Monoclonal antibodies against lens epithelial cells.

DESCRIPTION tai

<img file="GR77941B_D0001.tif" />

The invention declared by Baylor College of Medicine entitled:

Monoclonal antibodies against lens epithelial cells that prevent the development of lens epithelial cell residues after extracellular extraction.

SUMMARY

Monoclonal antibodies against epithelial cells of the lens are developed and methods for their production. Continuous cell lines are developed to produce monoclonal antibodies to lens epithelial cells. Human lens epithelial cells, producing antibodies, are fused with myeloma cells to produce a fusion hybrid, the hybrid is cultured, and antibodies specific to human lens epithelial cells are harvested.

The growth of lens epithelial cell residues after extracellular extraction is inhibited by the introduction of monoclonal antibodies specific for lens epithelial cells into the anterior chamber of the human eye, which are allowed to react with the lens epithelial cells. A supplement is then instilled in the anterior chamber to cause lysis or other damage to the lens epithelial cells, thereby preventing them from proliferating and migrating to cover the surface of the remaining lens cuff. This can be done at the time of extracellular cataract extraction or later to remove a second cascade caused by the growth of these cells. There is no damage to other parts of the eye except to the remnants of lens epithelial cells.

BACKGROUNDS OF THE INVENTION

Offshore cataract extraction has recently become a fairly common method of excluding cataracts possibly due to the lower incidence of post-operative complications such as macular cystic edema and possible retinal detachment. The advent of an improved extracorporeal export such as the emulsification of the lens and the requirement of an intact rear lens cage for transplantation from a wide variety of lenses have certainly played an important role in influencing such a trend. The only possible disadvantage of extracorporeal cataract extraction is the low frequency of opacity of the posterior lens of the lens, which requires additional surgical methods (posterior catheterization or retraction of the posterior lens of the lens) to achieve.

The pathogenesis of opacification of the lens of the lens after extracellular cataract extraction is known: the epithelial cells of the lens proliferate in the back of the lens to form aberrant fibrous and edema cells (ie Elschn's).

As announced in the Journal of Phthalmic Medicine Vol. 5 No 4, Oct / Dec 1979, Cataract: Studies of the chemist and radiologist and others, chemical and radiological means have been instrumental in finding a method associated with the cataract - it would decrease. * frequency of waterfall. As announced in this contact lens and endocell 175-178, After personal suspension, its. Rdy proceeded to the syringe of the chemist. posthumously? publishing pwb aquifers?

<img file="GR77941B_D0002.tif" />

were used (vincristine and vinblastine) an attempt was made to inhibit chemically subcutaneous epithelial cells because they were found to have direct inhibitory effect on cell mitosis (Goodman

LS and Gillman, A: The Pharmacological Action of Maximilan Therapeutics, New York, 1965, pp. 1373-1376). Vincristine and vinblastine were found to inhibit corneal wounds so that they healed poorly and due to the devastating effects on the cornea and iris it was the authors' view that these drugs should not be used in animal studies. . The authors further stated that radiation given the second day after surgery proved to be the most effective of all dosing regimens, however, indicated that there was a risk of injury and concluded that it was difficult to say if anyone was using radiation people whether there would be problems or not.

The authors further showed that if there was a pharmacological or chemical system that could selectively inhibit subcutaneous epithelial cells, this would be a useful way to help prevent post-cataracts.

Applicants are aware of the instillation of mitothexate and retinoic acid mitotic inhibitors or mixtures thereof in the anterior chamber of the eye in minimal effective doses at the end of the lens epithelial cell cycle, which instillation of the optic nerve effectively prevents exposure after extracurricular cataract extraction.

Methotrexate is a cyclo-dependent anti-metabolite, which inhibits the dihydrophilic reductase enzyme and thus interferes with the maintenance of the intracellular reservoir of diminished decolorized cells.

Retinoic acid, whose exact mechanism is unknown, appears to inhibit either cell division or DNA synthesis or both.

The present invention is an improvement from the production and use of monoclonal antibodies specific to the residues of lens epithelial cells, which can be used to selectively destroy these cells without damage to other parts of the eye at the time of primary cataract exclusion.

PREVIOUS TECHNICAL DECLARATIONS

Applicants are unaware of any technique involved in producing monoclonal antibodies specific to lens epithelial cells or in the use of such antibodies in selectively destroying lens epithelial cell residues without damage to other parts of the eye.

Representative examples of prior techniques related to the production of monoclonal antibodies are the following: Monoclonal antibodies, 1980, Plenum Press, New York, written by Roger H. Kenett, Thomas J. Mckearn, and Cathleen B. Bechtol * continuous fusion cultures. cells that produce predetermined antibodies; : 2.5 ^ Ί. .-August ^ ΐ 1375, '₇ 495 -437 and the following US patents related to the production of single, all-Z clonal antibodies No 4,271,145 * 4,196,265 * 4,172,124 * 4,195,125 *

4,262,090 and 4,294,927.

SUMMARY OF THE INVENTION

The present invention is directed to monoclonal antibodies specific to lens epithelial cells, methods for their production including continuous cell lines from which they are derived, and their use in destroying residues of lens epithelial cells selectively without damage to other portions of the lens. time of exclusion of primary cascade or later of exclusion of a meta-cascade. The monoclonal antibodies are instilled in the anterior chamber of the human eye and allowed to react with the lens epithelial cells. The supplement is then instilled in the anterior chamber to cause selective lysis or other damage to the epithelial cells of the lens without damage to other parts of the eye. This represents an obvious development in the use of mitotic inhibitors and other methods in that the destruction is specific only to remnants of lens epithelial cells, which otherwise multiply and migrate to cover the surface of the left capsule left behind. causes a secondary cataract resulting in loss of vision requiring a second operation.

Accordingly, it is an object of the present invention to provide monoclonal antibodies specific to lens epithelial cells.

It is yet another object of the present invention to provide a continuous cell line for the production of monoclonal antibodies specific for lens epithelial cells.

It is yet another object of the present invention to prevent the opacification of the lens capsule due to lens epithelial cell residues, which multiply and migrate to cover its surface after extracapsular extracellular cataract extrusion by monoclonal instillation. lens at the time the primary waterfall was excluded, allowing these antibodies to react with these lens epithelial cell residues and then instill a complement that solves these cells.

Still another object of the invention is the exclusion of post-cataracts caused by the growth and migration of lens epithelial cells, by instilling monoclonal antibodies in the anterior chamber of the eye, allowing these antibodies to react with these epithelial cells. after instilling a supplement that solves these cells. Other additional purposes, features and advantages are illustrated by the invention.

DESCRIPTION OF THE PREFERRED APPLICATIONS

The present invention is directed to methods for preventing the growth of lens epithelial cell residues after extracellular extraction by implanting monoclonal antibodies specific to these lens epithelial cells in the anterior chamber of the human eye and allowing them; -lab cell Lens Normally about autoclave antibodies were instilled and normally required about 30 / X

Minutes of time for these monoclonal antibodies to react with the epithelial cells of the lens. An anchor supplement is then instilled in an effective amount of approximately 100 µL, which causes lysis or other damage to the lens epithelial cell residues, thereby preventing them from proliferating and migrating to cover the surface of the lens capsule which remained. This can be done at the time of extracapsular cataract extraction, preferably immediately after cataract exclusion, or it can be done later to exclude a second cataract caused by the proliferation and growth of these cells on the surface of the lens capsule.

These monoclonal antibodies specific for lens epithelial cells are produced by fusion of human cells producing antibodies to the lens epithelium with myeloma cells to produce a fused hybrid, the hybrid is cultured, and the specialized epithelial cells are .

The complement is a stable complement, for example a standard complement, and its preparation, useful in the present invention, are described in Monoclonal Antibodies, supra, p. 391-2.

The following are methods for producing monoclonal antibodies against lens epithelial cells.

CELLULAR CULTIVATION

Human lens epithelial cells are recovered either from human eyes within 30 minutes of death or from tissues excluded during cataract surgery. The cells are grown as a layer in tissue culture incubator using well-tested techniques.

IMMUNIZATION WITH LENGTH CELL LENS

A mouse (BALB / c or other appropriate series) is injected intraperitoneally or intravenously with 5-10 million pure cells. Two weeks later a blood sample from each animal is tested for a specific antibody.

The higher-tailed animal is injected intraperitoneally or intravenously with 5-10 million pure cells.

SLEEEN OF IMMUNE SPLINE CELLS WITH MYELOMA CELLS

Three to four days after the (intravenous) immunization of the mouse, it is sacrificed by neck dislocation. Then it blows and the serum freezes. The mouse is purified with 70% ethanol and the spleen is sterilized. Using the sterile plunger of a sterile, easy-to-use 3-ml syringe, the tube is purged through a 50mesh stainless steel sieve with warm HBSS. The suspension is pipetted up and down sometimes with a 3ml syringe. A monocellular suspension is prepared by passing the suspension through a 200-mesh stainless steel sieve. The spleen cells are centrifuged for 10 minutes at 1200 rpm. The red blood cells were analyzed by placing 0.83% NH4Cl for 5 minutes at 40 degrees Celsius.

The spleen cells are washed twice in serum-free medium. Cells are counted and their viability determined by trypaSi-blue staining assay. '' · ·^! λ ·· '· - · -

<img file="GR77941B_D0003.tif" />

/5

The spleen cell suspension is prepared from non-immunized BALB / c mice as a trophic layer on hybrids. Myeloma cells are transferred to growth phase (5x10 ° cells / ml) in a 50ml polypropylene centrifuge tube. The spleen and myeloma cell suspensions are washed separately twice in serum-free medium. The cells are counted, pooled and washed once to obtain a mixed pellet (10 * spleen cells and 10? Myeloma cells). The centrifuge tube is gently closed to disperse the pellet in a 0.0ml suspension of 50% PEG added over one minute (37 degrees C). The suspension is allowed to stand for one minute. One ml of free serum medium is also added over one minute. 20 ml of free-standing medium is added over 5 minutes.

The cells are centrifuged and resuspended in 60-100ml of HAT-containing hybrid medium and 2-4x10x spleen cells from normal mouse BALB / C. Exact portions of 0.1 ml are divided into 96-well microbeads and incubated at 37 degrees Celsius in 10% CO2. An additional 0.1 ml of growth medium HT is added at 7 days when fast growth is observed. The HY medium is used until the sub-cultures are made. Changing the medium is repeated every 3 to 4 days. When colonies are visually observed (between 12 and 20 days) the clones are selected. 100ml of culture supernatants are collected for primary observation of antibody activity.

The materials used in the fusion of spleen immortal cells with myeloma cells are presented in Table I below.

TABLE I

A. MATERIALS:

1. 50% polyethylene glycol (PEG) 1540 (Polysciences) lml sterile PEG 1540 lml free-serum medium (SF-DMEM)

2. Thymidine (T) 1.6x10 ^ M 1.0x10-4; Hypoxacitin

4x10- ^ M. Aminopterin

a. 10Ox HT solution from stock

Dissolve: 0.01361g of hypoxanthine

0.0388 g of thimizine in 100ml of distilled water heated to 70-80 degrees Celsius

Sterile filters, split in equal parts and stored at -70 degrees Celsius.

b. 100x aminopterin solution from stock Dissolve: 0.0l8g in distilled water

Add D, IN NaOH dropwise if the aminopterin is not readily dissolved. Adjustment to pH 7.8

Sterile filters and refrigerated storage at -70 degrees. Celsius

c.

Hybrid medium / 6 \

MEM Dulbecco with High Glucose (4.5g / l) L-Glutamine Added to 4 Mm2% of 100 kmsnol 'Serum Type (Kappa; SexsntiJ; ic} imji Sodium Potassium (GIP co;' Non-essential amino acids (Gibco) 50 M B -Ethiocyanate

5ml HAT buffer.

\ What

Enzyme-conjugated Immunosuppressive Reaction (ELISA) for Cells

50µl of 5% glutaraldehyde in 0.1M NaHCO3 is added to each well of 96-2311 microtiter plate and left at room temperature for at least 30 minutes. A washed target cell suspension in HEES '- buffered saline (10HB).^G. cellulose is prepared. The plates are washed three times by filling the recesses with distilled water and knocking out the water. Rinse with 15M NaCl with 0MM Na2HPO4 (PBS-0), and the liquid is struck out. 50µl / recess of the cell suspension is added and the plates are centrifuged at 1500 rpm for 3 minutes at the time of stopping. 200µl / recess 1% formaldehyde in HbExE is added and left at room temperature for 15 minutes. The plates are centrifuged and the liquid is discarded. The plates are washed three times by pouring PBS-9 into the recesses and flushing out the liquid. 50µl / 1% BSA recess in PBS-9 is added to each plate and left for 10 minutes at room temperature. 50µl of hybrid media samples are added in double recesses of 5DMEM + 2% RS, added to row 1 of each plate and incubated for 90 minutes at room temperature or overnight in the refrigerator. The plates are washed 10 times with 0.05% Triton x-100 in distilled water. 50µl / recessive rat attachment, peroxidase-conjugated goat anti-mouse immunoglobulin IgG fraction dissolved 1:30 from the frozen stock is added to 0.5M NaCl, 0.5% Triton x-100, 0.5MM Na2 HPO4 and left for 10 minutes at room temperature. The recesses are washed 10 times with 0.05% Triton x-100. 100µl / recess of the substrate: O, IM yellow sodium containing 1/100 volumes 40mM 2.2¹-Azino-di- (3-ethylbenzthiazoline sulfonic acid) diairanonicum salt (ABTS) and 1/100 30% hydrogen peroxide is added. Substrate is added to row 1 of the clean plate.

The spectrophotometer turns on. After 30 minutes the plates are read at OD 414. Only medium readings (row 1) are determined for each plate. Mean values ​​and SD standard deviations are calculated and samples are considered positive if mean values ​​are SD 2 SD.

The mean of the controls is subtracted from each positive sample and the specific OD is indicated.

CYTALOLYSIS OF LENS CELLULAR CELLS

The supernatants for testing are divided into amounts of 1 to 5µl in micro-wells. The cells were washed in 0.1% BSA and suspended at approximately 2000 cells / µl. Img cells to be added are added to each well and incubated with the antibodies for 1/2 hour at room temperature. 5µl of rabbit serum, which gives a satisfactory solution with the control antibody and not a solution when added without additional antibodies and incubated at room temperature for 1 hour.

The percentage of killed epithelial cells of the lens is read under the microscope.

HISTORICAL CRITERIA AND CELLULAR FOR ANTISOMIC SPECIALTY

Cultures in a layer of lens epithelial cells are first produced with the antibodies and then supplemented (as described in the previous chapter). The cultures are then observed under the microscope to examine whether all lens epithelial cells have been resolved. From previous assays, antibodies from most promising clones are used to test whether these antibodies actually destroy monads, lens epithelial cells, and not other ocular tissues using lb

<img file="GR77941B_D0004.tif" />

intact human eyes or anterior chambers and observing the results histologically. The long-term cytotoxicity and efficacy of these antibodies are attested by injecting the antibodies and supplements into the anterior chambers of monkeys in. situ following extracapsular lens extraction. The long-term progress of the treated eyes will be compared to that of the untreated with ophthalmic observations and histological studies.

PRODUCTION OF LARGE-SCALE ANTIBODIES

Large-scale production of a single monoclonal antibody can be achieved by injecting about 10 hybridized cells into appropriate H-2 compatible mice. Ascotic tumors are introduced by the following method: To induce ascites, mice are injected intraperitoneally with 0.5ml pristane 2,6,10,14. tetramethylpentadecane, Aldrich and left for 1 to 2 months. 3-4 days before transfection of the intermediate classes of hybridomas, each mouse is injected with 50µl of non-lymphocyte serum. On the day of tumor metastasis, each mouse receives total body radiation (600 to 800 vads) followed by 6-8 hours later of congenital bone marrow (10? Cells / mouse). Hybrid cells (10 - 10?) In Dulbecco's Modified Eagle's medium are then injected intraperitoneally. As tumors begin to appear (10 to 30 days after injection) mice are bled and the presence and densities of antibodies in serum are constantly monitored. Appropriate antibodies are collected, purified and stored.

Modified methods for large-scale production of these antibodies include introduction of subcutaneous tumors using the method described above. The hybrid cells grow in tissue culture and the media containing the antibodies are constantly harvested.

The present invention, moreover, is well adapted to achieve the objects and purposes and has the features and advantages mentioned as well as other inherent in it.

While preferred embodiments of the invention have been provided for the purpose of development, modifications and modifications may be made in the spirit of the invention as defined by the scope of the appended claims.

What is claimed is