CA1200484A - Human monoclonal antibodies and lymphokines - Google Patents

Abstract

Human Monoclonal Antibodies and Lymphokines Abstract A method is described for separating fused cells, resulting from fusion of human cells known to produce a specific antibody or a specific lymphokine with malignant human partner cells, from the said partner cells which comprises addition of specific antiserum capable of identifying antigenic specificities unique to the clone and non-reactive with the non-fused partner cells. After reaction of the fused cell with the antiserum, the reaction product is separated within 24 hours by indirect rosetting.

Description

~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~

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 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 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 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 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'~

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 After two weeks, viable cell popula~ions are tested for the presence of anti-ideopath.ic antibody as above.

Claims (7)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of separating fused cells, result-ing from fusion of human cells known to produce a specific antibody or a specific lymphokine with malignant human partner cells, from the said partner cells which comprises addition of specific antiserum capable of identifying antigenic specificities unique to the clone and non-reactive with the non-fused partner cells.

2. A method of claim 1, wherein after addition of the antiserum and reaction of the fused cell therewith, separation of the reaction product with the antiserum is carried out within 24 hours by indirect rosetting.

3. A method of claim 1, wherein said malignant partner cell is of the T cell type.

4. A method of claim 1, wherein said malignant partner cell is of the B cell type.

5. A method of claims 1 and 2 wherein the specific antibody has the ability to bind to target cells derived from tumors obtained from patients with mammary carcinoma.

6. A method of claims 1 and 2 wherein the specific antibody has the ability to bind human thymus derived lymphocytes or subsets thereof.

7. A method of claims 1 and 2 wherein the specific lymphokine is leucocyte inhibiting factor.