Monoclonal antibodies specific for HIV and hybridomas for their production

Human IgG1 monoclonal antibodies are produced by hybridoma ATCC HB10074 and bind to gp120 of HIV.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to human monoclonal antibodies (abbreviated 
as MCAs hereinafter) specific for the human immunodeficiency virus, and 
the hybridomas which produce said MCAs. The objective of this invention is 
to provide human MCAs which are specific for HIV and which will be useful 
in the diagnosis, prevention and therapy of HIV infection. 
2. Discussion of the Background 
HIV is a virus which primarily infects helper T lymphocytes and brings 
about extreme immunological failure by destroying those cells, thereby 
causing AIDS (acquired immunodeficiency syndrome). In the early stage of 
HIV infection, some patients develop symptoms which resemble those of 
infectious mononucleosis, i.e., fever, fatigue, headache, etc. 
Subsequently, although the patient becomes asymptomatic, he/she becomes a 
carrier of anti-HIV antibodies in the blood. Then, after a latent period 
lasting a number of years, the patient develops AIDS-related complex 
(ARC). ARC patients exhibit various symptoms such as systemic swelling of 
lymph nodes, fever, general fatigue, weight loss, decreased platelet and 
lymphocyte levels, etc. As the disease progresses, the patient becomes 
susceptible to and develops Kaposi's sarcoma and various opportunistic 
infections such as Pneumocystis-carinii pneumonia, fungal infections, 
cytomegalovirusinfection, etc., which end in death. The most striking 
characteristics of AIDS are the decrease in helper T lymphocytes (T4), and 
a steady decrease in the ratio of T4 to suppressor T lymphocytes (T8), 
i.e., T4/T8, as the disease progresses. 
AIDS was first reported in the United States of America in 1981, and it has 
been estimated that today there are more than 20,000 AIDS patients in the 
USA alone. Carriers of the virus have been estimated to number one million 
persons in the USA. In addition to the USA, there are also many AIDS 
victims in Africa and Europe, and there is a huge amount of research being 
carried out today on methods for the diagnosis, prevention and treatment 
of AIDS. 
HIV, the causative agent of AIDS, is a retrovirus. This virus has been 
shown to be composed of RNA consisting of about 9,700 base pairs, three 
gag proteins (having molecular weights of 55,000, 24,000 and 
17,000daltons), a reverse transcriptase (molecular weights of 66,000 and 
51,000 daltons have been detected), three glycoproteins (two molecules 
having molecular weights of 120,000 and 41,000 daltons, and their 
precursor, a molecule with a molecular weight of 160,000 daltons; these 
glycoproteins are hereinafter abbreviated as gp120, gp41 and gp160) which 
comprise the envelope, and other components. Especially from the 
viewpoints of viral infection and the prevention thereof, the envelope, 
which is exposed as the surface of. HIV, carries particular importance. As 
a result of proteolysis, gp160 is cleaved into gp120 and gp41. Gp41 is a 
transmembrane protein which is incorporated into the lipid bilayer of the 
viral envelope, while gp120 is exposed on the outside of the envelope and 
some of it is released from the virus. Both gp41 and gp120 possess many 
sugar-binding sites, and about half of the gp120 molecule is comprised of 
sugars. The gp120 molecule binds to, or near to, the CD4 antigens which 
exist on the cell surface of helper T cells, etc., and in addition to 
bringing about infection of the cells by the virus, gp120 possesses 
activity which results in the syncytium formation in the cells. 
For example, M. Robert-Guroff et al. (J. Immunol. 138: 3731, 1987) reported 
that the progression of the disease was slower in patients whose blood 
contained viral-neutratizing antibodies in comparison with patients not 
having such antibodies. In addition, it has been reported that the 
neutralizing antibodies in the blood of AIDS patients bind to gp120 (L.A. 
Lasky et al.: Science 233: 209, 1986; and T. J. Matthew et al.: Proc. 
Natl. Acad. Sci. USA 83: 9709, 1986). 
Even more important there are reports of passive immunotherapy with high 
titer anti p-24 plasma in patients with HIV infection. This has cleared 
antigenemia and improved clinical prognosis. (A. Karpas et al.: Proc. 
Natl. Sci. USA 85: 9234, 1988; and G. G. Jackson: Lancet, 2, 647, 1988). 
In light of the above background information regarding HIV and AIDS, it is 
obvious that neutralizing antibodies specific for viral antigens exposed 
on the surface of the virus or infected cells have great significance in 
the prevention and/or treatment of said infection. 
A number of research groups have already reported successful development of 
mouse MCA specific for gp120. For example, T.C. Chanh et al. (Eur. J. 
Immunol. 16: 1465, 1986) reported that they chemically synthesized a 
portion of the peptide chain of gp120 and then prepared an MCA specific 
for that synthetic peptide. They employed that MCA in the indirect 
fluorescent antibody technique and reported that they were able to detect 
HIV infection with greater sensitivity than was possible with the reverse 
transcriptase determination technique. In addition, Gosting et al. (J. 
Clin. Microbiol.: 25, 845, 1987) reported that they solubilized HIV viral 
antigens, adsorbed them to a column of lentil lectin-Sepharose 4B, 
collected the glycoprotein fraction thereof and used it to immunize mice, 
and succeeded in producing anti-gp120 mouse MCA and anti-gp41 mouse MCA. 
Matsushita et al. (Medical Immunol. 14: 307, 1987) also reported-achieving 
viral neutralization with an anti-gp120 mouse MCA. These MCAs are useful 
in the diagnosis of HIV infection, but they are unfortunately unsuited for 
the tasks of prevention of HIV infection and treatment of established 
disease (ARC and AIDS). The reason for this is that, since those MCAs are 
mouse proteins, they are recognized as foreign by the human immune system 
if they are administered to the human body. As a result, not only would 
the MCA activity be inhibited by the anti-mouse MCA antibodies that would 
be produced by the human immune system, but anaphylactic side effects 
would also occur. Therefore, it is clear that, for the prevention and 
treatment of HIV infection in man, it is necessary to develop a 
human-origin MCA, not a mouse-origin MCA. 
In general, human-origin anti-HIV MCAs can be produced by (1) hybridomas 
obtained by fusion of human B lymphocytes having the ability to produce 
antibodies specific for HIV and cells of established lymphoid cell lines 
such as myeloma cells, and (2) lymphoblastoid cells obtained by 
Epstein-Barr (EB) virus-induced transformation of human B lymphocytes 
having the ability to produce antibodies specific for HIV. From about 1980 
up to the present time, much research has been carried out on the 
production of human MCAs, but none of those efforts have led to an 
established method such as in .the case of mouse MCAs because each of the 
approaches described above has its own special problems. 
In 1987, there were two reports concerning human MCAs specific for HIV. One 
was by L. Evans et al. (Proceedings of the Third Congress on AIDS, TP130, 
1987). They reported that they employed EB virus to transform lymphocytes 
from HIV-infected patients and obtained a human MCA which reacted with gag 
proteins having molecular weights of 55, 41 and 25 kilodaltons. That human 
MCA belonged to the IgG4 subclass, and it did not neutralize HIV. The 
second report was by B. Banapour et al. (ibid, TP114); they also employed 
EB virus to transform lymphocytes from anti-HIV antibody-positive 
subjects, fused the transformed cells with heteromyeloma cells, and 
obtained a human MCA which reacted with gp41. This MCA was IgG, but the 
subclass was not reported. This MCA also did not show HIV-neutralizing 
activity. Thus, in both of those reports, transformation by EB virus was 
employed. This technique, because it is very efficient at achieving 
immortalization of human B lymphocytes, is far superior to the cell fusion 
method. Nevertheless, the obtained lymphoblastoid cell lines produce EB 
virus, or even if they do not produce the virus particles, they contain 
the EB viral DNA which carries the potential for production of the virus. 
EB virus has the ability to transform lymphocytes, which means that this 
virus has tumorigenicity. Therefore, there is worry concerning the safety 
of using this EB virus transformation technique to produce a drug product 
for administration to humans. 
It is also known that lymphoblastoid cells resulting from transformation of 
lymphocytes by EB virus can be further infected by HIV, and there is thus 
the fear that a cell line producing human MCA might be infected by both EB 
virus and HIV. In addition, the antibody production by lymphoblastoid cell 
lines presents some disadvantages in view of the facts that it is usually 
lower and also less stable than the level of production by hybridomas. The 
reason that Banapour et al. performed additional cell fusion of 
lymphoblastoid cell lines was to attempt to improve the antibody producing 
ability of those cell lines. 
Accordingly, as seen above, if the immortalization of human B lymphocytes 
could be achieved with greater efficiency by cell fusion and if a 
hybridoma having the ability to produce human MCA specific for HIV could 
be obtained, then the resultant hybridoma would be very desirable on the 
basis of its having high productivity of an MCA which would moreover be 
safe for use as a drug. 
With regard to the subclass which would be the most desirable for human 
MCAs, it is evident that it would be advantageous for the antibody to be 
of a subclass which possesses the ability to activate complement and the 
ability to bind to the Fc receptors on macrophages and lymphocytes. It has 
been demonstrated that activation of complement by the classical pathway 
can be achieved by the IgG1 and IgG3 subclasses, whereas IgG2 and IgG4 
cannot carry out this activation (J. L. Winkelhake: Immunochem. 15: 695, 
1978). Furthermore, it has also been shown that the IgG1 and IgG3 
subclasses have a strong affinity for the Fc receptors of monocytes (Cosio 
et al.: Immuno. 44: 773, 1981). Therefore, for the objective of prevention 
of infection of cells, it is clear that the IgG1 and IgG3 subclasses are 
desirable. 
However, another consideration is necessary: that of purification of the 
produced human MCA. Affinity chromatography using protein A can be 
effective for the purification of MCAs, and since IgG1 binds to protein A 
whereas IgG3 does not, it is clear that the IgG1 subclass of human MCA 
would be the most desirable subclass from the viewpoint of ease of 
purification. 
SUMMARY OF THE INVENTION 
The inventors of the present invention, as a result of carrying out 
vigorous research aimed at obtaining an anti-HIV human MCA and employing a 
method involving fusion of mouse myeloma cells and lymphocytes from the 
lymph nodes or spleen of HIV-seropositive donors, succeeded in obtaining a 
hybridoma which produces a human MCA (IgG1 subclass) specific for gp121, 
and a hybridoma which produces a human MCA (IgG1 subclass) reacting with 
both gp120 and gp41. They also succeeded in culturing those hybridomas 
and/or cell lines originating from those hybridomas and were able to 
collect the anti-HIV human MCAs from the supernatants of those cell 
cultures. 
That is, the present invention consists of human monoclonal antibodies 
which are specific for HIV and belong to the IgG1 subclass, specifically 
an IgG1 antibody which binds with gp120 of HIV, and IgG1 antibody which 
binds with both gp120 and gp41 of HIV. In addition, this invention 
consists of the hybridomas which produce those human monoclonal antibodies 
and were formed by fusion between human lymphocytes and mouse myeloma 
cells. These hybridomas have been registered with the American Type 
Culture collection, 12301 Parklawn Drive, Rockville, Md. 20851USA as 
deposition numbers HB9669, HB9670 and HB10074 (Sl-1). In addition, another 
aspect of this invention is the method by which the inventors succeeded in 
efficiently forming those hybridomas, a method in which human lymphocytes 
were first treated with complement and anti-human T-lymphocyte mouse MCA 
or AET treated SRBC and Ficoll and then the treated human lymphocytes were 
fused with mouse myeloma cells.

BEST MODE OF CARRYING OUT THE INVENTION 
The human lymphocytes employed in the method of this invention can be 
obtained from the spleen, lymph nodes, peripheral blood, bone marrow, 
tonsils, adenoids, etc., of seropositive donors. To achieve the objective 
of this invention, use can be made of lymphocytes from any of those 
sources, but it is most desirable that they be obtained from the lymph 
nodes, spleen or tonsils of seropositive donors or patients with 
lymphadenopathy. 
As the mouse myeloma cells, it is advantageous to employ a cell line which 
is resistant to 8-azaguanine, and the following are some of the 
publicly-known cell lines from BALB/C mice: P3x65Ag8, P3-NS1/1-Ag4-1, 
P3x63AgU1, SP2/OAg14, P3x63Ag8.6.5.3, MPCll-45.6.TG1.7 and SP-1. 
In the method of this invention, prior to the fusion of the human 
lymphocytes and the mouse myeloma cells, it is desirable to treat the 
human lymphocytes with complement and an anti-human T-lymphocyte mouse MCA 
(e.g., OKT3, a product of Ortho Diagnostics Co., Ltd.) or to treat the 
human lymphocytes with AET (Aminoethylisothiouranium Bromide Hydrobromide) 
treated SRBC (sheep red blood cells) and Ficoll so as to eliminate the 
human T-lymphocytes. In the actual performance of the method of this 
invention, for example, a fixed lymphatic tissue is surgically excised 
from a seropositive human donor and gently dissected with scissors and a 
scalpel to obtain a liquid containing suspended cells. Then, to remove 
T-cells from these suspended cells, the following two methods were used: 
(1) This suspension is then layered onto a Ficoll-Paque solution, and the 
lymphocytes are separated and harvested by centrifugation. Then, the 
lymphocytes are treated with 0.5 ml of fresh serum as the source of 
complement and 1.0 ml of an anti-human T-lymphocyte mouse MCA to destroy 
the T-lymphocytes and resultant B-lymphocytes are? harvested by 
centrifugation. 
(2) This suspension is mixed with AET treated SRBC and then layered onto a 
Ficoll-Paque solution and B-lymphocytes are separated and harvested by 
centrifugation. If B-lymphocytes were used instead of nontreated 
lymphocytes, the hybridoma formation is increased. 
The thus-obtained human B-lymphocytes are then fused with mouse myeloma 
cells. The general conditions for cell fusion and culture of hybridomas 
are already known, but the inventors nevertheless carried out vigorous 
research to determine the most desirable combinations for achieving 
formation of hybridomas and propagation of them and as a result were able 
to achieve formation of one hybridoma for every 10.sup.4 lymphocytes 
treated by the method of the invention. 
Those conditions were determined to be as follows. For example, lymphocytes 
and mouse myeloma cells are mixed at a ratio of 10:1 to 1:100, preferably 1 
to 1:10, a suitable solution for cell fusion, such as RPMI 1640 containing 
ca. 35% polyethyleneglycol (molecular weight: about 1,000-6,000) and ca. 
7.5% dimethylsulfoxide is added, this cell suspension is stirred for one 
to several minutes at a temperature in the ambient to 37.degree. C. range, 
this suspension is gradually diluted and then washed with RPMI 1640 
containing 10% fetal calf serum (FCS), and finally it is adjusted with HAT 
(hypoxanthine-aminopterin-thymidine) selective culture solution to give a 
cell density of 1-5.times.10.sup.5 /ml. Mouse peritoneal exudate cells are 
added to a 96-well plate as a feeder layer, and the culture solution is 
removed immediately before the fused cells are introduced by dispensing 
0.2 ml aliquots of the suspension into the wells of the plate. These are 
then cultured for 2-3 weeks at 35-38.degree. C. in humidified air 
containing 5% CO.sub.2. Only hybridoma cells are present in the HAT 
culture solution, since the 8-azaguanineresistant myeloma cells and cells 
arising from fusion of myeloma cells cannot survive in the HAT solution 
(unfused antibody-producing cells die within a few days). 
After the culture of the hybridomas in the 96-well plates, the antibody 
titer of the culture fluid of each well containing cells is determined by 
the enzyme-linked immunosorbant assay (ELISA) technique, and only 
hybridomas which produce the desired antibodies are selected. Cells of 
each selected hybridoma are collected, cloning is performed by the 
limiting dilution method, and subclones which stably produce an MCA are 
established.. Then those hybridomas are further investigated by analyzing 
the antigens recognized by their produced MCAs by the Western blot 
analysis and/or Radioimmunoprecipitation analysis technique, and 
investigating the ability of the produced MCAs to bind to the surface of 
HIV-infected cells, and those hybridomas which are producing an MCA which 
binds to gp120 or gp160 and which is able to bind to the surface of 
infected cells are finally selected. 
The mouse-human hybridomas which were obtained by the method of this 
invention as described above and which produce anti-HIV human MCAs can be 
preserved by freezing. If these hybridoma cell lines and/or cell lines 
derived from them are cultured on a large scale by an appropriate method, 
it is possible to obtain from the culture supernatant the human MCAs which 
are the objective of the present invention. In addition, these hybridomas 
are transplanted into animals to form tumors, the produced human MCA can 
be obtained from the ascites or the serum of the animals. 
The anti-HIV human MCAs which have been obtained by the methods described 
above have been found to have the following characteristics. (1) In ELISA 
using fixed viral antigens obtained from HIV-infected cells, the MCAs were 
positive for binding, but they were negative for binding in ELISA using a 
plastic coated with substances obtained from uninfected cells by the same 
technique. (2) Since HIV is composed of many antigenic substances, the 
Western blot analysis technique and/or RIPA technique was applied to 
determine the nature of the structural components to which the human MCAs 
obtained in this invention bind. It was thus found that one of the human 
MCAs binds to a molecules having molecular weights of 120K and 160K (160K 
is the precursor of 120K and 41K molecules). The second MCA was found to 
bind to molecules having molecular weights of 41K, 120K and 160K. (3) The 
MCAs were investigated to determine whether or not they bind to the 
surface of HIV-infected cells. After the human MCA was reacted with 
unfixed HIV-infected cells, fluorescein-labeled antibody to human IgG was 
allowed to react, and strong fluorescence was observed on the surface of 
the infected cells. Therefore, it was learned that all of the human MCAs 
of this invention bind to the surface of infected cells. (4) Human IgG is 
known to have four subclasses, i.e., IgG1, IgG 2, IgG3 and IgG4, with each 
subclass having its own characteristic biological activities. Each of the 
anti-HIV human MCAs obtained in this invention was thus investigated for 
its subclass using a specific animal antiserum, and it was found that all 
of the MCAs of this invention belong to the IgGl subclass. 
Experimental Example 1 
A. Cell Fusion. 
1. Collection-of Lymphocytes 
A lymph node which was surgically excised from an ARC patient was finely 
minced using scissors and a scalpel. Cells therefrom were suspended in 
medium A (RPMI 1640 containing 10% fetal calf serum (FCS), 2 mM glutamine, 
1 mM sodium pyruvate, 20 .mu.g/ml L-serine, 0.05 .mu./ml human insulin and 
80 .mu.g/ml gentamicin sulfate). This cell suspension was layered onto a 
Ficoll-Paque solution and centrifuged at 1,500 rpm for 20 min. The cells 
which collected on the top of the Ficoll-Paque solution were harvested, 
centrifugally washed once with phosphate-buffered saline (PBS) and twice 
with RPMI 1640. Finally, the cells were resuspended in RPMI 1640 to a 
concentration of 1.times.10.sup.7 cells/ml. 
2. Treatment of Lymphocytes 
To reduce the amount of cell fusion that would take place with 
T-lymphocytes, the T-lymphocytes in the lymphocyte suspension were 
eliminated by either of the following two methods. 
(1) OKT3 (Ortho Diagnostics Co., Ltd.) was added to the above-mentioned 
cell suspension to give a final 200-fold dilution. After reacting at 
4.degree. C. for 60 minutes, the cells were precipitated by centrifugation 
(1,500 rpm for 5 min). Next, baby rabbit complement was diluted 3-fold 
(with RPMI 1640) and added to the cell pellet to obtain a suspension, 
which was then reacted at 37.degree. C. for 60 min. Then this cell 
suspension was twice subjected to centrifugal washing. 
(2) The same volume of 1.times.10.sup.8 AET treated SRBC suspension in 
medium A was added to the above-mentioned cell suspension. After gently 
mixing this at room temperature for 5 minutes, the cells were precipitated 
by centrifugation at 1000 rpm for 5 minutes. The cell pellet was incubated 
at room temperature for 20 minutes, then gently suspended, and layered 
onto. Ficoll-Hypaque. After centrifugation at 1500 rpm for 20 minutes, the 
B-lymphocyte fraction was collected from the interface layer of the medium 
and the Ficoll-Hypaque and subjected to centrifugal washing. 
3. Cell Fusion 
The OKT3-treated or AET rosette treated lymphocytes or untreated 
lymphocytes were each mixed with mouse myeloma P3U1 cells (both cell 
populations were 3.times.10.sup.7 cells) in RPMI 1640 medium. These cell 
mixtures were then precipitated by centrifugation (1,600 rpm, 5 min). The 
supernatant was discarded, and the cell pellet was broken up by tapping 
the tube. Then 1 ml of polyethylene glycol solution (35% v/v polyethylene 
glycol No. 1000 and 7.5% v/v dimethylsulfoxide in RPMI 1640) was slowly 
added to the tube, and this was allowed to stand for one minute at room 
temperature. Next, 2 ml of RPMI 1640 was added, and it was allowed to 
stand for one minute; another 2 ml of RPMI 1640 was added, and it was 
allowed to stand for 2 minutes. Then 4 ml of HAT medium (95 .mu.M 
hypoxanthine, 5.4 .mu.M aminopterin, and 16 .mu.M thymidine in medium A) 
was added, and the mixture was allowed to stand for 2 minutes; another 8 
ml of HAT medium was added and the mixture was allowed to stand for 2 
minutes; an additional 24 ml of HAT medium was added and the mixture was 
allowed to stand at 37.degree. C. for 30 minutes. Finally, the total 
volume was made up to between 75 and 150 ml by the addition of HAT medium. 
Aliquots of approximately 200 .mu.l were seeded into the wells of a 96-well 
flat culture plate. This culture plate had been pretreated by seeding ICR 
mouse (male) peritoneal exudate cells at 2.times.10.sup.4 cells/well; 
immediately prior to the seeding of the fused cells, the culture fluid was 
removed from the wells. This culture plate was then incubated at 37.degree. 
C. in a CO.sub.2 incubator. Once per week, half of the culture medium in 
each well was replaced by HT medium (HAT medium from which aminopterin had 
been left out), and the incubation was continued until hybridoma colonies 
became apparent. 
4. Cloning 
At the time when hybridoma colonies became apparent, each of the culture 
fluids was treated for the presence of antibody activity directed at HIV. 
The hybridomas of colonies which were found to be producing HIV-specific 
antibodies were then cloned. First, 96-well flat plates were seeded with 
only mouse peritoneal exudate cells at 2.times.10.sup.4 cells/well. Then, 
at various times from one hour to one day after the seeding, the culture 
medium was removed and the hybridomas were seeded into 96 wells each at 10 
cells/well. For the first cloning, HT medium was employed, while medium A 
was used for the second cloning. After 2-3 weeks of culture, the antibody 
activity was determined, and positive clones were picked up. 
B. ELISA (Enzyme-Linked Immunosorbent Assay) 
1. Viral Antigens 
a. HTLV-III (human lymphotropic virus type III) antigen (Bionetics 
Laboratory Products Co., Ltd.) 
b. CR10/N1T Antigens CR10/N1T is a cell line which was established by 
creating a persistent infection of CEM cells with the N1strain of HIV. The 
viral antigens were partially purified from this CR10 cell line. In brief, 
CR10/N1T cells were washed 3 times with PBS and then frozen at -70.degree. 
C. At the time of use, the frozen cells were thawed, and 10.sup.8 cells 
were suspended in 9 ml of distilled water; this cell suspension was 
vigorously agitated for one minute using a Vortex blender. This was then 
centrifuged for 10 minutes at 2,800 rpm, and the-supernatant was 
collected. One ml of 10-fold concentrated PBS was next added to the 
supernatant, centrifugation was performed at 15,000.times.g for 30 min, 
and the pellet was collected. The pellet was resuspended in 5 ml of PBS, 
sonicated 4 times for 15 sec each while chilling in ice and allowed to 
stand for a further 30 minutes while chilling in ice; the supernatant was 
then collected. The supernatant was subjected to ultracentrifugation at 
100,000.times.g for one hour, and the supernatant was employed as the 
viral antigen preparation. As the negative control, an antigen preparation 
was obtained by treating CEM cells (uninfected by HIV) in the same manner. 
2. Antigen-Coated Plates 
HTLV-III antigen (1 .mu.g/ml), CR10/N1T antigens (20-25 .mu.g/ml) and CEM 
antigens (20-25 .mu.g/ml) were each dispensed in aliquots of 50 .mu.l to 
the wells of separate microtiter plates (Coster, No. 3912), and the plates 
were then allowed to stand at 37.degree. C. for 60 min. The plates were 
then washed twice with HBSS-BSA (Hank's balanced salt solution, 0.5% 
bovine serum albumin and 0.1% NAN3), PBS (Ca.sup.++, Mg.sup.++) containing 
3% BSA was dispensed at 125 .mu.l/well, and the plates were allowed to 
stand at 37.degree. C. for 60 min and then at 4.degree. C. overnight to 
carry out blocking. 
3. ELISA 
The antigen-coated plates were washed twice with HBSS-BSA, and then 50 
.mu.l of each of the heated (56.degree. C. for 60 minutes) hybridoma 
culture fluids was added. After letting these react at room temperature 
for 60 minutes, the plates were again washed twice with HBSS-BSA. Then 50 
.mu.l of alkaline phosphatase-conjugated goat antibody to human IgG 
(diluted 1000.times.x; Tago Inc.) were added, and reaction was again 
allowed to take place at room temperature for 60 minutes before the plates 
were washed 4 times with HBSS-BSA. Next, 100 .mu.l of 0.05 M carbonate 
buffer containing 1 mg/ml p-nitrophenylphosphate and 1 mM MgCl.sub.12, pH 
9.5, was added to each well, and the plates were reacted at room 
temperature for 60 minutes or overnight. Finally, the optical density was 
measured at 405 nm using an ELISA Reader (Titertech Inc.). 
C. Experimental Results 
1. Lymph. node cells from Patient A were compared with and without OKT3 
treatment. 
TABLE 1 
______________________________________ 
Generation of Hybridomas Producing IgG Antibodies to HIV* 
Number of Anti-HIV IgG-Positive Wells 
Treatment 
High O.D.** Medium O.D. 
Low O.D. 
______________________________________ 
- OKT3 3 2 1 
+ OKT3 6 5 6 
______________________________________ 
*Indicates wells containing hybridomas which produce IgG that reacts with 
CR10/N1T antigens but not with negative control (CEM antigens). 
**"High" means that the optical density at 405 nm was larger than 1.0, 
while "Medium" indicates the 0.4-1.0 range and "Low" represents the 
0.2-0.3 range. Therefore, more hybridomas producing IgG antibodies to HIV 
were generated in the case of the lymphocytes which were treated with 
complement and antilymphocyte antibody. 
2. As reported above, hybridomas were obtained by fusion of mouse myeloma 
cells with OKT3-treated lymphocytes from the lymph nodes of patients with 
ARC, those hybridomas were cloned, and the inventors successfully 
established hybridomas No. 86 (aTCC No. HB9669) and No. 1, (aTCC No. 
HB9670which stably produce MCAs. On the other hand, hybridoma Sl-1 (aTCC 
No. HB10074) which stably produces MCA was obtained by fusion of mouse 
myeloma cells with AET rosette-treated lymphocytes from the spleen of 
patients with ARC. In ELISA, the MCAs produced by hybridomas No. 86, No. 
1, and Sl-1 reacted with HTLV-III antigen and CR10/N1T antigens but not 
with CEM antigens. The MCA production rates were 10 .mu.g/10.sup.6 
cells/day in the case of No.86, 20 .mu.g/10.sup.6 cells/days in the case 
of No. 1, and 5 .mu.g/10.sup.6 cells/day in the case of Sl-1. 
Experimental Example 2 
A. Purification of MCAs 
The Culture fluids (1.5-2 liters) of hybridomas No. 86, No. 1, and Sl-1 
were used as the starting materials. Ammonium sulfate was added to the 
culture fluids to 50% saturation, and the resultant protein precipitates 
were collected by centrifugation at 10,000 rpm for 30 min. The 
precipitates were then dissolved in a suitable volume of PBS, followed by 
dialysis against PBS. The dialyzed solution was next applied to a protein 
A-Sepharose column bed (bed volume: 6 ml; Pharmacia AB). The column was 
washed with saline, and then the IgG was eluted with HC1 in saline (pH 
2.5). The IgG eluted in this manner was confirmed to be pure by sodium 
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 
B. Identification of IgG Subclasses of MCAs 
1. Heavy Chains 
The purified MCA solutions were reacted with sheep antisera to human IgG1, 
IgG2, IgG3, and IgG4 (Serotec Inc.). The subclass of each MCA was 
identified on the basis of which antisera resulted in formation of an 
immunoprecipitation ring. It was thus found that No. 86, No. 1, and Sl-1 
MCAs reacted only with the anti-IgG1 and did not react with the other 
three antisera. Therefore, all of these anti-HIV MCAs were identified to 
be IgG1. 
2. Light Chains 
A microtiter plate was coated with goat antibody to human IgG (Tago Inc.). 
Each of the purified MCAs was then reacted with this anti-human IgG-coated 
plate. Next, in accordance with the method for ELISA described earlier in 
section B of Experimental Example 1, alkaline phosphatase-conjugated goat 
antibodies to human lambda chain and to kappa chain (Tago Inc.) were 
employed and the type of each MCA was identified. As a result, No. 86 MCA 
was shown to have a kappa chain, while No. 1 MCA and Sl-1 MCA were found 
to have lambda chains. 
C. Vital Antigens Recognized by the MCAs 
The Western blot method (Bio Rad Immunoblot Assay; Bio Rad Inc.)was 
employed to identify which vital antigens were recognized by MCAs No. 86 
and No. 1. MCA No. 1 has also been referred to as MCA 1.2 by the 
inventors; thus, MCA 1 and MCA 1.2 refer to the same cell line. The 
procedures of the assay technique are briefly described as follows. 
The HTLV-III strain of HIV was applied to SDS-PAGE, the separated viral 
antigens were blotted on nitrocellulose strips, and each of the 
semi-purified MCAs was reacted thereon. Next, peroxidase-conjugated 
antibody to human IgG was reacted with the strips, and finally, to develop 
color, an enzyme substrate was reacted with the strips. The results are 
shown in FIG. 1. In the figure, A is serum from an AIDS patient, B is 
serum from a normal human, C is No. 86, D to G are subclones of No. 86, H 
is the clone of No. 1, and I is Sl-1. 
MCA No. 86 reacted strongly with gp41 and reacted weakly with gp120. As the 
reason for reacting with both gp41 and gp120, it is possible that MCA No. 
86 was a mixture of one MCA which reacted with gp41 and another MCA which 
reacted with gp120. To investigate this possibility, the hybridoma 
producing MCA No. 86 was again cloned, yielding subclones 1, 2, 3, and 4, 
and the-MCA produced-by each of those subclones was also subjected to the 
Western blot assay. As seen in D, E, F, and G, the MCA from each of the 4 
subclones of hybridoma No. 86 reacted with both gp41 and gp120. 
This finding suggests that MCA No. 86 either recognizes an antigenic 
epitope which is present on both gp41 and gp120, or is an antibody 
directed at the cleavage site of gp41 and gp120. MCA No. 86 also reacted 
with gp160, and the reason for this is that this antigen is a glycoprotein 
constructed from gp41 and gp120. 
MCA No. 1 reacted with gp120. It, of course, also reacted with gp160, which 
is the precursor of gp120. 
MCA Sl-1 did not react with any antigen on Western Blotted paper. 
D. Radioimmunoprecipitation assay (RIPA) 
It sometimes occurs that some antigenic determinants recognized by MCAs are 
not detected by the Western blot analysis. This is thought to be due to the 
destruction of tertiary structure of antigens by strong detergent, heat, 
and methanol treatment of antigens used in the Western blot assay. This 
phenomenon was observed in the case of MCA Sl-1. Therefore, the antigens 
recognized by MCA Sl-1 was determined by RIPA as follows. 
.sup.35 S labeled cell extracts for RIPA were prepared as follows. MOT 
cells or HTLV. IIIb infected MOT cells (4 days after infection the MOT was 
equal to 10.sup.3 TCID.sub.50/5.times.10.sup.6 cells) were labeled with 
.sup.35 S-cysteine and .sup.35 S-methionine (50 .mu.Ci/ml total activity). 
The labeling media was RPMI 1640 containing 1/10 the normal concentration 
of methionine, 10% extensively dialized fetal calf serum, the other 
essential amino acids and the .sup.35 S labeled amino acids. Uninfected 
and infected MOT cells were cultured 14 hours in the labeling media and 
then washed with PBS(-). After washing, the cells were lysed with RIPA 
buffer (20 mM Tris-HCl pH 7.4, 1% Deoxycholate, 1% Triton X-100, 0.1% 
Sodium Dodecxysulfate and 1 mM (p-Amidinophenyl)methanesulfonyl fluoride). 
The lysate was clarified by high speed centrifugation (18,000 rpm, 60 
minutes) at 4.degree. C. The labeled antigens were divided and treated 
with 20 or 100 mM dithiothreitol (DTT) at 37.degree. C. for 30 or 60 
minutes or incubated in the absence of DTT for 30 or 60 minutes. The 
labeled antigens were immunoprecipitated by the MCA Sl-1 and HIV+human 
serum antibodies conjugated to protein-A sepharose beads in the presence 
of RIPA buffer. 
Labeled antigen-antibody complex conjugated to protein A-sepharose beads 
were washed eight times with RIPA buffer, twice with 10 mM Tris HCl pH 
6.8, and then suspended in sample buffer (62.5 mM Tris HCl pH 6.8, 1% SDS, 
20% glycerol, 0.2% bromphenol blue) in the presence or absence of 2% 
2-mercaptoethanol. After heating the suspension at 100.degree. C. for 3 
minutes, released labeled antigens were separated on a 10% acrylamide gel. 
After electrophoresis, the gel was fixed with 50% methanol-10% acetic acid, 
immersed in 1M salicylic acid - 3% glycerol, and dried using gel drier. The 
dried gel was autoradiographed at -80.degree. C. for 3 to 5 days. From 
FIGS. 2, 3, and 4, the following results were obtained: 
(1) MCA Sl-1 recognizes gp 120. 
(2) The antigenic determinant on gp120 (gp160) was easily destroyed by 
sulfhydryl reagents. 
E. Binding to Surface of HIV-Infected Cells 
The ability of MCAs No. 86, No. 1, and Sl-1 to bind to the surface of 
HIV-infected cells was investigated by the indirect fluorescent antibody 
technique. 
MOT cells (an HTLV-II transformed cell line), 5.times.10.sup.6 cells, were 
mixed with 2.5.times.10.sup.6 TCID.sub.50 of HTLVIIIb, and this mixture 
was incubated at 37.degree. C. for 2 hr to permit infection to proceed. 
These cells were then cultured for 3 days in RPMI 1640 medium containing 
10% FCS, after which the cells were washed 3 times at 4.degree. C. with 
PBS containing 0.1% NaN.sub.3. As, the negative control, MOT cells which 
were not infected with HIV were employed. 
These unfixed, cells were dispensed into conical tubes to give 
2.times.10.sup.6 cells/tube, and centrifugation was performed at 1,500 rpm 
for 5 minutes. The supernatant was discarded, and the cell pellet was 
suspended in 100 .mu.l of 50 .mu.g/ml MCA in 0.1% NaN.sub.3 -HBSS. This 
suspension was reacted at 4.degree. C. for 60 minutes, and then the cells 
were washed 3 times with 0.1% NaN.sub.3 -1 mM EDTA-PBS. Each cell pellet 
was suspended in 100 .mu.l of fluorescein isothiocyanate-labeled antibody 
to human IgG (50.times.dilution; Tago Inc.), followed by reaction at 
4.degree. C. for 60 minutes. 
The cells treated as described above were next analyzed by flow cytometry 
(FAC Scan; Becton Dickinson, Co.). The status of binding was investigated 
for the following combinations: HTLV-IIIb-infected MOT cells and serum 
(100.times.diluted) from an AIDS patient, uninfected MOT cells and serum 
(100.times.diluted) from an AIDS patient, HTLV-IIIb-infected MOT cells and 
MCA Sl-1 uninfected MOT cells and MCA Sl-1, HTLV-IIIb-infected MOT cells 
and MCA V1, and uninfected MOT cells and MCA V1. V1 was an IgG human MCA 
specific for an irrelevant antigen. 
The following results were obtained. MCA Sl-1 bound to the surface of 
HIV-infected cells, but it did not bind to the uninfected cells. The same 
results were Obtained with MCA No. 86 and MCA No. 1. MCA V1, which was not 
specific for HIV, did not react with the HIV-infected cells (FIG. 5). 
With an MCA which reacts with the surface of virus-infected cells, it can 
be speculated that it might be possible to destroy the infected cells in 
the presence of complement or in the presence of lymphocytes or 
macrophages, thereby stopping the production of new virus and permitting 
suppression of the spread of the infection. 
F. Neutralization assay 
The neutralizing assay of the MCAs was performed by two methods: neutral 
red dye uptake and p24 antigen capture. The neutral red dye uptake 
neutralization assay is based on the following premise: when HIV infects 
permissive cells, the cells lyse after a short time. Neutral red dye is 
incorporated into the cytoplasm of viable cells. In the neutral red dye 
uptake neutralization assay, if a MCA could bind to HIV and prevents it 
from entering permissive cells, the cells-would remain viable and they 
would take up neutral red dye, giving a colorimetric indication of cell 
survival which would be indicative of the neutralization of HIV. The 
protocol is given below. 
Protocol for Neutralization Assay: Neutral Red Dye Uptake 
Supernatant from HTLV-IIIb infected H9 cells was used as a virus source for 
the neutralization assays. A Multiplicity of Infection (MOI) of 20-25 was 
mixed with dilutions of anti-HIV antibody and incubated for 1 hour at 
37.degree. C. HIV mixed with an irrelevant MCA or HIV mixed with HIV 
positive serum were used as controls. After the virus-antibody incubation, 
a CD4+cell line (MOT) was added at 3.times.10.sup.4 cells/well. These 
plates, containing HIV, antibody, and MOT cells were then incubated for 
either 5 or 6 days. On day 5 or 6, the cells in the 96-well microtiter 
plates were suspended by micropipette action and 100 .mu.l was transferred 
into corresponding wells of a poly-L lysine coated plate containing 100 
.mu.l of 0.014% neutral red dye in media. The neutral red dye containing 
plates were incubated for 1 hour at 37.degree. C. All the cells attach to 
the poly-L lysine on the bottom of the well while only the viable, 
undamaged cells took up the neutral red dye. After 1 hour, the plate was 
washed free of excess dye and 100 .mu.l of 1% acetic acid in 70% ethanol 
was then added to the 96-well plates, The cells containing the dye lysed 
and released the dye into the supernatant, A colorimetric determination of 
cell survival was made using a Titertek ELISA reader at 540 nm, 
Results: Neutral Red Dye Uptake 
The following results were obtained in the neutral red dye uptake 
neutralization assay. MCA Sl-1 was observed to neutralize over 90% of the 
infectious HIV at a concentration of 100 .mu.g/ml as measured by the 
neutral red dye uptake, cell survival assay (FIG. 6). As the concentration 
of MCA Sl-1 decreased, it inhibited less HIV from infecting the permissive 
cell line, MOT. Normal serum did not inhibit HIV infection at all, while 
HIV-positive serum inhibited HIV infection to a 90-fold dilution. Neither 
MCA Sl-1 nor HIV-positive serum effectively neutralized HIV at 
concentrations less than 11 .mu.g/ml or at greater than 90-fold dilutions, 
respectively. 
Protocol for Neutralization Assay Antigen capture Assay 
Another HIV neutralization assay was performed that detects the p24 HIV 
core protein in an ELISA antigen capture assay. When HIV infects 
permissive cells, it replicates itself inside the cell and releases vital 
particles from the cell into the surrounding supernatant where they can be 
detected. Again, if a MCA were to bind to HIV and inhibit penetration into 
the cell, HIV could not replicate itself and would not release viral 
particles into the supernatant. This p24 antigen capture was performed as 
follows. 
Cell-free HTLV-IIIb infected H9 supernatant at a MOI of 20-25 was incubated 
with dilutions of anti-HIV antibody in 96-well microtiter plates for 1 hour 
at 37.degree. C. It is important to note that the amount of HIV inoculum 
could not be detected by this antigen capture assay. Only the viral 
particles produced by HIV infected cells are detected. HIV mixed with an 
irrelevant MCA or HIV mixed with HIV-positive sera were used as controls. 
After the virus-antibody incubation, a CD4+ cell line (MOT) was added to 
the plates at 3.times.10.sup.4 cells/well. These plates containing HIV, 
antibody, and MoT cells were then incubated for 7 days. Samples of the 
supernatants were taken from each well at 3, 5, and/or 7 days. These 
samples were heat-inactivated for hour at 56.degree. C. and then added to 
96-well ELISA plates coated with 5 .mu.g/ml of HIV-positive serum. After 1 
hour incubation at room temperature, the ELISA plates were washed with 
0.05% Tween-20 in phosphate buffered saline. Then a biotinylated MCA 
specific for p24 (Western Blot) was added to the plates at 2 .mu.g/ml, and 
the plate was incubated and washed as before. Streptavidin conjugated 
alkaline phosphatase was then added to the plate at a concentration of 1 
.mu.g/ml, incubated for 1 hour and washed free of excess reagent. One 
mg/ml of para-nitrophenyl phosphate in carbonate buffer pH 9.5 was added 
to the plate and the optical densities of the wells were read on a 
Titertek Multiskan ELISA reader at 405 nm. An increase in optical density 
indicated that more p24 was present in the original culture supernatant. 
Results: p24 Antigen Capture 
The following results were obtained from the HIV p24 antigen capture 
neutralization assay. MCA Sl-1 blocked HIV infection significantly as 
evidenced by low levels of the p24 HIV core protein in the presence of 
Sl-1 concentrations of from 100 to 25 .mu.g/ml (FIG. 7). Sl-1 did not 
maintain neutralization at lower concentrations. HIV-positive serum 
completely neutralized infection at 10-fold and 20-fold dilutions and 
partially neutralized HIV at a 40-fold dilution, but also did not maintain 
neutralization at higher dilutions as indicated by high levels of p24. 
Discussion 
MCA Sl-1 neutralizes HIV infection in permissible cells. The degree of 
neutralization depends on the concentration of the MCA. At high 
concentrations (100 .mu.g/ml) Sl-1 inhibits 93% of cell-free HIV from 
infecting cells in a cell survival assay. Although Sl-1 lost the capacity 
to neutralize HIV at lower concentrations, HIV-positive serum also lost 
the ability to neutralize at high dilutions. Consistent with the cell 
survival assay results, less p24 HIV core protein was produced in the 
presence of Sl-1, indicating that Sl-1 inhibits HIV infection. 
The results of the various experiments described above are compiled in the 
following Table 2. 
TABLE 2 
______________________________________ 
Property No. 86 No. 1 S1-1 
______________________________________ 
Isotype of MCA 
IgG1..kappa. 
IgG1..lambda. 
IgG1..lambda. 
Binding to HIV 
HTLV-IIIb HTLV-IIIb HTLV-IIIb 
in ELISA CR10/N1T CR10/N1T CR10/N1T 
Binding to HTLV-IIIb 
infected cells 
fixed + + + 
unfixed + + + 
Viral antigens 
gp120 gp120 gp120 
recognized by MCA 
gp41 
Neutralization 
- - + 
(90% at 
100 .mu.g/ml 
dye uptake 
method) 
MCA production rate 
10 20 5 
(.mu.g/10.sup.6 cells/day) 
______________________________________ 
Obviously, numerous modifications and variations of the present invention 
are possible in light of the above teachings. It is therefore to be 
understood that within the scope of the appended claims, the invention may 
be practiced otherwise than as specifically described herein.