This application is a continuation-in-part application of Serial No. 08/285,436, filed August 4, 1994.

Field of the Invention This invention is directed to protein produced by and secreted from human CD4-positive T cells which has potent anti-retrovirus activity.

Background of the Invention

In recent years, researchers have devoted significant attention to retroviruses and, more specifically, to diseases, such as Acquired Immune Deficiency Syndrome (AIDS) , ^caused by retroviruses. This attention primarily is due, of course, to the fact that AIDS has become a significant public health threat in recent years. A major breakthrough in AIDS research came in 1983-1984 when two groups independently isolated and identified a virus believed to be the causative agent of AIDS. The AIDS virus has been described by several names. It has been known as lymphadenopathy-associated virus (LAV) , AIDS-related virus (ARV) and human immunodeficiency virus (HIV) . Within the last few years, scientists have discovered that there are at least two distinct viruses, HIV-1 and HIV-2. HIV-1 is the virus originally isolated in 1983 {Ann . Virol . Inεt . Pasteur, 135E: 119-134 [1986] ) ;

HIV-2 was isolated by researchers in 1986 (see Nature, 326:662 [1987]) . As used herein, HIV refers to these viruses generically.

A distinguishing feature of HIV is its selective cytotoxicity for helper T lymphocytes. The virus also infects monocytes/macrophages, B lymphocytes and neural cells. Severe and diverse aberrations of the immune system significantly reduce the host defense against various opportunistic infections, ultimately resulting in host death.

There are a number of proteins secreted by human cells that can regulate the human immune system. These secreted proteins with regulatory activities generally are classified as interleukins or cytokines. The United States Food and Drug Administration (FDA) has approved the use of some of the interleukins/cytokines in clinical trials for the treatment of AIDS. Those approved for clinical trials include interleukin-2 (IL-2) , tumor necrosis factor- alpha (TNF-c.) , interferon-alpha (INF-α) , interferon- beta (INF-/?) , interferon-gamma (INF-γ) and colony stimulating factor for granulocytes and macrophages (GM-CSF) . Only the interferons have been shown to possess anti-viral activities before the initiation of the clinical trials. The high doses of interferon required in treatment often give undesirable short- term side effects that precipitate .the withdrawal of the interferon treatment. IL-2 has shown no anti-HIV activity, and data available thus far suggest that IL- 2 has no significant impact on the reconstitution of CD4-positive helper T cell populations in AIDS patients. Recent reports also have shown that TNF-α and GM-CSF can activate latent HIV in cells maintained in tissue culture. Thus, it is questionable if these

agents can be used long-term to treat AIDS patients. There thus remains an urgent need for methods of inactivating the AIDS virus and for the treatment of AIDS. It therefore is an object of the present invention to provide a novel protein having potent anti-retrovirus activity and, particularly, anti-HIV activity. It is a further object of the invention to provide a protein which can substantially inhibit the replication of HIV in HIV-infected cells. It is a further object of this invention to provide a protein which can substantially inhibit the replication of other retroviruses in cells infected with those viruses. Other objects of the invention will become apparent from the following description.

Summary of the Invention In accordance with the present invention, there is provided a method for obtaining a soluble protein, or proteins, having potent anti-retrovirus activity, secreted by a CD4-positive T cell line cultured in a tissue culture medium. It has been found that a soluble protein, or proteins, with potent anti- retrovirus activity, as determined by retention by a membrane with a defined pore size, has a molecular weight of at least about 30,000 daltons.

The protein, or proteins, can be recovered from the cell culture by first separating the cells from the medium. The resultant cell-free solution is passed through a 30 kD molecular sieve and the fraction which does not pass through the sieve is collected. The collected fraction comprises a protein having potent anti-retrovirus activity. The protein can be purified further by first passing it through a

cation exchange column and then an ion exchange column and collecting the fraction(s) which possess anti-HIV activity.

The protein can be used to inhibit the replication of a retrovirus in cells by exposing the cells to a substantially pure composition of the protein. The exposure and consequent virus inhibition can take place either in vi tro or in vivo .

The protein has potent activity against a number of retroviruses, including human T-cell leukemia and feline leukemia virus. In particular, the protein has activity against lentiviruses, including HIV and other viruses, such as simian immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus and visna virus.

This invention thus further provides a method of inhibiting the replication in cells of a retrovirus, particularly a lentivirus, and most particularly HIV, in cells by exposing said cells to a composition comprising the substantially pure protein of the invention.

Detailed Description of the Invention Commonly-assigned U.S. Patent 5,346,988, issued September 13, 1994, incorporated herein by reference, teaches obtaining a protein having potent anti-HIV activity by cultivating CD4-positive T cells in the presence of an activating agent and recovering from the culture medium a protein having a molecular weight in the range of 7,000 to 12,000 daltons. Applicants now have discovered that a larger protein, having a molecular weight of at least 30,000 daltons, which also has strong anti-HIV activity, also can be produced and secreted by the cells.

Suitable starting material for the process of this invention is a CD4-positive T cell line, such as CEM, available from the American Type Culture Collection (ATCC) , Rockville, MD, accession number CCL119. Also suitable are cells from the CEM cell line that were sub-cloned in microtiter wells as described in example 1 below. One subclone was designated Clone 4084 and a cell sample was deposited with the ATCC on August 3, 1994, in accordance with the provisions of the Budapest Treaty, and accorded accession number 11697. Applicants have discovered that CD4-positive T cells can produce a protein which has strong activity against HIV and other retroviruses, such as human T-cell leukemia and feline leukemia virus. In particular, the protein exhibits strong activity against lentiviruses, including HIV, simian immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus and visna virus. As a preferred embodiment of the invention comprises the inhibition of HIV replication, the protein is described in detail below primarily in terms of its anti-HIV activity.

To obtain the protein with a molecular weight of at least 30 kD having anti-HIV activity, CD4-positive T cells are cultured in a tissue culture medium. Generally, any commercially available medium is acceptable to support the growth of the cells, such as RPMI-1640, commercially available from Media Tech., Herndon, VA. Desirably, the medium is supplemented with 5% v/v fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

The culture medium alsc can be modified by the addition of an agent suitable for activating the cells. Activation of the cells is not necessary, but

larger amounts of the desired protein are found in the activated cell cultures than in non-activated cultures. It has been found that the yield of desired protein can be increased by up to about 10 fold if the cells are cultured in the presence of an activating agent. Examples of useful activating agents include PC6, an extraction from cones of conifer trees described by Lai et al . , AIDS Research and Human Retroviruses, 6: 205-217 (1990) , alkaline aqueous extracts from cones of conifer trees (Aπticaπcer

Research 7:1153-1160 (1987) , Klason lignin extracted from conifer cones or pulp waste {Methods of Wood Chemistry, B.L. Browning [ed.] Vol. II, p785, Interscience Publishers, NY) , alkaline lignin (Tokyo Kasei Organic Chemicals, Tokyo, Japan) , de-alkaline lignin (Tokyo Kasei) and lignin sulfonate (Tokyo Kasei) .

Generally, the activating agent is added to the tissue culture medium at a concentration of from about 1 to about 30 μg/ml of medium. The cells then are seeded at a concentration ranging from about 1 x 10 ⁵ to about 6 x 10 ⁵ cells/ml of medium.

The cells then are incubated, generally for a period of about 1 to about 7 days, under conditions which induce the cells to produce one or more proteins having anti-HIV activity. Generally, such conditions include an incubation temperature of about 37°C and a 100% humidified atmosphere of about 5% carbon dioxide. The length of the incubation time has not been found to be critical; under the conditions generally described above, the cells produce the desired proteins within about 24 hours of the start of the incubation, and high levels of protein still are detected in the medium after 7 days of incubation.

To recover the anti-retrovirus proteins, the cells are separated from the aqueous medium. Typically, this is done by centrifugation at, for example, 800 x g. The cell pellet is removed from the cell-free aqueous phase. The anti-retrovirus proteins are in the aqueous phase.

Throughout the remainder of the application, reference will be made to a relatively large protein and to a smaller protein, each of which has anti- retrovirus activity. It is to be recognized, however, that in fact what is produced and secreted by the cells may be a mixture of at least two proteins within each of the molecular weight ranges disclosed herein. The supernatant containing the anti-retrovirus protein is passed through a 30 kD molecular sieve.

General conditions include a pressure of 18 psi or 500 x g. Applicants have found that protein molecules which can inhibit replication of retroviruses are retained by this sieve. In addition, protein molecules which pass through this sieve but are retained with a 3 kD molecular sieve also have anti- retrovirus activity. Thus, this is suggestive that the cells produce at least one protein having a molecular size which is at least about 30,000 daltons, as well as at least one protein having a molecular size between about 3,000 and 30,000 daltons.

When cells infected with a retrovirus, such as HIV, are contacted with the protein, replication of the virus is inhibited. More specifically, when infected T-cells are contacted with the protein, HIV replication is inhibited by more than 80%. The protein can inhibit HIV replication in T-cells by over 90%, and desirably by over 95%. The degree of inhibition of HIV replication in myeloid cells, such

as U937 cells, typically is lower per given amount of protein than is observed in T-cells. The amount of protein that inhibits replication in T-cells by at least 80%, for example, typically inhibits HIV replication in myeloid cells by between about 60 and 80%.

Analysis of both the anti-retrovirus protein that is retained by the 30 kD sieve shows and the protein that passes through the 30 kD sieve shows that neither is interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, colony stimulation factor for granulocyte and macrophages (GM-CSF) , colony stimulation factor for granulocytes, colony stimulation factor for macrophages, interferon-alpha, interferon-beta, interferon-gamma, tumor necrosis factor-alpha, tumor necrosis factor-beta, or transforming growth factor-beta. Specific neutralizing antibodies to each of these factors fails to abrogate the anti-retrovirus activity of a medium comprising either protein.

If desired, the anti-retrovirus protein retained by the 30 kD sieve can be purified by ion exchange chromatography. Conventional liquid chromatography, high performance liquid chromatography, or fast performance liquid chromatography can be used. More specifically, to purify the protein, that fraction of the supernatant which was retained by the 30 kD sieve is added to an elution buffer to adjust the pH to be within the range of about 4.5 to about 5.6. A suitable elution buffer is 20 mM sodium acetate at pH 5.6, 10% glycerol, 0.1 mM of disodium salt of ethylene diamine tetraacetic acid (EDTA) at pH 6.0 and 0.1 mM dithiothreitol . The solution containing the anti-

retrovirus protein then can be added to a cation exchange column, such as a CM-cellulose or mono-S column. The flow-through fraction from this column contains the majority of the anti-retrovirus protein found in the retained portion of the medium. The pH of this fraction then is adjusted to be within the range of about 6.5 and about 7.8 in elution buffer. A suitable elution buffer is 20 mM Tris [hydroxymethyl] aminomethane at pH 7.4, 0.1 mM of disodium salt of ethylene diaminetetraacetic acid (EDTA) at pH 7.0, 10% glycerol and 0.1 mM of dithiothreitol. The solution containing the anti- retrovirus protein is added to an anion exchange column, such as a DEAE or mono-Q column. The anion exchange column then is eluted with the elution buffer containing 1 M sodium chloride. The eluate contains the anti-retrovirus protein.

The protein in aqueous solution can be stored at 4°C for up to about 4 weeks. The protein can be used to inhibit replication of a retrovirus, such as HIV, in cells infected with the retrovirus. For in vi tro inhibition, the amount of protein preparation needed to inhibit replication of the virus in virus-infected cells will vary depending upon the degree of purification of the protein.

Typically, however, if the infected cells are cultured in a medium comprising the crude protein (i.e., protein obtained by passing the protein-containing supernatant through a 30 kD molecular sieve) about 1 to about 5 mg of protein per ml. are sufficient to obtain at least 80% inhibition of the virus. If the protein to be used has been purified by passing it through cation and anion exchange columns, typically only about 10 to about 50 μg of protein per ml. are

needed to obtain at least 80% inhibition. Useful amounts of protein from any given preparation can be determined readily by one of ordinary skill in the art once he has determined the activity of the protein and the total amount of protein present in the preparation.

The protein can be administered to a mammal infected with a retrovirus, such as a lentivirus, so as to effect treatment of the virus. The treatment comprises administering the protein according to a treatment regimen sufficient to inhibit replication of the virus. Desirably, the protein is administered in an amount in the range of about 2,000 and about 20,000 units per kilogram of body weight of the mammal per day. Or.e unit is defined as the concentration of the anti-retrovirus protein needed to reduce virus replication in CD4 cells by 50% using the assay described in the examples below. The dosage regimen can be adjusted to provide the optimum therapeutic response. For instance, several divided doses can be administered daily. The protein can be combined with a pharmaceutically acceptable carrier and administered to a patient by any suitable and practical route, such as intravenously, intramuscularly, subcutaneously, intranasally or orally. Depending upon the route of administration, it may be desirable to coat the protein in a material which will protect it from the action of enzymes, acids, or other natural conditions which may inactivate the protein. For example, the protein could be destroyed by enzymes or acid hydrolysis in the gastrointestinal tract and stomach. If the protein is to be administered other than parenterally, it can be coated by, or administered in combination with, a material that will prevent the

protein's inactivation. For example, the protein can be administered in an adjuvant or in a liposome. The protein then can be administered with an inert diluent, encased in a capsule or compressed into tablets.

Pharmaceutical forms suitable for injectable administration include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions. The carrier must be sterile and sufficiently fluid that the formulation can be administered easily using a syringe. The carrier can comprise water, ethanol, a polyol such as glycerol or PEG, or vegetable oil. Fluidity can be maintained by using a coating such as lecitin, by the use of surfactants and, if the formulation is in the form of a dispersion, by maintaining the required particle size. The formulation further can comprise one or more antibacterial or antifungal agents or isotonic agents such as sugars or sodium chloride. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersions, media, coatings, isotonic agents, antibacterial agents, antifungal agents and the like. The use of such agents for pharmaceutically active substances is well known to persons of ordinary skill in the art. The administration of the protein typically inhibits replication of the virus in the mammal by at least about 80%, preferably by at least about 90%, and most preferably by at least about 95%. The present invention is further illustrated by the following examples, which are not intended to be limiting.

EXAMPLE 1 Preparation of Clone 4084

CEM cells at 1.5 x 10 ⁵ cells/ml were cultured at 37°C for 2 days in 50 ml of RPMI-1640 tissue culture medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin. The cells then were adjusted to 3 x 10 ⁶ cells/ml with fresh medium. One ml of this cell suspension was added to 9 ml of fresh medium to give 3 x 10 ⁵ cells/ml. After thorough mixing, 1 ml of this cell suspension again was added to 9 ml of fresh medium. This 10-fold dilution step was repeated serially to give a cell suspension containing 3 cells/ml. 100 μl of this cell suspension then were added to each well in a 96 well flat-bottomed tissue culture plate to give a frequency of 1 cell every three wells. A total of 20 tissue culture plates were seeded with this cell suspension. The plates were at 37°C and in a humidified atmosphere of 5% C0 ₂ . Wells in each plate were examined for signs of cellular proliferation every other day by use of an inverted microscope. At weekly intervals, 50 μl of fresh medium were added to each well to maintain the cells in culture. The plates were incubated for three weeks or when signs of proliferation were evident. Actively proliferating cells in each microwell then were transferred into wells in a 24 well flat-bottomed tissue culture plate. Each of these cloned cell cultures then were further expanded to 10 ml cultures in tissue culture flasks. One of the cloned cultures, clone 4, was recloned at 1 cell every three wells in flat-bottomed 96 well tissue culture plates as described above and again gave a large number of subclones. One of these was designated clone 4084.

This clone was a good producer of proteins having anti-HIV activity (see example 2, below) .

EXAMPLE 2 Production of Proteins Having anti-HIV Activity

Clone 4084 cells were cultured at 37 °C for 2 days in 13,000 ml. of RPMI-1640 tissue culture medium supplemented with 5% fetal bovine serum, 100 units/ml of penicillin, 100 μg/ml streptomycin and 10 μg/ml PC6 (obtained from Tampa Bay Research Institute, St. Petersburg, Florida) . The cells were cultivated in 100% humidified atmosphere of 5% C0 ₂ . The clone 4084 cells were removed from the culture medium by centrifugation at 800 x g for 10 minutes and subsequent removal of the resultant cell pellet. The supernatant was passed through a 30 kD molecular sieve, spiral filtration cartridge, type S10Y30 (Amicon, Beverly, MA) . The retentate contained the large protein with anti-HIV activity. The effluent was collected and allowed to pass through a 3 kD molecular sieve S10Y3 (Amicon) . The retentate collected contained the small protein with anti-HIV activity.

To determine the anti-HIV activity of the large and small proteins, the following procedure was used: The chloramphenicol acetyl-transferase (CAT) gene was introduced into HIV-1 (Langhoff, E. et al . , PNAS USA 88:7998-8002 (1991) . Expression of CAT was under the control of the HIV-1 promoter that controls the expression of all HIV-1 genes. Expression of CAT under the control of the HIV-1 promoter is a good measure of HIV-1 replication { Id. ) . CEM cells were

infected with this recombinant HIV-1 by adding 1 x 10 ⁶ cells to each 1 ml of the virus.

The infected cells then were placed in RPMI-1640 medium supplemented with 2% heat-inactivated newborn calf serum, 100 units/ml of penicillin, 100 μg/ml streptomycin and varying dilutions (1 in 5 to 1 in 500) of one of the three protein preparations shown in Table 1. After incubation for 3 days, the amount of HIV-l produced by the cells was measured by quantitation of CAT activity in each culture. CAT activity was measured by a mixed phase assay (Nielson, D.A., et al., Analyt. Biochejn. 179:19-23 [1989]) , wherein the ability to incorporate tritiated-acetyl group from tritiated acetyl Co-A onto chloramphenicol was quantitated in a liquid scintillation counter.

Percent inhibition of HIV-l replication was calculated from:

[ (P-B)/(C-B)] X 100 where P = CAT activity quantitated as counts per minute per microgram protein from infected cell culture treated with the protein preparation, C = CAT activity quantitated as counts per minute per microgram of protein from infected cell culture not treated with the protein preparation, and B is the background control given tritiated acetyl Co-A in the absence of chloramphenicol. ^■

The results are shown in Table 1.

TABLE 1

Inhibition (%) of HIV-l bv

Fraction through passed Fraction

30 kD retained

Supernatant sieve, but by the

Sample from Stimulated retained by 30 kD

Dilutions+ 4084 cells 3 kD sieve sieve

1 in 5 80% 83% 97%

1 in 25 65% 65% 94%

1 in 100 55% 21% 81%

1 in 500 N.T. N.T. 34%

Units/ml* 100 40 350

Total Units** 110,000 36,000 70,000 one unit is defined as the amount of protein r ^c -.uired to inhibit HIV-l replication by 50%.

** total unit is given by Units/ml x total volume of protein preparation the samples were concentrated lOx before testing in the varying dilutions listed below.

EXAMPLE 3 Inhibition of HIV Replication in U937 Cells

The following experiment was designed to show HIV-l replication in U937 myeloid cells could be inhibited by the protein retained by the 30 kD sieve.

CEM cells (ATCC Accession No. CCL119) or U937 cells (ATCC Accession No. CRL1593) were infected with the recombinant HIV-l as described in Example 2, above. These infected cells then were placed in RPMI- 1640 medium supplemented with 2% heat inactivated newborn calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin and a 1 in 10 dilution of the protein preparation retained by the 30 kD molecular sieve. After incubation for 3 days, the amount of HIV-l

produced was measured by quantitation of CAT activity in each culture. CAT activity was measured by a mixed phase assay as described in the preceding example, wherein the ability to incorporate tritiated-acetyl group from tritiated acetyl Co-A onto chloramphenicol was quantitated in a liquid scintillation counter. Percent inhibition of HIV-l was calculated as described in the preceding example. The results are set forth in Table 2.

TABLE 2

Percent inhibition of HIV-l replication in

CEM cells U937 cells

Protein retained by 30 kD sieve tested at 1 in 10 dilution 85% 64%

Example 4 Purification of Protein with Anti-HIV Activity

Clone 4084 cells were cultured at 37°C for 2 days in 13.8 liters of RPMI-1640 tissue culture medium, supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 10 μg/ml PC6. The cells were cultivated in 100% humidified atmosphere of 5% C0 ₂ . The cells were removed by centrifugation, the fraction that could be retained by the 30 kD molecular sieve and contained the large protein with retrovirus replication inhibiting was prepared as described in Example 2. The volume of this fraction was 3.3 liters. If one defines one unit (U) of anti-HIV activity as the amount of protein

needed to inhibit HIV replication by 50% in 1 ml culture of infected CEM cells, this fraction would have 10 U/ml or approximately 33,000 U of total anti- HIV activity (Table 3) . This fraction was added to an equal volume of acetate elution buffer [20 mM sodium acetate at pH 5.6, 10% glycerol, 0.1 mM disodium salt of ethylene diamine tetraacetic acid (EDTA) at pH 6.0 and 0.1 mM dithiothreitol] . The volume of this mixture was reduced to 3.3 liter by passing it through a 30 kD membrane (type S10Y30, Amicon) at 18 psi or

500 x g. This washing procedure was repeated 5 times to equilibrate this fraction with the acetate elution buffer. The equilibrated fraction then was added to CM-cellulose. Fractions that flowed through CM- cellulose were collected (3.3 liters) . Material bound to CM-cellulose was recovered by a single step elution with 0.3M NaCl in acetate elution buffer. As shown in Table 3, a total of 900 U were found in the 0.3M NaCl eluted fraction, whereas the CM-flow-through fraction has approximately 33,000 U. If one defines anti-HIV specific activity as the units of anti-HIV activity present in 1 mg of proteins, i.e. total anti-HIV units in a fraction divided by total proteins in mg in the fraction, the 0.3 M NaCl eluted fraction has a specific activity of 0.6, while the CM-flow-through fraction had a specific activity of 4.17.

The CM-flow-through fraction containing the large protein having anti-HIV activity that did not bind to CM-cellulose at pH 5.6 then was adjusted to pH 7.4 by the addition of Tris [hydroxymethyl] aminomethane and added to DEAE cellulose. The fraction that flowed through the DEAE-cellulose was collected. Material bound to DEAE-cellulose was recovered by stepwise elution with 0.3 M and 1.0 M NaCl in elution buffer of

20 mM Tris [hydroxyτnethyl] aminomethane at pH 7.4, 10% glycerol, 0.1 mM disodium salt of EDTA at pH 7.0 and 0.1 mM dithiothreitol. When tested for anti-HIV activity, the fraction that flowed through DEAE- cellulose and contained material not bound to DEAE- cellulose did not have anti-HIV activity. The 0.3 M NaCl eluted fraction had 2400 U of anti-HIV activity but with a specific activity of 0.87. In contrast, the 1.0 M NaCl eluate had 6000 U of anti-HIV activity and a specific activity of 95 (Table 3) .

Table 3

Amount of anti-HIV activity in

Specific Activity*** Units*/ml Total U** (U/mg)

Fraction retained by 30 kD molecular sieve 10 33,000 0.88

Fraction recovered by 0.3M NaCl elution from the CM-cellulose 900 0.66

Fraction not bound to the CM-Cellulose at pH 5.6 10 33,000 4.17 Fraction not bound to the CM-cellulose, and also not bound to DEAE-cellulose at pH 7.4 0 Fraction not bound to the CM-cellulose, but bound to DEAE- cellulose and eluted by 0.3 M NaCl 4 2,400 0.87 Fraction not bound to the CM-cellulose, but bound to DEAE-cellulose and eluted by 1.0 M NaCl 15 6,000 95.0

One unit (U) is defined as the amount of protein required to inhibit HIV-l replication by 50%

*★ Total units is given by U/ml x total volume of a protein containing preparation

*** Specific activity (U/mg) is given by total U in a protein preparation divided by the total amount of protein (mg) present in that preparation.

EXAMPLE 5

Clone 4084 cells were cultured at 37°C for two days in 7 liters of RPMI-1640 tissue culture medium, supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 10 μg/ml PC6. the cells wee cultivated in 100% humidified atmosphere 25 of 5% C0 ₂ . The cells were removed by centrifugation and the fraction retained by a 30 kD molecular sieve was allowed to pass through a CM-cellulose column at pH 5.6, bound to DEAE cellulose at pH 7.4 and eluted with 1.0 M NaCl in elution buffer of 20 mM Tris [hydroxymethyl] aminomethane at pH 7.4, 10% glycerol, 0.1 mM disodium salt of EDTA at pH 7.0 and 0.1 mM dithiothreitol as described in Example 4. As shown in the table, this fraction contained protein having anti-HIV activity. An aliquot of this fraction was dialyzed overnight at 4C°C against a buffer containing 25 mM Tris [hydroxymethyl] aminomethane and 10% glycerol at pH 7.4. This dialyzed preparation was added to a Superdex 75 gel filtration column

(Pharmacia) and eluted by fast performance liquid chromatography at 3 ml/minute with the elution buffer having 25 mM tris [hydroxymethyl] aminomethane and 10% glycerol at pH 7.4. Twenty 6 ml fractions were collected at 1 minute intervals between retention time 36 minutes to 76 minutes. Relative to the retention time of the molecular markers bovine serum albumin (66 kD) , carbonic anhydrase (29 kD) and cytochrome c (12.4 kD) eluted from the column under the same experimental conditions, the anti-HIV molecules in fractions 7 to 12 should have had an apparent molecular weight ranging from 18 to 45 kD. When tested, none of these fractions had significant anti-HIV activity. The

protein of interest was found only in the void volume and in fraction 1 obtained from the column.