Method of increasing natural killer cell population of cancer patients

An in vivo method is described for increasing the population of Natural Killer (NK) cells in the blood of patients suffering from cancer, such NK cells having known activity against tumor cells. The method broadly involves injecting a specially prepared biologic into the patient's bloodstream and allowing the biologic to activate the patient's immune system so as to achieve a desired NK cell population increase (preferably at least a twofold increase). The biologic is produced by injecting an animal such as a goat with a virus (preferably a normally immunosuppressive Parvovirus) and allowed to react to the virus for a period of time to generate the biologic in its blood serum; blood is then withdrawn from the animal and the serum fraction thereof, containing the desired biologic, can be used in fractionated or more highly purified form.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention is broadly concerned with an in vivo method for 
increasing the natural killer cell population in the peripheral blood of 
human patients, and especially patients suffering from cancer. More 
particularly, it is concerned with such a method which involves 
administering to such patients a biologic preferably derived by viral 
immunization of an animal such as a goat, and recovery of the biologic 
from the animal's blood. Advantageously, the goat or other test animal is 
treated with a virus such as a parvovirus which is normally 
immunosuppressive in a permissive host. 
2. Description of the Prior Art 
Vaccines have been in use since Edward Jenner (1749-1823) first recognized 
that people exposed to non-virulent strains of microorganisms could be 
protected against infection by the related virulent strains. His 
vaccination of patients with exudate of cowpox sores provided those same 
patients with partial protection against smallpox. Numerous vaccines have 
subsequently been prepared and used in both humans and animals. 
Immunization has been accomplished by vaccination with a suspension of 
live, attenuated, or killed organisms or specific protein, glycoprotein or 
surface material from various bacteria, rickettsiae, or viruses. These 
would include vaccines against anthrax, rabies, typhoid, cholera, 
smallpox, measles, mumps, pertussis, plague, and polio. The vaccine in 
each case has been intended to provide protection against a specific 
pathogen. While vaccination may provide long term specific protection it 
may or may not produce short term immunosystem modulation. The immune 
system responds to challenge via vaccination or infection by increased 
activity. This may result in the short term production of antigen 
non-specific immunoregulatory modulators such as interferon, interleukins, 
and other various lymphokines. 
The rationale for a system that becomes active only in response to a 
specific challenge is simple. If the immune system operated at a high 
pitch of activity all the time, it would age prematurely and no longer 
provide the systematic protection it was evolved to deliver. Another 
possible complication with an unusually active immune system could be the 
initiation of autoimmune reactions in which the system starts acting 
against itself, resulting in the destruction of normal tissue. Arthritis 
is exemplary of this particular condition. 
Increased activity of the immune system by virtue of improper regulation 
could furthermore result in an inability to elicit an appropriate 
response. As an example, a massive response to a splinter in a finger 
would be completely inappropriate, just as no response to a major 
infection would be equally inappropriate. 
Immunomodulation may provide a regulatory-directed approach at a 
self-healing, particularly with respect to such intractable diseases as 
cancer. For this to occur to minimal criteria must be met. The immune 
system must be sufficiently intact to respond to the regulator(s), and 
secondly, the system under appropriate stimulation must have T-cells, 
B-cells and Natural Killer cells present that are capable of responding to 
the target antigen(s) or cell(s). In such an instance, however, a 
nonspecific immunomodulator could potentially serve to "turn on" the 
immune system and initiate healing. 
A number of immunomodulators have previously been isolated and described 
and these appear to belong to one of three general groups, namely the 
interferons, the interleukins and the corticosteroids and leukotrienes. 
The interferons are a family of glycoproteins normally produced in response 
to a viral infection and are produced by leukocytes and fibroblasts. There 
are three main types, alpha, beta and gamma. They have molecular weights 
in the range of 15,000 to 40,000 daltons. Recently they have been produced 
by recombinant DNA techniques. The interferons have been found to have an 
absolute specie specificity, and are only effective in the specie that 
produced it. 
The second group of immunomodulators are the interleukins. They are a 
family of glycoproteins that are produced by white cells. It is believed 
that the lymphokines cause the activation of Natural Killer cells and 
B-cells, and act in the initiation and propagation of the specific 
sequences of cellular interactions that are not recognized as the immune 
response. These growth promoters and activators participate in the 
generation of immunoreactive cells. The lymphokines are antigen 
non-specific in that they activate all T and B cells in anticipation of 
the presentation of an antigen or a cell. The apparent molecular weights 
are in the range of 15,000 - 50,000 daltons. To date, treatment protocols 
for cancer patients involving interleukins have been of an in vitro 
character, i.e., blood is withdrawn from the patient, treated in vitro, 
and returned to the patient. This is a relatively cumbersome process and 
is subject to all of the typical problems attendant to in vitro, as 
opposed to in vivo, procedures. 
The third type of regulators are the corticosteroids and the leukotrienes, 
which act as regulators in inflammation. When produced in atypical amount, 
the leukotrienes can cause immediate type hypersensitivity and 
anaphylaxis. They are principally oxygenated products of archidonic acid. 
In contrast the corticosteroids and leukotrienes are small molecular 
weight compounds in comparison to the interleukins and interferons. 
All of the immunological regulators produced commercially are extracts or 
concentrates of tissue culture fluids from mitogen stimulated lymphoid and 
myleloid cells. The exceptions to this are interferons and IL-2 which have 
been produced by genetic engineering. The other immunological modulators 
that have been artificially produced are the interferons. The difficulty 
with each of these production methods is that the individual reagents 
being produced to not reproduce the plethoric effect seen in vivo. No 
practical method has heretofore been discovered to produce a plurality of 
the immunoregulators together in the same proportions as they would 
normally be produced in response to infection or cancer in situ. 
Additionally, the cost of prior known production methods has been 
considerable, thereby further limiting the utility of immunomodulators. 
SUMMARY OF THE INVENTION 
The present invention is broadly concerned with a unique method for 
significantly increasing the population of Natural Killer cells in the 
blood of human patients, and particularly those suffering from cancer. It 
has been established that increasing such Natural Killer cells is an 
important component of the immune system, and that accordingly the present 
method should be a decided advantage in cancer treatment. The method 
generally involves administering to the patient an amount of specially 
prepared biologic, and allowing the biologic to stimulate the patient's 
immune response and thus increase the Natural Killer cell population. 
Accordingly the method of the invention completely avoids in vitro 
treatment of the patient's blood. 
The patient's immune system should, of course, be sufficiently intact to 
respond to the biologic; moreover, if a cure or amelioration of metastatic 
disease is to be achieved, the patient must have Natural Killer cells 
present which can respond to the metastasis. Furthermore, it is believed 
at least a two-fold increase in Natural Killer cells should be effected in 
order to obtain meaningful treatment results. 
The biologic of the invention is advantageously obtained by a method which 
comprises the steps of injecting a virus such as a parvovirus into an 
animal, and permitting the injected animal to react to the presence of the 
virus for a period of time in order to develop in the animal's blood serum 
biologic in sufficient quantity and/or activity that 50 microliters of the 
animal's serum, when added to an in vitro human white blood cell culture 
containing 2-4.times.10.sup.5 white blood cells and followed by 3 days 
incubation at 37.degree. C. under a 5% CO2/95% air atmosphere, will give 
rise to at least about a 50% increase in T-helper and Natural Killer cells 
in the biologic-supplemented cell culture, as compared with an otherwise 
identical and identically cultured in vitro cell culture having added 
thereto 50 microliters of serum from a normal animal of the same species 
as the injected animal. The final step in the method involves recovering 
serum from the injected animal which contains the desired biologic. 
In particularly preferred forms the animal in question is a goat, and the 
virus injected is a Parvovirus of a type which is immunosuppressive in a 
normal permissive host animal (e.g., a cat or dog) for the Parvovirus. 
Thus, in the presently preferred method of the invention, use is made of a 
normally immunosuppressive virus in a non-permissive host for the virus; 
quite surprisingly however, this serves to develop an immunostimulative 
biologic in the injected animal's serum which has the salutory effects 
outlined above. 
In other forms of the invention, the injected animal can be selected from 
the group consisting of goats, horses, sheep, rabbits and monkeys, with 
the period of time after injection of the virus being at least about one 
month. 
The injected and immunized animal's serum can be used in relatively crude 
form, such as after only conventional ammonium sulfate fractionation. In 
other instances, however, the fractionated serum is subjected to further 
isolation procedures, in order to substantially purify the biologic. 
In actual practice, a number of test goats are normally immunized with 
Parvovirus, and the serum of these goats is tested after an appropriate 
time (e.g., about 3 months) for the presence of biologic using the 
standard outlined above. those animals which are positives, i.e., their 
serum exhibits the biologic in the manner defined, are then bled and their 
serum, containing the biologic, is recovered.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The following examples set forth the most preferred method of producing 
biologic in accordance with the invention. 
EXAMPLE I 
This Example sets forth the preferred procedures for the production and 
recovery of the biologic of the present invention. 
Goat Vaccination 
A total of five normal goats were initially hypervaccinated with Parvovirus 
in multiple sites on their dorsal rear halves, using a total of 1 cc. of 
commercially available Feline Pan Leukopenia vaccine (Dellen Laboratories, 
Inc., Westwood, Maine). Thereafter, at intervals of about one and two 
months after the initial injection, each goat was again injected with 1 cc 
total quantities of Canine Origin Parvovirus vaccine (Fromm Laboratories, 
Inc., Grafton, Wisconsin or Duramune Vaccine, Temple, Texas. At the end of 
about three months from the date of the initial injection, a sample of 
whole blood was withdrawn from the carotid artery of the neck of each 
goat, and the samples treated as hereinafter described. 
Goat Plasma Preparation 
The goat blood samples were separately centrifuged (about 500 ml. at a 
time) using a swinging bucket rotor centrifuge operated at 2000 rpm for 45 
minutes to pellet the red blood cells. The plasma was then removed from 
the packed red cells in each sample, and volumetrically measured. an 
absorbance reading (280 nm) of each plasma sample diluted 1:50 with PBS 
(pH 7.5) was then taken for record purposes. 
Serum Preparation 
Reagents Employed: 
1.0 molar calcium Chloride 
14.7 gm CaC1.sub.2 2H.sub.2 O in 100 ml distilled water 
Thrombin 
Miles Laboratories, Inc. 
Bovin Thrombin (500 units/ml) 
Reconstitute vial with: 
1.0 ml sterile saline 
1.0 ml glycerol 
Saline 
9 gms Sodium Chloride in 100 ml distilled water 
Procedure: 
Use 1.0 ml of 1. molar calcium chloride for each 100 ml of plasma. 
Use 100 ul of thrombin for each 100 ml of plasma. 
1. The required amount of calcium chloride and thrombin was added to each 
measured plasma sample in a beaker. 
2. The respective plasma/calcium chloride/thrombin mixtures were then 
stirred gently and thereafter each beaker was placed in a water bath at 
37.degree. C. for 30 minutes. 
3. The beakers of clotted plasma were then removed from the water bath, and 
the plasma in each beaker cut up into small pieces. 
4. Each mixture was then poured into a respective centrifuge tube, and the 
tubes were spun 10,000 rpm for 30 mintues. 
5. Serum was then carefully removed from the clot at the bottom of each of 
the tubes to give separate serum samples. 
6. The serum volume absorbance reading at 280 nm for each sample was then 
taken and recorded. 
Biologic Determination 
An in vitro assay was next performed on the serum samples derived from each 
of the test goats, in order to determine which of the goats produced the 
desired biologic in sufficient quantity. Normal human white cells were 
first cultured in RPMI-1640 media (a standard tissue culture media 
produced by K.C. Biological of Lenexa, Kansas) supplemented with 10% fetal 
calf serum. Fifty microliters of each of the goat test serums produced as 
described previously were then added to respective cultures each 
containing 2-4.times.10.sup.5 cultured human white blood cells in a total 
volume of 2 ml. The cultures, with goat serum added, were then incubated 
in an atomsphere of 5% CO.sub.2 /95% air at 37.degree. C. for 3 days. Each 
separate culture was then stained with two types of fluorescent-labeled 
monoclonal antibodies respectively specific against T-helper cells (OKT-4 
monoclonal antibody sold by Ortho Diagnostic of Raritan, New Jersey) and 
Natural Killer cells (LEU-7 monoclonal antibody sold by The 
Becton-Dickinson Co. of Mountain View, California). The strainings were 
performed following the supplier's directions, and after about one hour, 
the stained samples were analyzed using a flow cytometry device, namely a 
model 50H cytofluorograph (Ortho Instruments, Westwood, Mass.) to count 
both T-helper and Natural Killer cells in each culture. The test animal 
serums were compared against a stained and counted control sample 
comprising cultured human white blood cells and 50 microliters of normal 
goat shich exhibited as outlined at least about a 50% increase in the 
number of T-helper cells and Natural Killer cells, as compared with the 
control, were considered positives for the biologic of the invention. In 
this specific test three of the five goat serum samples gave positive 
results; one sample gave about a 50% increase, whereas the other two 
samples gave an increase on the order of 125%. 
The above described in vitro determination assay is used throughout the 
purification and isolation protocol in order to identify which of the 
separated fractions in each instance contained the desired biologic. 
The three positive serum samples were each further treated as described 
below (the procedure for only a single serum sample is set forth, but the 
method of treating all the smples was the same). In this example, the 
respective positive serum samples were treated separately; however, such 
samples could be pooled if desired. 
Ammonium Sulfate Fractionation 
Reagents Employed: 
Ammonium Sulfate (Sigma S-5182, Sigma Chemical Co., St. Louis, Missouri) 
0.01m Sodium Phosphate (pH 7.6) 
Procedure: 
1. 24.3 gm of ammonium sulfate was weighed out for each 100 ml of serum to 
be fractionated. 
2. The serum sample was placed in a beaker and stirred. During stirring 
small amounts of ammonium sulfate were added, making sure that all was 
dissolved before adding more ammonium sulfate; this procedure was 
continued until entire amount of ammonium sulfate was added to the sample. 
The sample was then gently stirred for another 30 minutes. 
3. The serum sample was then placed in a centrifuge tube and spun at 10,000 
rpm for 30 minutes, whereupon supernatant was carefully removed from the 
pellet. The supernatant serum fraction was tested for the presence of the 
desired biologic using the in vitro cell culure assay described above 
under "Biologic Determination" and was found to be negative; 
this supernatant was therefore discarded. 
4. The centrifuged pellet from step 3 was then redissolved in 0.01m sodium 
phosphate (pH 7.6), with the final volume of redissolved pellet being the 
same amount as that of original serum volume. 
5. Steps 2 and 3 were then repeated for the serum sample, in order to 
effect another separation; again, the in vitro determination assay 
confirmed that the supernatant did not contain the desired biologic. 
6. The pellet was then redissolved to one-half of the original serum 
volume, using 0.01 M sodium phosphate (pH 7.6). 
7. The resuspended material sample was then placed into a section of 12,000 
mw cut-off dialysis tubing. 
8. The sample was then dialyzed against .01 M sodium phosphate (pH 7.6), 
using three changes of 4 liters in the dialysis. 
9. The sample was then removed from the dialysis tubing and put into a 
centrifuge tube. 
10. The tube was next spun at 10,000 rpm for 30 minutes. 
11. The supernatant from the centrigue tube was then removed and the volume 
measured and recorded; another in vitro determination assay as described 
above was performed on the supernatant, to confirm that it did contain the 
desired biologic. 
12. The absorbance at 280 nm for the supernatant was taken and recorded. 
13. The supernatant material resulting from the fractionation of the sample 
was stored in a sterile bottle. 
First Stage Chromatography (using DE-52 anion exchange resin, Whatman, 
Inc., Clifton, N. J.) 
1. An appropriate quantity of DE-52 resin was swelled and equilibrated in 
0.01 molar Sodium Phosphate, pH 7.6 
2. A column (4.4 cm x 83 cm) was packed with swelled DE-52 resin. The 
column was washed with 2 liters of 0.01 M NaH.sub.2 PO.sub.4 (pH 7.6) at 
75 cm pressure. 
3. 40 ml of the ammonium sulfate fractionated serum from the sample were 
loaded onto the DE-52 column at the same pressure as the wash. The 
material was then eleuted with 0.01 M NaH.sub.2 PO.sub.4 (pH 7.6) wash. 
4. Liquid from the column was collected in separate tubes (150 
drops/tube--approximately 5.0 ml) until all protein fractions were 
completely eluted. About 1.0 liter of .01 molar 
NaH.sub.2 PO.sub.4 was passed through the column. 
5. The column was then washed with 1.5 liters of 0.01 molar NaH.sub.2 PO4 
containing 0.5 m NaC1 (pH 7.6) to remove bound material from the column. 
Liquid from the columns was collected in separate tubes (150 drops/tube) 
until the buffer was expended. 
6. The absorbance at 280 nm was taken for all tubes collected. A plot of 
absorbance (A.sup.280) vs. tube number was prepared to locate the peaks. 
This graph is reproduced as FIG. 1. 
7. The material within tubes located in each separate peak off the plot 
were then pooled and assayed using the described in vitro determination 
assay. 
8. The pooled material from the first peak (peak A of FIG. 1) was found to 
contain the biologic of the invention. This material was concentrated by 
placing the material into a section of dialysis tubing and tying off the 
ends; the tubing was then placed into a tub of polyethylene glycol (20,000 
mw, Sigman Chemical Co., St. Louis, Missouri) and concentrated to about 
10% of the original pooled volume. The tubing was then rinsed well with 
water. 
9. The pooled material within the tubing was then dialyzed against PBS (pH 
7.5). Three changes of 4 liters were used to remove polyethylene glycol 
and salt. 
10. The dialyzed material was then removed from tubing and the volume 
measured. Absorbance readings at 280 nm were taken and recorded along with 
volume. 
Second Stage Chromatography (using S-200 size exclusion chromatography 
resin, Sigma Chemical Co., St. Louis, Missouri) 
1. An elution column (4.4 cm x 83 cm) was packed and equilibrated with 
S-200 resin, and the column was washed with 2 liters of PBS (0.2 M 
NaH.sub.2 PO.sub.4 containing 0.15 m NaC1, pH 7.5) at 60 cm. pressure. 
2. 15 mls. of the pooled, dialyzed material from the first concentrated 
peak (peak A, FIG. 1) off of the DE-52 column was next loaded onto the 
S-200 column, and the sample was eluted using the PBS of step 1. The 
liquid off the column was collected in separate tubes (100 drops/tube, 
approximately 3.0 ml). 
3. Absorbance readings at 280 nm were then taken for all of tubes 
collected. A plot of A.sup.280 vs. tube number was then prepared; this 
plot is reproduced as FIG. 2. 
4. The material within tubes located in each peak were pooled and 
concentrated with polyethylene glycol as set forth in steps 7 and 8 of the 
first stage chromatography procedure. The pooled samples were each then 
tested using the in vitro determination assay. 
5. The pooled material from the largest peak (peak B, FIG. 1) was found to 
contain the biologic. This material was then dialyzed using dialysis 
tubing against 3 changes of 4 liters of PBS (0.01 M K.sub.2 HPO.sub.4 
containing 0.15 M NaC1, pH 7.5). 
6. The dialyzed material was then removed from the tubing and the volume 
measured and recorded. An absorbance (A.sup.280) was then taken on the 
dialyzed material, and the reading recorded. 
Third Stage Gel Chromatography (using CM Blue AFFI gel cation exchange 
resin, Bio-Rad Corp., Richmond, California) 
1. A column (1.5 cm x 38 cm) was packed and equilibrated with the CM Blue 
AFFI gel, and the column was washed with 2 liters of 0.01 M K.sub.2 
HPO.sub.4 containing 0.15 M NaC1, pH 7.25, at 45 cm pressure. 
2. 8 ml of material concentrated from the largest peak off the S-200 column 
(peak B, FIG. 2) was then loaded onto the column. 
3. The column was then washed with 0.01 M K.sub.2 KPO.sub.4 containing 0.15 
M NaC1 (pH 7.25) until all peaks were eluted, using approximately 150-200 
ml PBS for the column. Liquid off the column was collected in separate 
tubes (90 drops/tube, about 2 ml). 
4. The column was again washed with 100 ml of .01 M K.sub.2 HPO.sub.4 
containing 0.5 m NaC1 (pH 7.25 to further elute the bound material; liquid 
from the column was collected in tubes (90 drops/ tube). 
5. Absorbance readings at 280 nm were taken for all tubes and a plot of 
A.sup.280 vs tube number from both elutions was prepared; this plot is 
reproduced as FIG. 3. Material within each peak was pooled, and these were 
assayed using the in vitro determination assay. 
6. Material from the first peak (peak C, FIG. 3) was found to contain the 
biologic; this material was concentrated with polyethylene glycol as 
described above in the first stage chromatography procedure. 
7. The concentrated material was next dialyzed against PBS, using three 
changes of 4 liters each. 
8. The material was next removed from dialysis tubing. Absorbance at 280 nm 
was read and recorded. 
9. The material was filtered using a 0.2 um filter and stored. 
As is evident from the foregoing, after a determination is made for the 
positive serum samples (i.e., those containing adequate amounts of 
biologic) successive fractionation and separation techniques may be 
employed to obtain the relatively purified biologic material, with 
appropriate assays being performed on separated fractions in order to 
ensure that the biologic is retained throughout. The final biologic 
product comprises proteinaceous component(s) which have not to date been 
completely characterized. However, the product is presently believed to be 
an interleukin protein or proteins having an apparent molecular weight in 
the range of 100,000 to 120,000 daltons; the components also appear to be 
glycosylated. A sample of the purified biologic made in accordance with 
the present invention has been deposited with the American Type Culture 
Collection; such sample has been accorded accession number 40105. 
Obviously, however, there is no desire to be bound to any preliminary or 
partial characterizations, and such are offered only in an effort to 
disclose all presently available pertinent information. 
While the above described separation and isolation procedures are in many 
cases preferred, it should be understood that the biologic product derived 
from the methods of the invention can be used without such purifications, 
particularly if used in connection with animals. In the latter case, the 
fractionated positive goat serum can be used, inasmuch as such a 
relatively crude material contains the desired proteinaceous component(s), 
while potentially interfering interleukins have been removed. 
EXAMPLE II 
EFFECT OF BIOLOGIC ON HUMAN CANCER PATIENT 
A terminally ill cancer patient undergoing chemotherapy was treated with 
the purified biologic described in Example I. A whole blood sample was 
collected from the patient before her first treatment, and the patient was 
tested by intradermal challenge skin scratch test (50 ug) for 
hyper-reaction to the goat-derived biologic prior to treatment. The first 
course of therapy involved injection of fifty (50) micrograms of purified 
stimulant (diluted to a volume of 5 ml. in saline solution) given each day 
for three successive days, starting on Dec. 19. The saline/biologic was 
given by slow intravenous push. The patient was sampled again after the 
third treatment. The fourth and fifth treatments were identical and given 
on Jan. 4 and Jan. 5. On Jan. 6 the patient's blood was sampled. The 
samples were collected using a vaccutainer containing EDTA. Thereafter, 
the patient was treated with 1 mg/5 ml injections on Jan. 4 and Jan. 6. 
The patient's condition continued to deterioriate and she was again 
treated (10 mg/ml) on April 10. On Apr. 13 the patient's blood was again 
sampled. 
At the end of the therapy the patient had noted no unusual qualitative 
differences during the treatment period. However, the patient contained to 
deteriorate and died in June. The major change that occurred during 
treatment was a 3.5 fold increase in the patient's Natural Killer cell 
population. Historically, the Natural Killer population has been 
demonstrated to be active against virally injected cells and tumor cells. 
Accordingly, the biologic served as an immunostimulant in the patient. 
TABLE I 
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TREATMENT OF HUMAN WITH PURIFIED BIOLOGIC 
Lymphocyte (% of Each Population) 
Biologic Sam- Natural 
Date Saline ple T-Helper 
T-Suppressor 
Killer 
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12/19 50 ug/5 ml X 53.2 29.1 10.7 
12/20 50 ug/5 ml 
12/21 50 ug/5 ml X 54.7 30.8 18.9 
12/29 -- X 47.7 24.6 24.2 
1/04 1 mg/5 ml X 50.8 29.3 25.8 
1/06 l mg/5 ml X 52.5 31.6 24.6 
3/28 -- X 34.0 23.2 26.5 
4/10 10 mg/10 ml 
4/13/ -- X 50.7 42.0 35.2 
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