-2 1)
spleen and thymus together with pertussis vaccine 1 x 109.
Additional immunizations were carried out every 7 days for
13 times under the same conditior~ but without pertussis vac-
cine. On the fourth day after the last immunization~ the
spleen was dissected out of the immunized mouse and dispersed
into single cell suspension by being passed through stain-
less steel nets.
Cell fusion was performed by Kohler and Milstein meth-
od. Spleen cells 1 x 108 in 1.O ml of RPMI 1640 medium con-
taining 35% polyethylene glycol [PEG 2000] and 5% dimethyl-
sulfoxide were fused with 2 x 10' myeloma P3X63-Ag8.653 cells.
These myeloma cells do not secrete immunoglobulin light chain.
B) Select'ion' an'd' growth of hyhridomas
Fused cells were cultured in HAT medium at 37C in 5%
CO2. After a few weeks of growth, supernatants of hybridoma-
containing cultures reacted with human peripheral monocytes
and human B cells strain (EBV-Wa) and were analyzed by double
fluorescent staining method. Reaction-positive fused cells
were grown in RPMI 1640 medium supplemented with 15% fetal
calf serum in the presence of feeder cells and cloned twice
by a limiting dilution method to finally obtain a monoclonal
antibody-producing cell strain, clone HAK-75. In the limiting
dilution the medium was used both with and without HAT.
Complement-dependent cytotoxicity to human peripheral
monocytes and Epstein-Barr virus (EBV)-transformed human B
cell strain (EBV-Wa) was assayed for the antibody activity
of hybridomas. Test cells, 20 ~1 of 2 x 106 cells/ml and 20
~1 of hybridoma culture supernatant were incubated at 37C
for 15 min. and washed once with MEM medium (minimal essential
--10--
~3~
medium) containing 2% fetal calf serum. For complèment
supply, Z-fold diluted rabbit serum, 20 ~1, was added to
the cells and incubated at 37C for 90 min (in other experi-
ments where mouse test cèlls were used, rabbit serum was di-
]~cd 10- to 12-fold for use and the incubation time was 30
min). After complement reaction, the cells were washed once
with the same and cold MEM medium as above, stained with
trypan blue ana observed under a microscope for the number
of dead cells. Cytotoxicity (%) was calculated by the fol-
lowing equationO Normal mouse serum was used as the negativecontrol.
Cytotoxicity (%)
= 100 x [cytotoxicity % - control cytotoxicity %]
~100~ - control cytotoxicity %]
Clone HAK-75, 1 x 10 cells (in 0.2 ml), was injected
intraperitoneally into mouse BALB/c pre-treated with 2,6,10,
14-tetramethylpentadecane (purchased as the trade name
Pristine from Aldrich Chemical Company). Ascitic ~luid
from the mouse thus treated gave similar results to those ob-
tained for culture supernatants of clone HAK-75 in its char-
acterization as described below. Monoclonal antibody HAK-75
was shown/ by the standard technique, to be IgG2b subclass.
EXAMPLE II
Characterization of monoclonal antibody HAK-75
A) Cytotoxicit~ to mouse spleen cells
The spleens were taken out of H 2 haplotype k, b,d, f,
r and s mice, dispersed into single cell suspensions by the
usual method, reacted with culture supernatants of clone
HAK-75 and tested, as in the Example-I, for complement-dep-
endent cytotoxicity. It was shown from the test results
that culture supernatants of clone HAK-75 possessed specific
--11--
8~
cytotoxicity to spleen cells from the mouse where H 2 locus
is of haplotype k (H~2 ). H-2 recombinant mice were also
used, in similar cytotoxicity tests r to examine specificity
toward the I-region. In Table 1 are shown the results of
cytotoxicity tests of clone HAK-75 culture supernatants to
spleen cells from various H-2 haplotyoe and H-2 recombinant
mice.
Tahle 1 Murine strain distribution of the determinant
recognized by HAK-75 using mice with different
` H-2 haplotypes and recombinant mice
.
Mouse H-2 HaplotypesMaximal
Strains Subregionscytotoxicity
K I-A I-B I-J I-E I-C D ( )
.
A.TL .s k k k k k d 45
A.TH s s s s s s d c5
C3H/HeN k k k k k k k 47
BlO.A k k k k k d d 51
BlO.A(4R) k k b b b b b 53
20 BlO.A(5R) b b b k k b b 9
C57BL/6 b b b b b b b <5
BlO.S s s s s s s s <5
BlO.S(9R) s s . k k d d 6
BALB/c d d d d d d d 11
BlO.RIII r r r r r r r <5
BlO.M f f f f f f f 8
In additicn, cytotoxicity patterns of clone HAK-75 culture
supernatant in its various dilutions are shown in Fig. 1.
As it is clear from the above Table 1, clone HAK-75
culture supernatant exhibited maximal cytotoxicities 51% and
53%, to the spleen cells from H-2 recombinant mice BlO.A and
BlO.A (4R), respectively, but less than 5%, less than 5~ and
6% o~ cytotoxicities, respectively, to the spleen cells from
-12-
~ z~
similar recomhinant mice C57BL/6, BlO.S, and BlO.S`(9R).
Thus the supernatant was not cytotoxic to the spleen cells
from the last 3 recombinan~ mice. It was concluded that
among subregions I-A, I-B, I-J, I-E and I-C, monoclonal anti-
body HAK-75 specifically recognized an antigen expressed by
the gene in I-A subregion.
In order to next examine the expression of the anti-
gen in the subclass of lymphocytes, the spleen cells of
H-2k haplotype mouse C3H/HeN (Table 1) were separated, by
means of a nylon-wool column, into a free-passing T cell
fraction and a column-adsorbing B cell fraction. The B cell
fraction obtained by this method contained about 50~ of B
cells. Antibody HAK-75 was then tested for its cytotoxicity
to these subclass cells and shown to be an antibody against
the Ia (I-A subregion) antigen mainly expressed in B cells.
Antibody HAK-75 did not exhibit clear reactivity with T cells
(nylon wool colu~n-passing fraction) from the spleen cells
of mouse C3H/HeN (Table 1).
B) Cytotoxicity to *he class of human lymphocytes
.
Heparin-added human peripheral blood was obtained from
healthy voluntary donors (50, male and female, Japanese,
Asians and Caucasians) for the isolation of lymphocytes by
Ficoll-Hypa~ue density gradient centrifugation 2S described
by Boyum [Scand. J. Clin. Lab. Invest. 21 (suppl. 97~, 77
(1969)]. Lymphocytes were separated into B cell- and T cell-
containing fractions by a nylon-wool column. Alternatively,
plastic dish (Falcon)-adhering cells were isolated from
unfractionated lymphocytes. Plastic dish-nonadhering cells
were then subjected to E rosette formation in the presence
of 5% neuraminiaase-treated sheep red blood cells (SRBC). E
rosette cells were layered onto Ficoll-Hypaque solution and
centrifuged to recover E pellets. E pellets were treated
with 0.155 M NH4Cl (10 ml/10 cells) to obtain T cells. Fur-
ther plastic dish-nonadhering cells were adsorbed with a dish
coated with pepsin-treated rabbit and anti-human immunoglobu-
lins [anti-F(ab )2~ to isolate adsorbed surface Ig (B~ cells.
-13-
Th~ reaction o~ the culture supernatant of Glone HAK-75
with these purified classes of lymphocytes is presented in
the Table 2 together with that of other test cells.
.
Table 2 Distribution of monoclonal antibody HAK-75
reactive determinant on cells fractionated
from human peripheral blood lymphocytes
.
. _ .
Cells Tested Percent cytotoxicity
_ . . . .
Nylon-wool adherent ) 30 - 70
Nylon-wool nonadherentl) <5
10 Plastic dish adherent 75
Purified B ) 50 - 60
Purified T3) <5
.
1) 50 individuals were tested.
2) Cells were recovered from a plastic dish coated with
rabbit anti-human immunoglobulins ~F(ab')2j.
3) S~BC rosette-forming T cells.
As shown in Table 2, B cell- and T cell-fractions obtained by
a nylon-wool column exhibited 30 - 70% and less than 5~ cyto-
toxicities (or distribution of monoclonal antibody HAK-75
reactive determinant), respectively. For purified classes
of lymphocytes, T cells obtained by rosette method exhibited
less than 3% cytotoxicity whereas plastic dish-adhering cells
~macrophages) and surface Ig B cells exhibited 75% and
50 - 60~ cytotoxicities, respectively.
It was further examined whether or not the Ia antigen
expressed in human T cells reacts with the culture superna-
tant of clone HAK-75. Human fibroblast T cells (activated T
cells) were obtained either by mixed culture of human T
cells and mitomycin C-treated lymphocytes from another indi-
vidual (one-way allogeneic MLR); by mixed culture of human
T cells and mitomycin C-treated B cells/macrophages from
the same individual for 6 days (autologous MLR), or by cul
ture of human T cells with concanavalin A for 6 days~ The
mi~xed culture contained a total of 1 x 106 cells and the
. ., --1~--
`` ~LZ~3~
medium used was RPMI 1640 containing 10% fetal caI~ serum
and 20 mM HEPES Buffer (pH 7.2). Reactivity between acti-
vated T cells and antibody HAK-75 was examined by a double
fluorescent staining method. In the mixed lymphocyte reac-
tion (MLR), a total of 1 x 10 cells reacted with 60 ~1 of
the culture supernatant of clone HAK-75 for 30 min at 4C
and were washed 3 times with MEM medium containing 2~ fetal
calf serum. The washed cells again reacted with 10 ~1 of
FITC-labeled rabbit anti-mouse immunoglobulins for 20 min
at 4C, were washed 3 times with the above MEM medium and
observ~d unaer a microscope. 20 - 40% of activated T cells
were reaction-positi~e. In one-way alloyeneic MLR cell cul-
ture was examined at intervals for reactivity with clone
HAK-75 antibody. Reaction-positive cells increased to a
peak, or 27 - 42~ of whole cells, in 6 days (Fig. 2).
From the results described above, it was concluded
that antibody ~K-75 recognized a non-polymorphic antigen
expressed mainly in human B cells, macrophages and activated
T cells.
EXA~PLE III
Analysis of soluble cell antigen reactive with monoclonal
. _ _ _ . .
antibody HAK-75
Human B cell strain EBV-Wa was grown in RPMI 1640
medium containing 10% fetal calf serum. Cells (1 x 108)
were washed with physiological phosphate buffer-saline (PBS)
solubili~ed with 0.5% Nonidet P-40 (NP-40) for 15 min at
4C and centrïfuged at 4,000 x g for 1 hr. The supernatant
fluid was applied onto a lentil-lectin column and eluted with
0.1 M mannose. The mannose eluate contained purified and
concentrated Ia antigen. This fraction was dialyzed against
PBP containing 0.1% NP-40 and concentrated further in vacuo
with Amicon~ membrane to 100 ~1. The soluble cell antigen was
labeled with 125I by chloramine T method, gel-filtered
through Sepnade~ G 25 and reacted with the culture supernatant
-15-
~LZ~3~
o clone HAK-75. Radioactive immuno-complexes thus;formed
were precipitated with rabbit anti-mouse immunoglo~ulins
by indirect immunoprecipitation method. These precipitates
were dissolved and electrophoresed in sodium dodecylsulfate
(SDS)-12.5~ polyacrylamide gel electrophoresis. The gel
was cut and counted for radioactivity. As shown in Fig. 3
two peaks were detected at -the molecular weight range of
30,000 (30K) and 35,000 (35 K). These peaks apparently cor-
respond to Aa and A~ chains of the gene product of mouse
I-A subregion. Similar results were also obtained when hu-
man B cells (EBV-Wa) were labeled with 125I by lactose per-
oxidase method, solubilized and analyzed as above.
Alternatively, human B cell strain EBV-Wa, 5 x 10
cells, were labeled with 0.5 mCi of 35S-methionine ~or 76
hrs. Labeled cell antigen solubilized as described above
was precipitated with antibody HAK-75 by an indirect immuno-
precipitation method and analyzed similarly. Two similar
peaks were also detected. Incidentally, when normal mouse
serum (NMS) was used in the above experiment, the said peaks
were not detected ~Fig. 3~.
Immunological and biochemical analysis up to the moment
indicates that human HLA-DR antigen molecule corresponds
to the gene product of mouse I-E subregion, and especially
that the antibody against mouse Ia.7 cross-reacts with human
HLA-DR antigen. In fact, anti-I-EkCkSk antiserum obtained by
immunizing mouse [B10 . S (7R) x A.CA] with spleen and thymus
cells from mouse BlO.HTT reacted with mouse Ia.7, and
cross-reacted with human HLA-DR antigen which corresponds
to the gene product of mouse I-E subregion. Ten ~1 of 1 5I-
labeled soluble cell antigen from human B cells (EBV-Wa)
reacted with 70 ~1 of clone HAK-75 culture supernatant for
30 min at 4C and immunoprecipitates were removed by Sepha-
rose beads coated with rabbit anti-mouse immunoglobulins.
To the remaining antibody HAK-75 non-reactive mat~rial was
added anti-I-EkCkSk antiserum and immuno-complexes thus formed
were precipitated with rabbit anti-mouse immunoglobulins. As
the re~erence, normal mouse serum or the culture supernatant
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~2~3~
of clone HAK-75 was added to antibody HAK-75 non-réactive
material. These immunoprecipitates were dissolved, electro-
phoresed in SDS-12.5% polyacrylamide gel and counted for
radioactivity. Two peaks of the molecular weight range of
29,000 ~29 K) and 34,000 (34 K) were detected (Fig. 3).
Even when 125I-labeled cell antigen reacted with anti-I-Ek
CkSk antiserum before the treatment with antibody HAK-75,
immunoprecipitates gave rise to similar two peaks (Fig. 3).
Thus monoclonal antibody HAX-75 recognizes an antigen
that differs from human HLA-DR antigen corresponding to
the gene product of mouse I-E subregion. This antigen cor-
responds rather to the gene product oE mouse I-A subregion
and is expressed selectively in specific classes of human
lymphocytes.
For the convenience of explanation, reference ~7as
made only to a single hybridoma producing a single monoclonal
antibody against the antigen expressed in the lymphocyte
class. However, the present invention is not limited to
that exten-t. Antibody HAK-75 of the present invention was
determined to belong to IgG2b subclass' of 4 mouse IgG sub-
classes. These subclasses of immunoglobulin G differ inso-called "constant" regions but antibodies against a spec-
ific antigen (determinant) possess so-called "variablel' re-
gions which are the same regardless of which subclass of im-
munoglobulin G the antibodies belong to. Accordingly, mono-
clonal antibodies with such specificities as described here
may be of subclass IgGl, IgG2a, IgG2b, o g 3
IgM or IgA, or o-E even other Ig class~ The difference'among
these classes or subclasses should not affect the selectiv-
ity of reaction patterns of antibodies butthe reactivity ofthese antibodies with othersubstances such as complement or
anti-mouse antibody may be influenced. The antibody de-
scribed above is confined to the one of class IgG2b. How-
ever, antibodies of the reaction pattern in the above example
can be included in'the prese~ invention regardless of immuno-
globulin class or subclass they belong to.
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Furthermore, the method to produce the aforementioned
monoclonal antibody by the hybridoma technique, as it is
described here, is an object ofthe present invention. Hy-
bridomas producing antibodies of the characteristic reac~iv-
ity as described above may be obtained by the immunization,
fusion and selection procedures presented clearly here in
detail. Individual hybridomas derived from known myeloma
cell lines of known mouse species cannot be identified ex-
cept by referring to the antibodies produced. All hybri-
domas producing antibodies of the reaction specificities
as described above as well as the production method of anti-
bodies by these hybridomas can also be included in the present
invention.
Summarizing, the present invention, in one o~ its em-
bodiments, provides for a complement-fixing monoclonal anti-
body of class IgG produced by a hybridoma formed by fusion of
a mouse myeloma line and spleen cells from a mouse previously
immunized with spleen and thymus cells from a congenic mouse
of different immune responsiveness, wherein said antibody:
a) is specifically cytotoxic to H-2 haplotype mouse spleen
cells;
b) exhibits cytotoxicity patterns to spleen cells from H-2
haplotype and recombinant mice as show~ in Table 1 and
Figure 1 and recognizes the antigen expressed by the gene
in mouse I-A subregion;
c) reacts with the Ia antigen expressed in mouse lymphoid
B cells, but not with mouse lymphoid T cells;
d) exhibits complement-dependent cytotoxicities 30% to 70%
and less than 5~ to B cell and T cell fractions, respect-
ively, for peripheral lymphocytes from human adult male
and female donors;
e) exhibits cytotoxicities, among human peripheral monocyte
fractions, less than 3% to T cells obtained by neuramini-
dase-treated SRB~ rosette method, about 75% to plastic-
dish adherent cells ~macrophages) and 50% to 60% to sur-
face Ig B cells nonadherent to a plastic dish but adsorbed
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~Z~4~
by a dish coated with pepsin-treated rabbit anti-human
immunoglobulins;
f~ reacts with 12% to 14% of T cells from one-way allogen-
, .
eic mixed lymphocyte reaction, autologous mixed lympho-
cyte reaction and concanavalin A culture, and exhibits
time-course reaction patterns with cells from one-way
allogeneic mixed lymphocyte reaction;
g) reacts with spleen cells, but not with thymus cells, from
human, monkey, goat, dog, rabbit, guinea pig, rat and
mouse; and
h) gives two immunoprecipitates of the molecular weight
range 30,000 to 40,000 from cells of human B cell line
EBV-Wa by indirect immunoprecipitation method.
The invention also provides a monoclonal antibody as
described above which is of subclass IgG2b.
The present invention also provides a monoclonal anti-
body as described above which is produced from a hybridoma
formed by the fusion of P3X63-Ag 8-653 myeloma cells and
spleen cells from A.TH (H~2t ) mouse immunized with A.TL
(H-2 ) mouse lymphocytes.
The invention provides a monoclonal antibody which is
produced from a hybridoma having the identifying character-
istics of hybridoma clone HAK-75.
More specificall~, the present invention provides for
an IgG complement-fixlng monoclonal-antibody-producing hybri-
doma formed by fusion of a mouse myeloma line and spleen
cells from a mouse previously immunized with spleen and thy-
mus cells from a congenic mouse of different immune respon-
siveness, wherein said anti~ody has the properties indicated
in a) to h) shown above.
The present invention also provides an IgG complement-
fixing monoclonal-antibody producing hybridoma wherein the
antibody produced thereby is of subclass IgG2b.
This hybridoma is formed by fusion of P3X63-Ag~ 653
myeloma cells and spleen cells from A.TH (H-2 ) mouse im-
munized with A.TL (H-2 ) mouse lymphocytes.
--1~--
3~
The invention also provides a hybridoma havAing the
identifying characteristics of hybridoma clone HAK-75.
The present invention also proviaes a method for pre-
paring complement-fixing IgG monoclonal antibody,whereIn said
antibody has the properties of a) to h) as described above,
and which comprises the steps of:
i) immunizing mice with spleen and thymus cells from con-
genic mice of different immune responsiveness;
ii) removing the spleens from said immunized mice and making
a suspension of spleen cells;
iii) fusing said spleen cells with mouse myeloma cells in
the presence of a fusion promoter;
iv) diluting and culturing the fused cells in separate wells
in a medium which will not support the unfused myeloma
cells;
v) evaluatiny the supernatant in each well containi.ng a
hybridoma for the presence of the desired antibody;
vi) selecting and cloning hybridomas producing the desired
antibody; and0 vii) recovering the antibody from the supernatants of abovesaid clones.
More specifically, the method is used wherein said
immunized mice are of strain A.TH (H-2 ), said congenic mice
supplying spleen and thymus cells for said immunization are
of strain A.TL tH-2 ) and said myeloma cells are P3X63-Ag~-653.
The present invention also applies to a method for pre-
paring complement-fixing IgG monoclonal antibody, which anti-
body has t'ne properties recited in a~to h) above, and com-
prises the steps of: -
i) immunizing mice with spleen and thymus cells from
congenic mice of different immune responsiveness;
ii) removing the spleens from said immunized mice and
ma~ing a suspension of spleen cells;
iii) fusing said spleen cells with mouse myeloma cells
in the presence of a fusion promoter;
iv) diluting and culturing the fused cells in separate
,
-20-
wells in a medium which will not support the un-
fused myeloma cells;
v) evaluating the supernatant in each well containing
a hybridoma for the presence of the desired antibody;
vi) selecting and cloning hybridomas producing the de-
sired antibody;
vii) transferring said clones intraperitoneally into mice;
and
viii) harvesting the malignant ascites or serum from said
mice.
More specifically this method is also used wherein
said immunized mice are strain A.TH (H-2 ), said congenic mice
supplying spleen and thymus cells for said immunization are
of strain A.TL (H-2 ) and said myeloma cells are P3X63-Ag8.653.
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