CA1261255A - Process for producing human interferon and method for assaying the interferon productivity of blood - Google Patents

Abstract

Abstract of the Disclosure A process for producing HuIFN using whole human blood and a method for assaying the blood HuIFN productivity are disclosed. The whole blood is incubated in the presence of an anticoagulant (e.g. heparin, ACD, and CPD) and a viral inducer under the conditions appropriate to accumu-late a substantial amount of HuIFN. The blood HuIFN productivity deter-mined by titrating the accumulated HuIFN with a suitable procedure (bio-assay, radioimmunoassay, or enzyme-linked immunosorbent assay) is useful in clinical test to detect cancer in its early stage. The HuIFN per se is recovered, and purified prior to its prophylactic and therapeutic uses.

Description

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PROCESS FOR PRODUCINC ~IUMAN INTERFERON
AND METIIOD FO~ ASSAYING THE INTERFERON PRODUCTIVITY OF BLOOD

Field of the Invention The present invention relates to a process for producing human-specific interferon (abbreviated as " HuIFN" hereinafter) using whole human blood and to a method for assaying the HuIFN productivity of human blood.

Description of the Prior Art In recent years, clinical tests whereby the level of a blood en-zyme or its metabolite is determined chemically have been in a wide use.
Since HuIFN, a blood component, exhibits antiviral- and antitumor activities, it has been proposed to include the level of serum HuIFN in the items of clinical test. Such proposal, however, has not been realized because serum HuIFN is minute.
Blood consists of a fluid, plasma, in which are suspended formed elements such as erythrocyte, leukocyte and platelet. One mm3 blood generally contains, in addition to 7 . 4X103 leukocytes and 3X105 platelets in adult, 5,4X106 erythrocytes in man or 4.8X106 in woman.
It is well known that HuIFN is produced by human leukocyte.
In conventional processes to produce HuIFN, viable leukocytes separated from human blood are used. For example, as is evident from .. ...
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Hans Strander and Kari Cantell, Ann. Med._ex~. Fenn., Vol.
44, pp.265 273 (1966), and the specifications of Japan Patent Kokai Nos.6,111~74 and 94,008/78, leukocytes are separated from other element3 present in whole blood, and then allowed to produce HuIFN.
Detailed studie~ on these conventional processes confirmed that they, however, give a low recovery yield of leukocytes ~rom blood, i.e. 30-50~, and damage leukocytes during the separation to lower the viability to 40-60~ and, eventually, the overall recovery yield to 10~30%, as well as that the separated leukocytes give an inconsistent HuIFN
production; these facts render the estimation of the HuIFN
productivity of blood very difficult, and cause an obstacle in mass-production of HuIYN.
Summary of the Invention According to an a~pect of tha invention there is provided a clinical assay for detacting human cancer, comprising:
collecting whole blood from a subject; incubating in vitro a predetermined amount of the whole blood in the presence of an effective amount of an anticoagulant and an effective amount of a virus under conditions appropriate for the production of interferon; and determining the interferon level in the resultant culture, whereby the determination of an interferon level below a predetermined amount is indicative of the possible presence of human cancer~
According to a further aspect of the invention there rm/ h~l " ' ~ ' ' ~ ~ti~5 - 2a -is provided a clinical a~say for detecting human cancer, comprising: collecting whole blood from a subject; removing the pla~ma from the donated whole blood; suspending the residual solid in a member selected from thle group con~isting of saline, .isotonic buffer, and nutrient culture medium; incubating in vitro a predetermined amount of thle resultant cell suspension in the presence of an effective amount of an ant~coagulant and an effective amount of a virus under conditions appropriate for the production of interferon; and determining the interferon level in the resultant culture, whereby the determination of an interferon level below a predetermined amount is indicative of the possible presence of human cancer.
Detailed Descri~ion of the Invention .

As the results of my researches for the ~ass-production of HuIFN using preciou~ human blood, as well as for the assay of the HuIFN productivity by use of a donated blood, the present inventor eventually found that a large amount of HuIFN can be produced with an ea~e by incubating a whole blood in a veqsel while exposing the whole blood to an anticoagulant and a viru~.
The present inventor also found that the HuIFN productivity of a whole blood can be determined with an ease and a high reproducibility by incubating a whole blood in a vessel while exposing the whole blood to an anticoagulant and a virus, and titrating the accumulated HuIFN.
Detailed studies confirmed that the exposure to an intact or inactivated virus of an amount of 20-200,000 HA/ml whole blood is favor-,;~, ,.
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, -3- ~ ti~ ;i5 able . " I IA " represents the unit of the haemagglutination titer of a virus .
The wording of "whole blood" means fresh blood preparations collected from donors, and also suspensions which are obtained by removing plasma liquid from such blood preparations and suspending the residual formed elements in an suitable non-plasma liquid, e . g . physiological saline, buffer solution or nutrient culture medium.
Any anticoagulant capable of preventing the coagulation of such whole blood does not affect HuIFN production is usable in the invention.
For example, heparin, acid citrate-dextrose (ACD) and citrate-phosphate-dextrose ( CPD ) are favorable .
The viruses usable in the invention are those which are capable of inducing HuIFN production in the whole blood. For example, Sendai virus or Newcastle disease virus may be used intact or after inactivation.
The inactivated viruses usable in the invention include those whose repro-ductivities are partially or completely suppressed, for example, by uv-irradiation, heating or treatment at an extreme pH. An appropriate range for inoculum of the virus is 20-200,000 HA/ml whole blood.
The step of incubating the whole blood in a vessel while exposing the whole blood to the anticoagulant and virus is carried out in such a manner that the whole blood is exposed in the vessel to the anticoagulant and virus to produce HuIFN. For example, to the prescribed amounts of the anticoagulant and virus in the vessel is added an appropriate amount of the whole blood, and the mixture is incubated therein. Alternatively, a mixture of the anticoagulant and whole blood is placed in a vessel, added with the virus, and incubated. In this incubation step, a suitable medium, e. g . physiological saline, isotonic buffer solution or nutrient culture me-S~

dium, may be used adclitionally.
Tank, jar, flask, test tube, ampul and micro plate well of anyshape and volume may be used as the vessel in the invention.
The incubation conditions are those under which HuIFN is pro-ducible: for example, temperature range of 30-~0C; and incubation time, 5-50 hours. In this case, priming or superinduction may be carried out if necessary .
After incubation to produce HuIFN and an optional dilution with physiological saline or isotonic buffer solution, the whole blood is then separated with suitable procedure(s), such as centrifugation or filtration, to remove formed elements such as blood cells, and the resultant supernat-ant or filtrate containing HuIFN is subjected to purification or titration.
The HuIFN can be purified to obtain an HuIFN preparation having a possible highest purity by combination of conventional procedures, e. g .
salting-out, dialysis, filtration, concentration, adsorption and desorption by ion exchange, gel filtration, affinity chromatography using a suitable ligand such as antibody, isoelectric point fractionation and electrophoresis.
The obtained HuIFN is advantageously feasible as injection or drug for an external or internal use in the prevention and treatment of human diseases alone or in combination with one or more substances.
The HuIFN productivity of human whole blood can be determined according to the invention by titrating the HulFN level in the above de-scribed supernatant or filtrate. For the purpose of such titration, any assay can be used as long as the HuIFN production by the whole blood is titrated therewith; e.g. bioassay, radioimmunoassay and enzyme-linked immunosorbent assay.

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~.. -, In recent years, enzyme-linked immunosorbent assay ha~ been developed as a highly safe, convenient and speedy assay. Any enzyme-linked immunosorbent assay capable of titrating IFN as the antigen is em-ployable in the invention. For example, double antibody sandwich tech-nique and modified double antibody sandwich technique are favorable.
It was confirmed that the HuIFN productivities determined in this way are very useful for clinically testing the individual donor.
The method according to the invention confirmed that the blood collected from a cancer patient is much lower in blood HuIFN productivity than those collected from healthy volunteers.
The following experiments further explain the present invention.

Experiment 1 Effect of pretreatment on the HuIFN productivit~of blood The effect of pretreating blood on the productivity of HuIFN was studied. In this Experiment, fresh blood samples from three healthy volun-teers were used after heparini~ation.
The treated bloods used in this Experiment were as follows: a plasma-free suspension, obtained by centrifuging blood to remove plasma liquid and suspending the residual formed elements in RPMI 1640 medium to give the same element density as that in blood, and an ammonium chloride-treated suspension, obtained by treating blood with Tris-HCl buffer (pH
7.2) containing 0.7596 ammonium chloride in usual way to effect the haemoly--6- ~ 5~

sis of the erythrocytes, centrifuging the mixture and suspending the re-sultarlt erythrocyte-free formed elements in RPMI 1640 medium to give the same element density as that in blood.
One ml aliquots of the heparini~ed or treated blood were placed in different plastic test tubes which were then added with 0.1 ml aliquots of physiological saline containing Sendai virus in respective amount of 0, 100, or 1,000 HA, followed by 16-hour incubation at 37C. The incubated mixtures were uv-irradiated to completely inactivate the Sendai virus, and centrifuged to obtain supernatants which were then assayed for HulFN
titers per ml whole blood.
The HuIFN titer was determined by the dye uptake assay report-ed in Anne L . R . Pidot, Applied Microbiology, Vol .22, No.4, pp .671-677 (1971). The haemagglutination titer (HA) was determined by the method as reported by J.E. Salk, The Journal of Immunology, Vol.49, pp.87-98 (1944) with slight modification.
The results are given in Table 1.
As is evident from these results, the whole blood and plasma-free suspension containing the whole formed elements of blood are favorable for assaying the HuIFN productivity because of its high and consistent HuIFN
productivity. It was also confirmed that the ammonium chloride-treated suspension wherein the erythrocytes were haemoly~ed and removed gives a low and inconsistent HuIFN productivity.

7~ 55 Table 1 Healthy volunteer TreatmentSenclai virus _ _ A B C
. _ _ No treatment 100 3,800 2,500 4,200 1,000 3,700 2,600 ~,500 ~ . . _ , _ . _ _ suspension1 ~ ooooo 4 16000 22 84oo 4 200 _ . _ . . _ _ _ treated suspension 1, 000 0 3000 1070 . _ . . _ Experiment 2 Effect of virus_inoculum on the productivity of HuIFN

The effect of virus inoculum on the productivity of HuIFN was studied. Fresh blood samples from three healthy volunteers and two cancer patients were used after heparini~ation.
According to the method as described in Experiment 1, 1 ml aliquots of either heparinized blood sample were placed in different test tubes, added with O.1 ml aliquots of physiological saline containing Sendai virus in respe~tive amount of O, 2, 20, 200, 2,000, 20,000, or 200,000 HA, -8- 3L~ ti~

incubated, and assayed for HuIFN titers per ml b:lood.
A series of experiments using 2,000,000 HA Sendai virus per ml blood was scheduled, but not clone because preparation of such a high-titer Sendai virus was unsuccessful.
The results are given in Table 2.

T able 2 Healthy volunteer Cancer patient Sendai virus (HA) D E F G H

2 130 70 30 10 0 2,600 1,~00 5,400 10 30 200 2,~00 2,400 5,800 20 70 2,000 4,100 2,100 6,300 20 190 20,000 3,500 2,300 8,800 10 140 200,0004,400 2,200 7,300 . 10 180 2,000,000 ND ND . ND ND ND

Note: ND means not done.

As ;s evident from these results, virus inocula in the range of 20-200,000 ~lA/ml blood are favorable.
It was confirmed that the blood collectetl from a cancer patient is much lower in blood HuIFN productivity than those collected from healthy volunteers. This suggests that the assay of blood HuIFN productivity is helpful for the detection of cancer in its early stage.
Several embodiments of the present invention are disclosed here-inafter .

Production of HuIFN

Example 1 One ml of a heparinized fresh blood from a healthy volunteer was placed in a plastic test tube, added with 1,000 HA of Sendai virus, incu-bated at 37C for 20 hours, and uv-irradiated to completely inactivate the virus. After centrifuging the mixture, the resultant supernatant was assayed for HuIFN titer.
The HuIFN production was about 3, 600 units per ml blood .

Example 2 One ml of a hepar;nized fresh blood from a healthy volunteer was added with 2, 000 HA of Newcastle disease virus wherein 90~ of the repro-ductivity had been inactivated. After incubating at 37C for 15 hours, the -:: :

mixture was assayed for HuIFN titer similarly as in Example 1.
The l-luIFN production was about 2, 800 units per ml blood .

Example 3 A heparinized fresh blood from healthy volunteers was centrifuged to remove plasma. The formed elements so obtained were then centrifugally washed in physiological saline, and suspended in RPMI 1640 medium to give the same element density as that in blood.
The resultant suspension was placed in a mini jar, and added with 500 HA of Sendai virus per ml suspension. After incubating at 37C
for 16 hours, the mixture was treated and assayed for HuIFN titer similarly as in Example 1.
The HuIFN production was about 3, 000 units per ml blood .

Example 4 A suspension containing the formed blood elements was prepared similarly as in Example 3.
The suspension was placed in a mini jar, added with 300 units of HuIFN per ml suspension, and incubated at 37C for 6 hours. Thereafter, the suspension was further added with 1, 000 HA of Sendai virus per ml suspension, and incubated at 37C for additional 16 hours. The resultant was treated and assayed for HuIFN similarly as in Example 1.
The HuIFN production was about 27, 000 units per ml blood .

5 ~

Assay of blood HuIFN productivity Example 5 One ml of a heparinized fresh blood from a healthy volunteer, 28-year old man, was treated similarly as in Example 1, and subjected to bioassay for HuIFN titration.
The HuIFN productivity was about 3, 600 units per ml blood O

Example 6 One ml of a heparinized fresh blood from a healthy volunteer, 33-year old woman, was treated similarly as in Example 2, and assayed for HuIFN titration similarly as in Example 5.
The HuIFN productivity was about 2, 800 units per ml blood .

Example 7 A heparinized fresh blood from a healthy volunteer, 61-year old man, was treated similarly as in Example 3 to obtain a suspension containing the formed blood elements.
One ml of the suspension was placed in .a plastic test tube, and added with 1,000 HA of Sendai virus. After incubating at 37C for 16 hours, the mixture was subjected to double antibody sandwich technique, an enzyme-linked immun~sorbent assay, for HuIFN titration.
The HuIFN productivity was about 3, 400 units per ml blood .

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This value was consistent with that obtained by bioassay.

Example 8 A heparinized fresh blood from a cancer patient, 68-year old man, was treated similarly as in Example 5 to obtain an HuIFN productivity of about 140 units per ml blood.

Example 9 A heparinized fresh blood from a cancer patient, 55-year old woman, was treated similarly as in Example 5 to obtain an HuIFN productiv-ity of about 70 units per ml blood.

It will be obvious to those skilled in the art that various changes and alterations may be made without departing from the scope of the inven-tion and the invention is not to be considered limited to what is described in the specification.

Claims (19)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A clinical assay for detecting human cancer, comprising:
collecting whole blood from a subject;
incubating in vitro a predetermined amount of the whole blood in the presence of an effective amount of an anticoagulant and an effective amount of a virus under condi-tions appropriate for the production of interferon; and determining the interferon level in the result-ant culture, whereby the determination of an interferon level below a predetermined amount is indicative of the possible presence of human cancer.

2. The assay of claim 1, wherein the amount of the virus is in the range of 20-200,000 haemagglutination titers/ml whole blood.

3. The assay of claim 1, wherein said anticoagulant is a member selected from the group consisting of heparin, acid-citrate-dextrose, citrate-phosphate dextrose, and mixtures thereof.

4. The assay of claim 1, wherein said virus is a member selected from the group consisting of Sendai virus, Newcastle disease virus, and a mixture thereof.

5. The assay of claim 1, wherein the incubation step is carried out at a temperature in the range of 30 to 40°C

for 5 to 50 hours.

6. The assay of claim 1, wherein the determination step is effected by bioassay.

7. The assay of claim 1, wherein the determination step is effected by radioimmunoassay.

8. The assay of claim 1, wherein the determination step is effected by enzyme immunoassay.

9. The assay of claim 1, further including the step of comparing the interferon level obtained in said determining step with a standard to determine whether the interferon level is sufficiently low to warrant a positive indication of the presence of cancer.

10. The assay of claim 1, consisting essentially of said collecting, incubating and determining steps.

11. A clinical assay for detecting human cancer, comprising:
collecting whole blood from a subject;
removing the plasma from the donated whole blood;
suspending the residual solid in a member selected from the group consisting of saline, isotonic buffer, and nutrient culture medium;
incubating in vitro a predetermined amount of the resultant cell suspension in the presence of an effective amount of an anticoagulant and an effective amount of a virus under conditions appropriate for the production of interferon;

and determining the interferon level in the result ant culture, whereby the determination of an interferon level below a predetermined amount is indicative of the possible presence of human cancer.

12. The assay of claim 11, wherein the amount of the virus is in the range of 20-200,000 haemagglutination titers/ml whole blood.

13. The assay of claim 11, wherein said anticoagu-lant is a member selected from the group consisting of heparin, acid-citrate-dextrose, citrate-phosphate-dextrose, and mixtures thereof.

14. The assay of claim 11, wherein said virus is a member selected from the group consisting of Sendai virus, Newcastle disease virus, and a mixture thereof.

15. The assay of claim 11, wherein the incubation step is carried out at a temperature in the range of 30 to 40°C
for 5 to 50 hours.

16. The assay of claim 11, wherein the determination step is effected by bioassay.

17. The assay of claim 11, wherein the determination is effected by radioimmunoassay.

18. The assay of claim 11, wherein the determination step is effected by enzyme immunoassay.

19. The assay of claim 11, further including the step of comparing the interferon level obtained in said deter-mining step with a standard to determine whether the interferon level is sufficiently low to warrant a positive indication of the presence of cancer.