Anti-tumor agent from human serum and process

The invention relates to an anti-tumor agent prepared from human serum by the process steps of multiple salting out, centrifuging, desalting and gel filtration.

BACKGROUND 
This invention relates to an anti-cancer agent and process for preparing 
same. More particularly, this invention relates to an agent obtained from 
human serum which inhibits and/or prevents the growth of tumor cells. 
The presence of an anti-tumor factor (TNF, tumor necrosis factor) in the 
serum of animals has been described in Cancer Letters, 6: 235-240 (1979). 
Following the injection of bacterial endotoxin, TNF is found in the serum 
of mice inoculated with Corynebacterium parvum (CP). TNF was purified 
about 50 fold and is believed to be a .alpha..sub.2 globulin having a 
molecular weight of about 150,000. TNF was detected by an in vitro assay 
using mouse L-cells and by an in vivo bioassay, using female BALB/c mice 
bearing a methyl cholanthrene-induced fibrosarcoma. 
TNF-like activity has been found in extracts of liver microsomes from 
CP-treated mice and in very low, but measurable amounts in microsomes from 
livers of normal mice. TNF has also been found in the serum of normal 
mice. 
A fraction of the .alpha..sub.1 - .alpha..sub.2 globulins from serum of 
normal humans has been found to be cytotoxic for mouse L cells in culture 
and Meth A tumors in mice (Cancer Letters, 6: 235-240 1979) and inhibited 
the growth, in vitro, of human colon cancer, melanoma and neuroblastoma 
cells lines (J. Cell. Biol., 79: 67, 1978). 
SUMMARY 
The present invention provides a process for preparing an anti-tumor agent 
from human serum. Blood is coagulated naturally and the serum is separated 
from the coagulated components. The serum is centrifuged to remove fats. 
The serum is diluted with an isotonic buffer and a first portion of 
proteins is removed by salting out, for example from a 35% saturated 
solution of ammonium sulfate. A second portion of proteins is recovered 
from the same serum by further salting out, for example from a 70% 
saturated solution of ammonium sulfate. The second portion of proteins is 
collected, dialyzed to remove the salt and gel filtered. A gel filtered 
active fraction having a molecular weight of more than 150,000 is 
collected. 
DESCRIPTION 
The blood employed in the process of the present invention is human blood 
and all steps should be carried out under sterile conditions. The blood is 
allowed to coagulate naturally and the coagulated components are removed 
from the blood serum by centrifugation. 
The isotonic buffer employed in diluting the serum is preferably an aqueous 
solution of about 0.05 molar in potassium phosphate and containing about 
0.9 percent of sodium chloride. The dilution can be achieved by admixing 
about one-half to two-fold volume of isotonic buffer solution with the 
serum. 
Dialysis is preferably carried out using a semi-permeable casein membrane 
having a pore size for excluding molecules having a molecular weight of 
more than 10,000. 
The recovered protein portion is preferably gel filtered through a 
polyacrylamide-dextran gel. The gel filtered protein is separated into 
fractions depending on the running time through the filter. The active 
fraction (called "nHG") is further separated by electrophoresis on a 
semi-solid gel comprising acrylamide and a cross-linking. 
The present invention further provides an anti-tumor agent from human serum 
comprising a protein fraction (nHG) having a molecular weight of more than 
about 150,000 which is stable to heat at 90.degree. C. for at least one 
hour, non-dialyzable and inhibits the growth of HeLa tumor cells in vitro. 
The nHG fraction can be dispersed in an aqueous solution to a final 
concentration from 0.5 to 2.5% and is preferably dispersed in an isotonic 
saline solution. The nHG fraction can be further concentrated by freeze 
drying. 
The blood is allowed to clot naturally and the coagulated material is 
separated by centrifuging. The serum is then further processed in an ultra 
centrifuge. The centrifugal force can range from about 10,000 g to about 
200,000 g and is preferably about 105,000 g. The centrifuging time can 
range from about 2 minutes to 2 hours. Preferred centrifuging time is 15 
minutes. 
The remaining serum is diluted with a buffer which contains a salt or salts 
from the group of potassium phosphate and sodium chloride. The 
concentration of the buffer solution is preferably about 0.05 molar in 
phosphate and about 0.9% sodium chloride. A 0.9% concentration of sodium 
chloride provides an isotonic solution. The serum is diluted with about 
from 1/2 to 2 times the volume of the serum with the buffer solution. 
Preferably the amount of buffer added to the serum is of the same volume. 
The following steps are carried out at 4.degree. C. using a water and ice 
mixture. 
One hundred ml of the buffered serum solution are taken and under stirring 
anhydrous ammonium sulfate is added slowly. The amount of anhydrous 
ammonium sulfate is 24.5 g which brings the solution to about 35% 
saturation. During the addition of the ammonium sulfate, ammonium 
hydroxide is added at the same time to counteract the acidity of the 
ammonium sulfate and to keep the pH value constant at 7. The addition of 
the ammonium sulfate causes a first portion of proteins to precipitate. 
This solution is allowed to stand at 4.degree. C. for one hour. The serum 
is now centrifuged under conditions similar to those of the prior 
centrifugation step, e.g. at a force of 105,000 g for about 15 minutes. 
The resulting precipitate is discarded and another 24.5 g of ammonium 
sulfate is added to the solution in the manner described above along with 
ammonium hydroxide to keep the pH value at about 7. The solution is 
allowed to stand for about 1 hour at 4.degree. C. and is centrifuged 
again. At this point the serum is discarded and the precipitate is 
collected. 
The precipitate is dialyzed to remove the salt in a commercially available 
membrane made from semi-permeable casein. The pores of the membrane 
exclude passage of materials having a molecular weight of more than about 
10,000 and preferably 20,000 and above. The dialyzing medium is an 
isotanic aqueous solution containing 0.9% sodium chloride. 
The solution resulting from the dialyzing process is then subjected to gel 
filtration. The gel employed can be Sephacryl S-200 which contains beads 
having a diameter of about 40-105 .mu.m which are swelled with water and 
consists of polyacrylamide plus dextran. Every 100 drops of the filtrate 
are collected separately and if desired, concentrated by lyophilization 
(freeze drying). After the lyophilization the resulting concentrate is 
dialyzed to remove the salt. The resulting fractions are then contacted 
with tumor cells such as HeLa cells which have originally been obtained 
from humans having cervical cancer. The cells (500) are placed in a Petri 
dish. Then the cells are contacted with a fraction obtained by the process 
described. Thereafter, nutrient solution medium is added to allow the 
cells to grow. In a control or blank test where no nHG is added, numerous 
cells grow and can be stained with a dye such as Amido Black. 
The initial fraction of the gel filtration containing the highest molecular 
weight proteins is the most active agent. The molecular weight of this 
material is at least 150,000 and is believed to be derived from an 
.alpha.-globulin of the human serum. The active fraction remains stable to 
heat at 90.degree. C. for at least one hour. The nHG fraction can be 
subjected to dialysis with a Visking membrane excluding the passage of 
molecules having a molecular weight of higher than about 20,000. The agent 
remains active. 
The active fraction was further analyzed by electrophoresis on a semi-solid 
gel comprising acrylamide and a cross-linking agent. The resulting 
fractions of proteins in the gel were stained with Coomassie Blue G-250. 
For each gel in the analysis about 4 mg of substance can be employed. The 
speed of passage through the gel depends on the size and the charge of the 
molecules. Albumin moves relatively fast having a molecular weight of only 
about 70,000 and appears at the bottom of the gel. A pattern evolved 
showing at the top about 4 rings of stained proteins which indicated the 
various components of the active composition. The active nGH fraction can 
be extracted from slices of the gel. 
The active nHG fraction can be further analyzed by immunoelectrophoresis. 
In the immunoelectrophoresis it is found that the nHG fraction included 
albumin, haptoglobulin (an agent active against inflammation), 
.alpha..sub.1 -anti-trypsin and an .alpha..sub.2 -macroglobulin. 
The immunoelectrophoresis also indicates that the active nHG fraction does 
not contain immunoglobulin, IGG, IGA, IGM and Kappa chains and Lambda 
chains.

EXAMPLE 
Preparation of active nHG fraction from serum. All procedures were carried 
out at 4.degree. C. One hundred milliliters of serum was sterilized by 
filtration through a sterile 0.22 .mu.m millipore filter and was diluted 
with 100 ml of sterile phosphate-buffered saline (PBS). Forty-nine grams 
of solid (NH.sub.4).sub.2 SO.sub.4 was added slowly with stirring. The pH 
was maintained at 7.0 by addition of NH.sub.4 OH. The precipitated protein 
was removed by centrifugation at 105,000.times.g for 15 minutes and 49 g 
of (NH.sub.4).sub.2 SO.sub.4 was added to the supernatant solution with 
stirring. The insoluble material [35-70% saturation with respect to 
(NH.sub.4).sub.2 SO.sub.4 ] was collected by centrifugation, taken up in a 
small volume of sterile water, dialyzed against sterile PBS containing 
penicillin and streptomycin from the Sloan-Kettering Institution Media 
Laboratory until free of (NH.sub.4).sub.2 SO.sub.4 and diluted with 
sterile PBS to a final protein of 500-700 mg/ml. 
Gel filtration of the 35-70% fraction was carried out on a column of 
Sephacryl S-200 (superfine), from Pharmacia, Upsala, Sweden, previously 
equilibrated against Sterile PBS. The bed height was 85 cm., the eluant 
was PBS and the flow rate was 15 ml/h. The gel column was first calibrated 
for determination of molecular weights with dextran blue (void volume), 
purified aldolase (160,000), bovine serum albumin (67,000), soybean 
trypsin inhibitor (21,600), and cytochrome c (12,500). Ten milliliters 
aliquots of the dialyzed 35-70% fraction from each test serum were 
filtered through the column and the protein which was eluted between 195 
and 236 ml of PBS was pooled, concentrated by lyophilization and dialyzed 
against 0.15 M NaCl until free of phosphate. The concentrated material, 
called nHG, was sterilized by filtration through a sterile Nalgene 0.22 
.mu.m filter. 
Protein was determined by the method of Lowry et al. (1951): Protein 
measurement with the Folin phenol reagent, J. Biol. Chem., 193: 265-275, 
agarose electrophoresis was as described in the Corning Operations Manual 
for Agarose Electrophoretic Systems No. 47166, Corning 490; San Antonio 
Road, Palo Alto, Calif. 
A purified fraction (nHG) of the .alpha..sub.1 -.alpha..sub.2 globulins was 
prepared from serum by ammonium sulphate fractionation, Sephadex 
filtration, PAG-electrophoresis and affinity chromatography. Its effect on 
two normal fibroblast cell lines (W138) and one from human skin, 
maintained at (MSKCC) and on HeLa cells were determined. Survival of the 
HeLa cells is disclosed in terms of colony formation by single cells, 
following a 24-hour exposure to 50, 100, 150, 200 or 250 .mu.g nHG/ml. At 
these doses, cell survival relative to controls was 90%, 78%, 16%, 3% and 
0.5%, respectively. In contrast, W138 and skin fibroblast cells grew 
normally after 24 hours of exposure to 500 .mu.g nHG/ml. Whole human 
serum, buffer extracts of the Sephadex column and non-globulin proteins 
had no anti-NeLa cells activity. Dialysis of nHG in the presence and 
absence of EDTA did not alter its activity. Up to 100 .mu.g E. coli 
endotoxin/ml did not inhibit HeLa cell growth. These effects were recorded 
by time lapse cinematography. Five hundred .mu. g nHG/ml was seen to be 
first cytostatic then cytotoxic; 50% of the HeLa cells were dead after 41 
hours and more than 95% were dead in 44 hours. Under identical conditions 
no adverse effects were seen in the normal fibroblast cell cultures in 60 
hours.