Note: Descriptions are shown in the official language in which they were submitted.
~0~ ~L?~
Technical Field
The present invention relates to a new method of
produci.ng lymphokines and monoclonal antibodies oE high
speciEicity useul in diagnosis and therapy using a
human-human hybridoma technique which does no~ require
l.0 the use of enzyme deficient malignant partner cells.
Disclosure of the Invention
More specifically, the invention relates to a
novel method of separa~ing fused cells resulting from
fusion Oe a human cell known to produce a specific
antibody with a malignant human partner cell, which
does nvt need to be enzyme deEicient, from the said
partner cell and subsequent culturing of the fused
cells. This separati.on technique utilizes -the reaction
of ~he fused cell with antiserum and separation of the
fusion product with the anti~erum wi~hin ?4 hours by
indirect rosetting. A new method of subculturing is
provided using multiple fractionations of putative
clones limiting the number of cells per well to about
10, 000.
Instead of the cell producing a speci.fic antibody,
there may also be used a human cell producing a
speciFic lymphokine (immunomodulator) such as leucocyte
8~
--2--
inhibitory factor, interferon and the like.
Best Mode or Carrying Out the Inventi_
In practice of this invention, patients are
selected for their abili~y to produce particular
lymphokines or certain antibodies. ~mong the
antibodies are those with speciEicity useful in
diagnosis and therapy of human disease. Among the
diseases in which these human monoclonal antibodies
will be useful for diagnosis are those in which there
- 10 is a shedding of antigen into the peripheral system.
Useful specificities are exemplified by the
carcinomas and especially clinical types of mammary
carcinoma, as well as viral conditions such as herpes
(e.g., Type I and II), and tetanus.
A group of disease conditions in which the anti~T
cell antibodies produced by this invention are of value
are immunoregulatory disorders, exemplified by
autoimmune diseases and immunode-Eiciency states and
particularly adult and juvenile rheumatoid arthritis,
systemic lupus, severe combined immunodeEiciency as
well as hyper- and hypogammaglobulinemia.
~ nother field of diagnostic and therapeutic
utility compr:ises the field of organ transplants. By
use of anti-T cell antibodies, it is possible to
monitor cells involved in graft rejection and to
modulate the number of cells, thereby eliminating in
many cases the onset and severity o~ graft rejection
crises.
The technique of this invention can also be used
~2~
~o produce a hybrid clone which secretes a variety of
immune modulators, lymphokines, such as the leucocyte
inhibiting Eactor (LIF) and Interleukin II. Up to now,
it has been difficul~ to purify such lymphokines. The
availability of hybrid clones of this invention
producing dis-tinct lymphokines opens new pathways to
their produc-tion and characterization.
Lymphocytes are taken from the patient producing
antibodies or lymphokines of a specificity as described
above, typically from the peripheral blood, and fused
with a malignant human partner cell. This partner cell
can be selected from cell cultures such as those
available from RPMI (Rosewell Park Mernorial Institute,
Buffalo, New York). Preferred are cells with
characteristics of rapid growth, good stability and
hi~h fusion efficiency.
As a result of fusion of the antibody producing
cell with the said partner, there results a mixture of
1) fused cells;
2) non-fused antibody producing cells; and
3) non-fused malignant partner cells.
E'or the separation of the fused cells from this
mixture, the prior art has taught ~le need to employ an
enzyme deficient fusion partner, specifically an HAT
(hypoxan~hine-aminopterin-th~nidine) sensitive cell.
The disadvantages of use of such partners are:
1) a decrease in the efficiency of fusion to
produce hybrid clones;
2) loss of rapid growth characteristics;
3) increased genetic instability; and
4) logistical difficulty associated with the
selection and maintenance of enzyme
~AP~
-4~
deficient mu~ant malignant fusion partner
cells which is time consuming and expensive.
The present invention avoids the neecl to use such
enzyme deficient fusion partners. Insteacl, there i5
used the technique of positive selection oE the clones
~rom the non-fused partner by the addition of a
specific antiserum which identifies antigenic
specificies unique to the clone and is non-reactive
with the non-fused partner cells. These antisera are
available as HLA (Human leucocyte antigen) typing
~ reagents. It will be obvious to those skilled in the
art that in selecting a partner cell, one of a
diferent HLA t~pe than the non-fused antibody or
lymphokine producing cells must be used. Differences
of at least one or more HLA alleles between antibody or
l~mphokine producing cell and partner cell are normally
sufficient to allow efficient separation of fused clone
frorn non-fused partner cell. In other words, some
cross-reactivity between the antibody or lymphokine
producing cell and the partner cell is permitted. Even
one out of ten alleles difference is sufficient to
allow separation. Reaction of the fused cell Wit}l the
antiserum is typically completed within 60 minutes.
The positive selection of the fused clone cell
from the non-fused partner cell, which has not reacted
with the antiserum, is carried out within 24 hours by
the conventional indirect rosetting techni~ue, using
density gradient centrifugation.
There is thus separated a mixture of the fused
clone cells and the non-fused antibody or lymphoXine
producing cells. These non-fused cells have a
relatively short life, typically no more than lO days
--5--
while the fused clone cells survive and multiply in
culture.
In sub-culturing îndividual clones, a technique
different from that employed with murine cultures is
used~ While in the case of the murine culture,
individual cells may be set into subculture and made to
~row, this technique has not been found effective with
human clones. It has been found useful to proceed by
multiple fractionations of putative clones, so that the
limiting number oE cells per well during subculturing
is approximately 10,000.
There are thus obtained cultures of clones which
selectively produce specific antibodies useful in
diagnosis and therapy as discussed above.
The specific antibodies are obtained from batch
cultures using conventional methods such as affinity
column chromatography or preparative isoelectric
focusing. The isolated antibodies are used as such or
are incorporat~d in ~er se known manner into
pharmaceutical compositions such as solutions, test
kits, or radioimmune assa~ materials.
In a fur~her aspect of the present invention/
there is provided a human monoclonal anti~ody which is
useful for the identification of various malignancies,
with specific testing in vitro to show the presence of
human mammary cancer antigens. Tests conducted with
human monoclonal antibodies designed to "recognize" the
presence oE human mammary cancer have been done under
the microscope using conventional indirect fluoresence
and shown to be useful in reacting wi-~h the human
mammary cancer. In a generic embodiment for the
--6
identification of mammary cancer there is thus provided
a method which comprises contacting serum or ductile
secretion from the mammary region of said subject with
a human monoclonal antibody, said human monoclonal
antibody being derived from the fusion of a normal
human hlood lymphocyte producing an antibody with
specificity for mammary carcinoma, and a malignant
partner cell. A positive reaction between said human
monoclonal antibody and said serum or ductile secretion
indicates the presence in said subject of -tumor
antigens, suggesting the presence of mammary carcinoma
- cells.
In a preferred embodiment the malignant partner
cell is an acute lymphocytic leukemia cell of the ~
type. The positive reaction whereby the indication of
human mammary cancer is suggested may be, Eor example,
through precipitation of the human monoclonal antibody
with the serum or ductile secretion, or through
indirect fluorescence.
~0 Althouyh T lymphocytes do not produce immunoglo-
bulins themselves, they have been found to be highly
effective fusion partners for purposes of this
invention. Thus the use of B cells as malignant fusion
partners is not a re~uirement for production of
antibody secreting clones. The selection of particular
malignant cell lines is not critical, provided that
they are vigorous and of long life. The special
advantage of the use of T cells is the availability of
more stable hybrids which are relatively resistant to
genetic change and long lived.
The invention also has special utili~y i~ the
field of juvenile rheumatoid arthritis where during
8 ~
periods of e~acerbation cer~ain antibodies are present,
which are absent during remission. Human hybridoma
cells lines are provided herein using lymphocytes from
patients durin~ exacerbation with lymphoblastoid T
cells, which have been found especially effective as
fusion partners; the resulting clones secrete antibody
which identifies a su~set of normal human peripheral
blood T lymphocytes similar to those identified by
autoimmune an~ibodies found in sera of such patients.
These antibodies have applicability as specific probes
Eor examination of the T cell population and
potentially for modulating speciEic immune response in
vlvo .
The Eollowing examples are provided for purposes
of illustrating the invention in further detail. They
are not to be construed as limiting the invention in
spirit or in scope. Persons skilled in the art will
recognize that equivalent antigens, reagents, subjects,
cells, and procedures can be adopted without departing
from the scope oE the invention.
EXAMPL.E I
A group of patients is screened for reactivity
against long term cell lines derived from mammary
carcinoma tissue. Selected patients with serum
reac-tivity against par-ticular lines are bled and then
HLA typed. The lymphocytes are separated using a
polysaccharide density ~radient such as Ficoll-Hypaque.
20,000,000 isolated lymphocytes are mixed with
10,000,000 malignant fusion particles, such as Ball-l,
a cell line derived from a patient with acute lymphatic
leukemia of a B cell variety in the presence of
~2~
polyethylene glycol. The mixture is centrifuged at 400
g and incubated for a total of 8 minutes at which time
the cells are washed and placed in culture for a period
of about 20 hours.
At this time, the cells are washed and incubated
with the anti~HLA reagent appropriate according to the
resu]t of the typing. ~fter 60 minutes, the cells are
washed and rosetted with human red blood cells which
are coated with affinity column puriEied anti-IgG. The
indirect rosetted mixture is carefully layered on-to a
Ficoll-Hypaque density gradient and centrifuged at 1400
g for 15 minutes. The non-rosetted, non-fused
malignant paxtner cells are located at ~he interface
and are rernoved.
The rosetted fused and non-fused antibody
producing cells are located in the pellet. These are
treated with buffered ammonium chloride to remove the
red cells and the cells are washed and placed in
culture a~ a concentration of 2,000,000 cells per well
in 24 well p]ates. The culture is maintained at 37C
in 5% carbon dioxide atmosphere until maximum growth is
observed, typically in 5-7 days. Each well is then
sub-cultured so that 100-500 cells are placed into each
new subculture well. These subcultured cells are
allowed to grow to a concentration of approximately
100,000, af~er which subculturing is repeated. The
specificity of the antibody being produced is
advantageously ascertained after each subculturing
step.
Subcultures with the appropriate specificity are
then grown to large levels and supernatants are
collected routinely. The antibody is isolated from
these supernatant by conventional immunochemical
techniques. ~ typical subculture producing antibody to
one Eorm of mammary carcinoma as evidenced by
reactivities to ~he mammary carcinoma cell line S.W.
5 527 is A.T.C.C. HB 8143.
EXAMPLE II
Lymphocytes are obtained from a group of pa~ients
who have been diagnosed as having auto-immune
disease. These patients are pre-screened for the
presence of antibodies directed agains~ ~hymus derived
lymphocytes (T cells). These patients are also HLA
typed. The lyml~hocytes are processed as in Example I.
There are thus obtained cultures producing
an~ibody with a specifici~y for the 1' lymphocyte
popuLation. In the case of blobd from certain
patients, the specificity of hybridoma antibodies is
against functionally and an-tigenically distinc~ subsets
of the T cells.
A specimen of a cell line producing antibodies
with the specificities for llelper-T cells has been
deposited as A.T.C.C. HB 8145.
EXAMPLE III
For the production of leucocyte inhibiting factor,
LIF, it is desirable, using known specific
identification techniques based on the difEering
affinities of subsets of human T lymphocytes for sheep `
red blood cells, to isolate cells responsible Eor the
production of leucocyte inhibitory factor. Thus human
peripheral blood mononuclear cells are isoia~ed on
--10--
Ficoll~Hypa~ue gradients, washed and rosetted with
sheep erythxocytes. E+ cells are rosetted through
Ficoll-Hypaque gradients and treated with buffered
ammonium chloride to remove the red cells and washed
thoroughly. E+ cells are then sensitized with 1:he
monoclonal antibody Leu 3a (Becton-Dickinson), washed
and rosetted (800 ~ for lO minutes) with human red
cells coupled with affinity column purified rabbit
anti-mouse Ig. Rosetted mix-ture are la~ered on to
Ficoll-Hypaque gradients and centrifuged at lO0 g for
15 minutes. The Leu negative T cells remain at the
interface while the Leu 3a~ rosetted cells are formed
in the pellet.
The Leu 3a negative cells are cultured at a
concentration of 5xlO6/ml in one ml aliquots for 48
hours with the lectin known as concanavalin A (O.Ol
mg/ml) at 37C in a humid atmosphere with 5% CO2. Then
the supernatants of concanavalin A stimulated Leu 3a
negative cells are tested for inhibitory activity to
ensure LIF production. By first isolating such a LIF
producing subset, the development of the human clone is
greatly improved as compared to techniques using
general stimulation of T cells to produce LIF. Cells
fro~n strongly positive wells are pooled and used in the
fusion. ~he cells are washed thoroughly with
commercial RPMI 1640 medium containiny lO~ fetal calf
serum and then used as a fusion partner with a human
malignant cell.
In this case, since LIF is not an antibody
molecule, a malignan-t fusion partner of the T cell type
is used, which is not capable of producing LIF. A
mixture of 20,000,000 of these concanavalin A
stimulated cells and lO,000,000 cells of such
V~
lymphoblast T cells, e.g., of the line designated J.M.
by Rosewell Park in polyethylene glycol is centrifuged
and then further treated as in E~ample I to effect
fusion, separa~ion, culturing and subculturiny.
The following Table shows results of an assay of
the potency of L,IF produced by a human T cell
hybridoma, (A.T.C.C. HB 8144) thus produced in 3 tests
at a dilution of 1:1 to 1:1000, compared to
a) J.M. supernatant, previously tested to
ensure inactivity;
b) Eiuman anti-T cell hybridoma superna~ant' and
c) Positive con-trol supernatants of freshly
isolated T cells stimulated with
concanavalin A for 48 hours and diluted to
1:10.
The first determinations a) and b) were made to
minimize the possibility that the cell fusion produces
a non-specific inhibitory factor.
Potency o LIF Produced
by a Human T Cell Hybridoma*
Clone lB2E12 Dilution Mi~ration Index
_ Test 1 Test 2 Test 3
1:1 0.53 0.51 0.45
1:2 0.43 0.67 0.63
1:10 0.55 0.38 0.52
1:100 0.40 0~4~ 0.50
1:200 0.43 0.78 0.61
1:400 0.40
J.M. supernatant 1.15 1.08 0.96
Human anti-T cell 0.89 0.98 1.03
hybridoma supernatant
Positive control 0.65 0.57 0.66
* Indicator cells [polymorphonuclear leucocytes (PM~)~
were isolat~d by dextran sedimentation (molecular
weight 500,000). 20~ by volume of a 6% dextran solu-
tion prepared in normal saline was added to heparinized
blood in a 50-ml syringe. m e syringe was incubated
at room temperature for 30 minutes in an upright
position, and the buEfy coat cells were carefully
expressed. The cells were diluted in HBSS 1:2 and
centrifuged through a Ficoll-diatrizoate gradient. The
pelleted PMN were washed three times in EIBSS, and, when
necessary, any contaminating erythrocytes were lysed by
hypotonic shock. The PMN were suspended in an agarose
medium containing 10~ horse serum and 0.1% agarose.
Droplets ~0.002 ml) containing cells at 108/ml were
dispensed with a Elamilton syringe into flat-bottomed
microtitre plate wells, and 0.1 ml of hybridoma
supernatant or compared superna-tant was added to each
of three wells. After incubation Eor 4-6 hours at
37C, the areas of migration outside ~he droplets were
calculated using an inverted microscope with a
-13-
calibrated lOx ocular. The zone of migration from the
edge of ~he droplet to the border oE the migrating
cells ~Jas measured in Eour perpendicular directions;
the radius of the droplet was subtrac~ed from the area
of the migration zone. Results were expressed as a
migration index calculated as area of migration in
presence of mitogen divided by area of migration in
absence of mitogen.
EXAMPL~ IV
~n antibody prepared in Example I is mixed with
serum or ductile secretion from a woman sus~ected of
having mammary carcinoma. There is added a
precipitating agent such as goat anti-human antibody,
which has been radio-labeled. The mixture is
centriEuged at high speed to bring down the
precipitate. The precipitate is washed to remove
excess radioactivity and the resulting precipitates are
counted in a gamma counter.
EXAMPLE ~
Lymphocytes are obtained from the blood oE
patients in an active stage of juvenile rheumato.id
arthritis (JRA). The sear of these patients are pre-
screened by an assay for bindin~ to T cells from normal
donors and their lymphocytes are HLA typed. The
lymphocytes are then separated and subjected to the
-Eusion technique as in Example 1 using as ~he malignant
fusion partner lymphoblastoid T cells, e.g., the cell
line from J.M. RPMI (other T or B cell lines may also
be used).
After fusion, separation, culture and subculture
of the clone is conducted as in Example I. A desirable
culture medium consists of 90~ cor~nercial RPMI medium
plus 10% fetal bovine serum. Assays of the
supernatants obtained from the subcultures and sera of
the donor patients comparing reactivity to isolated T
cells from normal donors prove that ~.he clones make the
same type of antibody to JR~ as the patient's serum.
In order to elimina-te the possibility of non-specific
binding caused by products resulting from the fusion
process, supernatant from a human clone producing
leukocyte inhibiting factor was also tested on T cells
- from these normal donors; a negative result was
obtained.
EXAMPLE VI
Nionatal mice less than 2~ hours old are injected
interperitoneally with 0.03 ml of a mixture of 90~
cornmercial RPMI culture medium and 10~ fetal bovine
serum, the culture medium used in Example V for
subculturing. Thirty days later the mice are tested
for reaction to this mixture and only the toleriæed
mice, which do not react, are used. These tolerized
mice are injected with 0.5 ml of the supernatant
mixture frorn the JRA clone subculture of Example V.
Fourteen days later, the mice are given a booster shoot
of 0.5 ml of ~he same supernatant.
Fourteen days later the mice are bled from the
ocular sinus and the serum is tested for anti-ideotypic
antibody (antibody to JRA antibody). In a first test,
positive mouse serurn reacts with clone superna-tant to
cause precipitation; care should be taken to run a
control with clone~free medium which should be
ne~ative.
L'~
-15-
In another available test, the anti~ideotype serum
is tested with active serum from a patient in an active
stage of JRA to obtain precipitation, while negative
results are obtained from patients not exhibiting
disease activity. Spleens from mice giving a positive
-test for anti-ideotype antigens are then used for
fusion.
Plasmacytoma (e.g., NS-l Erom the Salk Institute)
is maintained in continuous culture at 37C in C02 and
used for the hybridizations. The growth medium
consists of a high-glucose modified Eagle's medium
(DMEM) (Gibco - Grand Island Biological, Inc., NY)
with 10~ fetal calE serum (FCS) and 2% antibiotic
mixture containing penicillin, streptomycin, and
amphotericin B. Cells are cultured in flasks or multi-
well culture plates and split, with new medium added
every other day. Immunoglobulin is not secreted by
this line, thereby alleviating the problem c,f
nonspecific secretion of immunoglobulin. Feeder layers
of macxophage are obtained by flushing the peritoneal
cavity with 5 ml 0.34M sucrose. Cells are washed in
medium with 10% FCS, resuspended to 2-3x104 ml in HAT
medium and then 1 ml is added to each well of a 24-well
culture plate. Incubation at 37~C in 10~ C02 is
carried out for 1 hour to allow feeder cells to adhere.
Sterile spleen cells from immuno-tolerant or
control mice are obtained by teasing in 10 ml Hank's
balanced salt solution (HBSS). Cells are transferred
into 15 ml centrifuge tubes, dispersed by pipetting,
allowed to stand 10 minutes, transferred to 50 ml
cen-trifuge tubes, washed twice with HBSS, resuspended
and counted. Approximately 108 lymphoid spleen cells
are combined with 107 washed myeloma cells and
16~
centrifuged at 400 g for 5 minutes. After removal of
the supernatant, the cell pelle-~ is gen~ly resuspended
and 300 ml of polyethylene glycol (PEG 4000) in HBSS
wlth 5% DMS0 are added, mixed for 30 seconds, then
cen-trifuged at 600 rpm for 6-7 minutes at room
temperature. Af~er B minutes in PEG, 5 ml of Hy medium
(the hybridoma medium shown below) is carefully added,
Eollowed by 5 ml medium with 20~ FCS. After incubation
for 1 minute at room temperature, the tubes are gently
swirled and then centriuged at 1000 rpm for 5
minutes. The supernatant is removed and 5 ml HAT
medium is added. After incubation at room temperature
or 5 minutes the tubes are gently resuspended and the
cells brought up to 48 ml in HAT medium and distributed
at 1 ml in each well of 24-well cluster p:Lates
containing macrophage feeder layers. Cells are
incubated at 37C in 10~ CO2. On days 1, 7, 10 and
every second day up to 3 weeks, one ml of medium is
removed from the wells and replaced by fresh HAT medium
up to day 14 and by hybridoma medium without HAT ater
that.
Hybridoma Medium - used for fusions contains:
- Dulbecco's MEM (modified Eagle's medium) wi~h
4.5g/L glucose
- 20% FCS (fetal calE serum)
- 10~ NCTC (Nat'l Collection of Type cultures~ 109
medium
- 584 rng/L L-glutamine
- 50 mg/L sodium pyruvate
- 132 mg/L oxaloacetate
- 20 units/L bovine insulin
- 1% pen-strep (penicillin~dehydrostreptomycin)
- 25 m/L lM Hepes
-17-
After two weeks, viable cell popula~ions are
tested for the presence of anti-ideopath.ic antibody as
above.
