Monoclonal antibody against ovarian cancer cells (OVB-3)

Monoclonal antibodies are produced which specifically bind to human ovarian cancer cells. These antibodies are conjugated to Pseudomonas exotoxin in order to produce an immunotoxin suitable for the chemotherapeutic treatment of human ovarian cancer.

BACKGROUND AND GENERAL DESCRIPTION OF THE INVENTION 
Current approaches to cancer chemotherapy and other immunological therapies 
focus on the use of cell-specific therapeutic agents. Ideally, 
immunotoxins should discriminate to a high degree between target and 
non-target cells. The present invention discloses an immmunotoxin 
conjugate formed between monoclonal antibody OVB-3 (which specifically 
binds to human ovarian cancer cells) and Pseudomonas exotoxin. 
Since the advent of the monoclonal antibody methodology disclosed in 
Koprowski et al (U.S. Pat. No. 4,172,124) many monoclonal antibodies 
designed to treat a wide variety of human ailments have been developed. 
However, two problems have prevented this methodolgy from being practiced 
more extensively. First, many monoclonal antibodies to hmman target (e.g. 
cancer) cells also bind to non-target (normal) human cells. Secondly, many 
of the toxin delivery systems reduce the effectiveness of the toxin, 
reduce the entry capacity of the toxin into the target cell, or are not 
specific enough to deliver a sufficent amount of toxin to the target site. 
The present invention discloses an immunotoxin which differentiates 
between normal and cancerous cells, is highly specific for human ovarian 
cancer cells, and is capable of carrying and delivering a toxin to the 
cancer cell without destroying the toxin's entry and lethal activity. In 
this manner, the present invention satisfies a long felt need in the area 
of cancer chemotherapy: very few ovarian cancer monoclonal antibodies 
exist, and those that do are not capable of delivering an exotoxin in 
condition for the cancer cell to internalize the endotoxin. Furthermore, 
the present invention uses recently developed technology to produce an 
effective immunotoxin which specifically binds to ovarian cancer cells 
without binding to normal human cells. 
The present invention builds on the discovery disclosed by the same 
inventors in U.S. Pat. No. 4,545,985, titled "Pseudomonas Exotoxin 
Conjugate Immunotoxins." This patent is hereby incorporated by reference 
because it discloses the method used in the present invention for 
modifying Pseudomonas exotoxin so that the toxin will selectively kill 
target human tumor cells. 
The present invention is an improvement on this process; with the 
production of highly specific monoclonal antibodies, human ovarian cancer 
may be targeted and treated by the products of this invention. 
Pseudomonas exotoxin (PE) has been conjugated to a variety of monoclonal 
antibodies recognizing certain human tumors and to a monoclonal antibody 
recognizing the human y antigen blood group substance [Richert et al, J. 
Biol. Chem., 258:8902-8907 (1983); and Fredman et al, J. Biol. Chem., 
258:11206-11210 (1983)]. The toxin conjugates specifically kill the 
appropriate target cells. PE can now be conjugated to a variety of 
peptides, proteins, and growth factors that react with specific receptors 
on cells. These include sarcoma growth factors, melanocyte stimulating 
hormone (MSH) somatostatin, glucagon, insulin, transferrin, low density 
lipoprotein, calcitonin, alpha.sub.2 -macroglobulin, and 
lysine-bradykinin. Pseudomonas exotoxin is particularly preferable to 
other toxins (such as ricin or diphtheria toxin) because large amounts are 
easily prepared, because humans do not usually have neutralizing 
antibodies against it (as is the case with diphtheria toxin), and because 
it does not need to be separated into subunits before being conjugated (as 
does ricin toxin). 
DEPOSIT STATEMENT 
The subject matter of this invention, monoclonal antibody OVB-3 has been 
deposited in the American Type Culture Collection in Rockville, Md., under 
ATCC No. H89147 , and will be maintained for a term of thirty (30) years 
or five (5) years after the last request for such deposit or for the 
effective life of the patent, whichever is longest. The deposit will be 
replaced if the culture mutates or becomes nonviable during the term of 
the deposit.

SPECIFIC DISCLOSURE 
Pseudomonas exotxin (PE) is a known and readily available toxin isolated 
from Pseudomonas aeruginosa. The particular exotoxin used in this 
invention is commercially available through the Swiss Serum Company. 
PE was chosen for this invention because it acts in the cytosol of the cell 
to inhibit protein synthesis by catalyzing the enzymatic 
(ADP-ribosylation) inactivation of Elongation Factor Two. 
Monoclonal antibodies (Mabs) specific for ovarian cancer cells exist [see, 
for example, Bast et al, J. Clin. Invest., 68:1331-1337 (1981); and Tsuji 
et al, Cancer Research, 45:2358-2362 (1985)], but either are poorly 
internalized by human ovarian cancer cells or do not exhibit the degree of 
specificity shown by the monoclonal antibody of the present invention. 
Monoclonal antibody OVB-3 is produced by conventional methods. In general, 
mice are immunized with OVCAR-3, a human ovarian cancer cell line 
[described in Hamiliton et al, Cancer Research, 43:5379-5389 (1983)]. 
Spleen cells from the immunized mouse are then fused with a myeloma cell 
line, thus forming hybridomas which are capable of producing monoclonal 
antibodies which specifically bind to human ovarian cancer cells. One such 
Mab, OVB-3, is highly specific for human ovarian cancer, and is used to 
instruct the Pseudomonas exotoxin conjugate. 
The Table shows the reactivity of monoclonal antibody OVB-3 with a variety 
of ovarian cancer cell lines and with normal human cells. This Table shows 
that the monoclonal antibody of this invention is highly specific for 
human ovarian cancer cells. 
Pseudomonas exotoxin-monoclonal antibody OVB-3 conjugates (PE-OVB-3) are 
constructed either using a disulfide exchange reaction or by forming a 
thioether bond. Generally, PE is treated with 2-iminothiolane (formally, 
methyl-4-mercapto-butyrimidate) in order to introduce two thiol groups per 
molecule of toxin. This step is optimally conducted in 0.15 M KPO.sub.4 
(pH 8.0), 1 mM EGTA. Derivatized PE from the above step is then reacted 
with dithiobis(2-nitrobenzoic acid), DTNB. Purified OVB-3 is also treated 
with 2-iminothiolane in order to introduce one sulfhydryl group per 
molecule. The treated antibody is then mixed with excess treated PE at pH 
8.0 and allowed to incubate for 2 hours at 23oC. At the end of the 
reaction, the pH is adjusted to 7.0 and cysteine is added to displace the 
TNB from any PE molecules that have not formed conjugates or that have one 
unreacted --SH group. 
Alternatively, the antibody can be modified with m-maleimidobenzoyl N 
hydroxy-succinimide ester (MBS) and the resulting activated antibody is 
reacted with SH--PE--SH to produce a conjugate containing a thioether 
bond. This conjugate is more stable in an animal environment since it 
cannot be inactivated by reduction of the disulfide bond. 
The resulting PE-OVB-3 conjugate is purified by gel filtration. Typically, 
1-5 ml of conjugate at 3-5 mg/ml is passed over an HPLC gel filtration 
column, TSK-250 (600.times.21.5 mm). Aggregates in the void volume exhibit 
low activity and are discarded. Two conjugate peaks, of higher molecular 
weight than native antibody, are included in the column. These correspond 
to 2:1 and 1:1 antibody-toxin conjugates. Both peaks have high activity, 
but usually only the 1:1 conjugate is used for further studies. The 
conjugate is assayed by adding it to human ovarian cancer cells and 
measuring inhibition of protein synthesis or cell death. The 
ADP-ribosylating activity of the conjugates is also assayed in cell-free 
extracts, usually reticulocyte lysates, using .sup.14 C-NAD as described 
in Fitzgerald et al, Cell, 32:607 (1983). All experiments were conducted 
either in vivo in mice, or in vitro on human cells in tissue culture. 
Approximately 50.times.10.sup.6 ovarian cancer cells in 0.5 ml saline are 
injected intraperitoneally into nude mice. These cancer cells are allowed 
to establish for 4-5 days and then PE-OVB-3 is administered in 4 daily 
injections. Untreated mice die after 40 days with massive malignant 
ascites. PE-OVB-3 is cytotoxic for these ovarian cancer cells and kills a 
high enough percentage of them to allow the mice to live 40-50 days longer 
than mice receiving no treatment, mice receiving the antibody alone, or 
mice receiving an irrelevant antibody-PE conjugate. Furthermore, PE-OVB-3 
is cytotoxic for cells expressing the OVB-3 receptor, but is inactive 
against receptornegative cells. 
THE TABLE 
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Summary of Localization of OVB-3 
Tissue Reactivity Detected 
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Ovarian Tumors (NIH): 
#1 (O.S.) (++) 
#2 (G.) (++) 
Ovarian Tumors (Alabama): repeat: 
86-M (poorly differentiated 
(++) #2- (++ var) 
adenocarcinoma) 
86-R (mucinous well-differ- 
(++) #2- (++) 
entiated adenocarcinoma) 
81-N (mixed Mullerian poorly 
(++) #2- (++) 
differentiated carcinoma) 
86-O (serous cystadenocarcinoma) 
(-) #2- (+/-)-(+) 
Breast Tumors (Cetus): 
BCNA (invasive lobular or ductal 
(+-+++) 
carcinoma) 
BCBA (invasive ductal carcinoma) 
(+-+++) 
BCUA (Paget's disease of breast) 
(-) 
BCTA (invasive scirrhous carcinoma) 
(+/-) 
Pituitary (-); occ. cells 
reactive in pars 
intermedia 
#2 repeat:(-); occ. 
cells in anterior 
lobe 
Thyroid (++) epithelial 
follicular cells 
Parathyroid (-) 
Breast (++) ductal 
epithelium 
Heart (-) 
Lung (-); ?(+) 
peribronchiolar 
dendritic cells 
Liver (-) 
Gallbladder (- ) 
Lymph Node (++) scattered 
endothelial & 
germinal cells 
Spleen (-) 
Bone Marrow (-) 
Adrenal (+) chromaffin cell 
granules?; (-) 
cortex 
Kidney (++) apical surface 
of proximal tubule 
cells 
Prostate (+++) secretion; 
(+/-) epithelial 
cells 
Testis (-) seminiferous 
tubules 
Ovary (-) 
Uterus (-) 
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