CA1213841A - Hybrid cell line for producing complement-fixing monoclonal antibody to mouse i-a antigen and to i-a analogous antigens on human and other animal lymphocytes, antibody and methods - Google Patents
Hybrid cell line for producing complement-fixing monoclonal antibody to mouse i-a antigen and to i-a analogous antigens on human and other animal lymphocytes, antibody and methodsInfo
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Abstract
ABSTRACT OF THE DISCLOSURE
Hybrid cell line for production of monoclonal antibody to mouse I-A antigen and to I-A analogous antigen on human and other animal lymphocytes is described. The hybrid is formed by fusing P3X63-Ag8-653 myeloma cells with spleen cells from A.TH (H-2t2) mouse immunized with A.TL (H-2t1) mouse lymphocytes. The monoclonal antibody reacts with I-A analo-gous antigens in a wide range of animal species.
Hybrid cell line for production of monoclonal antibody to mouse I-A antigen and to I-A analogous antigen on human and other animal lymphocytes is described. The hybrid is formed by fusing P3X63-Ag8-653 myeloma cells with spleen cells from A.TH (H-2t2) mouse immunized with A.TL (H-2t1) mouse lymphocytes. The monoclonal antibody reacts with I-A analo-gous antigens in a wide range of animal species.
Description
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-:~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.
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 ~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 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 ~ 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.
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.
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-:~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.
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 ~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 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 ~ 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.
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 ~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 ~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.
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 ~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; and 0 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 , 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.
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 ~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 ~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.
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 ~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; and 0 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 , 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.
Claims (20)
1. A process of preparing a complement-fixing IgG
monoclonal antibody which:
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 shown 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 frac-tions, respectively, from 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 neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig+ B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,000 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method, which process comprises the steps of:
i) immunizing mice with spleen and thymus cells from congenic mice of different immune responsi-veness;
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) evaluating the supernatant in each well containing a hybridoma for the presence of the desired antibody;
vi) selecting and cloning hybridomas producing the desired antibody; and vii) recovering the antibody from the superna-tants of the above said clones.
monoclonal antibody which:
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 shown 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 frac-tions, respectively, from 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 neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig+ B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,000 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method, which process comprises the steps of:
i) immunizing mice with spleen and thymus cells from congenic mice of different immune responsi-veness;
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) evaluating the supernatant in each well containing a hybridoma for the presence of the desired antibody;
vi) selecting and cloning hybridomas producing the desired antibody; and vii) recovering the antibody from the superna-tants of the above said clones.
2. The process of claim 1, wherein the antibody produced by the hybridoma is of subclass IgG2b.
3. The process of claim 1, wherein said immunized mice are of strain A.TH (H-2t2), said congenic mice supplying spleen and thymus cells for said immunization are of strain A.TL (H-2t1) and said myeloma cells are P3X63-Ag8?653.
4. The process of claim 1, wherein the hybridoma has the identifying characteristics of hybridoma clone HAK-75.
5. A process of preparing a complement-fixing IgG
monoclonal antibody which:
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 shown 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 frac-tions, respectively, from peripheral lymphocytes from human adult male and fernale donors;
e) exhibits cytotoxicities, among human peripheral monocyte fractions, less than 3% to T cells obtained by neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,009 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method, which process comprises the steps of:
i) immunizing mice with spleen and thymus cells from congenic mice of different immune responsi-veness;
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) evaluating the supernatant in each well containing a hybridoma for the presence of the desired antibody;
vi) selecting and cloning hybridomas producing the desired antibody;
vii) transferring said clones intraperitoneally into mice; and viii) harvesting the malignant ascites or serum from said mice.
monoclonal antibody which:
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 shown 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 frac-tions, respectively, from peripheral lymphocytes from human adult male and fernale donors;
e) exhibits cytotoxicities, among human peripheral monocyte fractions, less than 3% to T cells obtained by neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,009 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method, which process comprises the steps of:
i) immunizing mice with spleen and thymus cells from congenic mice of different immune responsi-veness;
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) evaluating the supernatant in each well containing a hybridoma for the presence of the desired antibody;
vi) selecting and cloning hybridomas producing the desired antibody;
vii) transferring said clones intraperitoneally into mice; and viii) harvesting the malignant ascites or serum from said mice.
6. The process of claim 5, wherein the antibody produced by the hybridoma is of subclass IgG2b.
7. The process of claim 5, wherein said immunized mice are of strain A.TH ( H-2t2), said congenic mice supplying spleen and thymus cells for said immunization are of strain A.TL (H-2t1) and said myeloma cells are P3X63-Ag8?653.
8. The process of claim 5, wherein the hybridoma has the identifying characteristics of hybridoma clone HAK-75.
9. A complement-fixing monoclonal antibody of class IgG produced by a hybridoma formed by fusion of a mouse myeloma line and spleen cells from a mouse pre-viously 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 shown 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 frac-tions, respectively, from 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 neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig+ B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,000 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method, whenever prepared by a process according to claim 1 or its obvious chemical equivalents.
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 shown 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 frac-tions, respectively, from 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 neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig+ B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,000 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method, whenever prepared by a process according to claim 1 or its obvious chemical equivalents.
10. The monoclonal antibody of claim 9, which is of subclass IgG2b, whenever prepared by a process according to claim 2 or its obvious chemical equivalents.
11. The monoclonal antibody of claim 9, whenever prepared by a process according to claim 3 or its obvious chemical equivalents.
12. The monoclonal antidoby of claim 9, whenever prepared by a process according to claim 4 or its obvious chemical equivalents.
13. A complement-fixing monoclonal antibody of class IgG produced by a hybridoma formed by fusion of a mouse myeloma line and spleen cells from a mouse pre-viously 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 recomhinant mice as shown 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 frac-tions, respectively, from 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 neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig+ B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,000 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method, whenever prepared by a process according to claim 5 or its obvious chemical equivalents.
a) is specifically cytotoxic to H-2 haplotype mouse spleen cells;
b) exhibits cytotoxicity patterns to spleen cells from H-2 haplotype and recomhinant mice as shown 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 frac-tions, respectively, from 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 neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig+ B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,000 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method, whenever prepared by a process according to claim 5 or its obvious chemical equivalents.
14. The monoclonal antibody of claim 13, which is of subclass IgG2b, whenever prepared by a process according to claim 6 or its obvious chemical equivalents.
15. The monoclonal antibody of claim 13, whenever prepared by a process according to claim 7 or its obvious chemical equivalents.
16. The monoclonal antibody of claim 13, whenever prepared by a process according to claim 8 or its obvious chemical equivalents.
17. An IgG complement-fixing monoclonal-antibody-producing 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, which 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 shown 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 frac-tions, respectively, from 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 neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig+ B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,000 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method.
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 shown 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 frac-tions, respectively, from 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 neuraminidase-treated SRBC rosette method, about 75% to plastic-dish adherent cells (macrophages) and 50% to 60% to surface Ig+ B cells nonadherent to a plastic dish but adsorbed by a dish coated with pepsin-treated rabbit anti-human immunoglobulins;
f) reacts with 12% to 14% of T cells from one-way allogeneic mixed lymphocyte reaction, autologous mixed lymphocyte 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 mole-cular weight range 30,000 to 40,000 from cells of human B cell line EBV-Wa by indirect immunoprecipitation method.
18. The hybridoma of claim 17, wherein the antibody produced thereby is of subclass IgG2b.
19. The hybridoma of claim 17, which is formed by fusion of P3X63-Ag8?653 myeloma cells and spleen cells from A.TH (H-2t2) mouse immunized with A.TL
(H-2t1) mouse lymphocytes.
(H-2t1) mouse lymphocytes.
20. A hybridoma having the identifying charac-teristics of hybridoma clone HK-75
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP89804/1982 | 1982-05-28 | ||
| JP8980482A JPS58206532A (en) | 1982-05-28 | 1982-05-28 | Monoclonal antibody and its preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1213841A true CA1213841A (en) | 1986-11-12 |
Family
ID=13980899
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000429023A Expired CA1213841A (en) | 1982-05-28 | 1983-05-27 | Hybrid cell line for producing complement-fixing monoclonal antibody to mouse i-a antigen and to i-a analogous antigens on human and other animal lymphocytes, antibody and methods |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS58206532A (en) |
| CA (1) | CA1213841A (en) |
-
1982
- 1982-05-28 JP JP8980482A patent/JPS58206532A/en active Pending
-
1983
- 1983-05-27 CA CA000429023A patent/CA1213841A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58206532A (en) | 1983-12-01 |
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