Synergistic cytotoxic composition

Compositions comprising an interferon and a cytotoxic protein designated tumor necrosis factor exhibit a synergistic cytotoxic effect on tumor cells.

This application relates to lymphokines. In particular, it relates to 
cytotoxic factors secreted by lymph cells and methods for making same in 
recombinant cells. 
Immune cells such as B cells, T Cells, natural killer cells and macrophages 
are known to elaborate substances that exert cytotoxic activity toward 
tumor cells but which are innocuous to normal cells. These substances have 
been variously named, for example, lymphotoxin, tumor necrosis factor, NK 
cell cytotoxic factor, hemorrhagic necrosis factor, macrophage cytotoxin 
or macrophage cytotoxic factor. At the present time the identities of the 
proteins associated with these names are unclear. The principal 
difficulties have been that the biological assays employed to detect the 
proteins do not discriminate among them, the proteins appear to be found 
in nature as aggregates or hydrolytic products, and the amounts heretofore 
obtained have been so small that the high degree of purification needed to 
fully characterize the proteins has not been reached. 
Typically, such cytotoxic substances are found in the sera of intact 
animals, or in the culture supernatants of lymph cells or cell lines after 
the animals or cells had been treated with a substance known to stimulate 
the proliferation of immune cells (an "inducer"). Thereafter the serum or 
supernatant is recovered and assayed for cytotoxic activity towards a 
target tumor cell line. A standard target is L-929, a murine tumor cell 
line. This cell line and others used in bioassays of this type are 
nonspecific in their lytic response because a variety of apparently 
discrete lymph cell products are able to effect lysis. Similar nonspecific 
responses are observed in in vitro tumor necrosis assays. Thus, cytolytic 
assays which observe for the lysis of cell lines in vitro or tumor 
necrosis in vivo are inadequate to distinguish among the various cytotoxic 
lymph products. 
Cytotoxic factors tentatively have been classified on the basis of the 
lymph cells from which they are induced. For example, lymphotoxin is a 
name commonly applied to the cytotoxic secretory products of B or T 
lymphocytes, or cell lines derived therefrom, while tumor necrosis factor 
often is used to describe cytotoxic products of macrophages or their 
derived cell lines. This classification system, however, has not been 
developed to the point where there is any assurance that only a single 
protein is being referred to, or that proteins referred to by different 
name are in fact different. 
Attempts have been made to purify and characterize the cytotoxic factors 
secreted by each cell type. To the extent that reports vary as to a 
property of a cytotoxic factor, or are completely inconsistent as to a 
given property, it could be concluded either that the characterization was 
erroneous or that a plurality of discrete cytotoxic factors are secreted 
by each cell type. For example, the cytotoxic products derived from 
macrophages, monocytes or monocytic cell lines, while sometimes generally 
referred to as tumor necrosis factor, have been reported to have 
properties that appear inconsistent with a theory of a single cytotoxic 
product. See for example the following literature: R. MacFarlan et al., 
1980, "AJEBAK" 58(pt 5): 489-500; D. Mannel et al., 1980, "Infection and 
Immunology" 30(2): 523-530; H. Ohnishi et al., UK patent application No. 
2,106,117A; and J. Hammerstrom, 1982, "Scand J. Immunol." 15: 311-318. 
On the other hand C. Zacharchuk et al., 1983, "Proc. Nat. Acad. Sci. 
U.S.A.", 80: 6341-6345 suggest that guinea pig lymphotoxin and a cytotoxic 
factor from guinea pig macrophages are immunochemically similar, if not 
identical. Similar conclusions are advanced in Ruff et al., 1981, 
Lymphokines Vol. 2 pp. 235-272 at pp. 241-242. 
The attempts at characterization of immune cytotoxic factors also have 
focused on using as starting material the sera or peritoneal fluid of 
animals that have been exposed to immunogenic antigens. These sources 
contain the entire cornucopia of the stressed immune system, in contrast 
to the product or products of a single cell type or line. The following 
should be consulted as examples of publications of this type: S. Green et 
al., 1982, "J. Nat. Cancer Inst." 68(6): 997-1003 ("tumor 
necrosis-inducing factor"); M. Ruff et al., 1980, "J. Immunology" 125(4): 
1671-1677 ("tumor necrosis factor"); H. Enomoto et al., European Patent 
Application No. 86475 ("antitumor substance"); H. Oettgen et al., 1980, 
"Recent Results Cancer Res." 75: 207-212 ("tumor necrosis factor"); F. 
Kull et al., 1981, "J. Immunol." 126(4): 1279-1283 ("Tumor Cell 
Cytotoxin":); D. Mannel et al., 1980, "Infection and Immunity" 28(1): 
204-211 ("cytotoxic factor"); N. Matthews et al., 1980, "Br. J. Cancer: 
42: 416-422 ("tumor necrosis factor"); S. Green et al., 1976, "Proc. Nat. 
Acad. Sci. U.S.A.:, 73(2): 381-385 ("serum factor"); N. Satomi et al., 
1981, "Jpn J. Exp. Med." 51(6): 317-322; N. Matthews, 1979, "Br. J. 
Cancer" 40: 534-539 ("tumor necrosis factor"); K. Haranaka et al., 1981, 
"Jpn. J. Exp. Med." 51(3): 191-194 ("tumor necrosis factor"); and L. Old 
et al., European Patent Application No. 90892; T. Umeda et al., 1983, 
"Cellular and Molecular Biology" 29(5): 349-352; and H. Enomoto et al., 
1983, European Patent Application No. 86,475. 
Further literature which should be consulted is J. Nissen-Meyer et al., 
1982, "Infection and Immunity" 38(1): 67-73; J. Klostergaard et al., 1981, 
"Mol. Immunol." 18(12): 1049-1054; N. Sloane, U.S. Pat. No. 4,359,415; and 
H. Hayashi et al., U.S. Pat. No. 4,447,355; K. Hanamaka et al., 1983, 
European Patent Application No. 90,892; and G. Granger et al., 1978, 
"Cellular Immunology" 38: 388-402. 
European Patent Application Publn. No. 100641 describes a cytotoxic 
polypeptide which was purified substantially free of impurities from a 
human lymphoblastoid cell culture. This polypeptide was designated 
lymphotoxin, although its relationship to other reported cytotoxic 
polypeptides under the name lymphotoxin is conjectural. It was not known 
whether this was the sole cytotoxic polypeptide elaborated by immune 
cells, as suggested by Zacharchuk et al. (Id.), or whether it was one of a 
potential family of cytotoxic factors. 
The polypeptide of the '641 Application has two amino termini, a larger 
variant ending with Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr 
Ala Arg Gln His Pro Lys Met His Leu Ala His Ser Thr . . . and a smaller 
variant with the truncated amino terminus His Ser Thr Leu Lys Pro Ala Ala 
. . . The amino acid sequence of the lymphotoxin of the '641 Application 
is disclosed in copending U.S. Ser. No. 616,503, filed May 31, 1984, 
wherein the term "lymphotoxin" is defined. 
According to the prior literature the interferons, which exhibit some tumor 
inhibitory activity, and a poorly characterized protein having an AlaAla 
amino terminus (U.K. Patent Application Publn. No. 2,117,385A), were 
candidates for non-lymphotoxin cytotoxic factors. As will be seen, the 
tumor necrosis factor of this invention is not an interferon, is not 
lymphotoxin and does not have an AlaAla amino terminus. 
It is an object of this invention (a) to conclusively determine whether or 
not another tumor necrosis factor than lymphotoxin exists and, if so, to 
identify it in such a way as to clearly distinguish other such factors; 
(b) to produce such a factor by methods other than induced cell culture, 
which is expensive and yields product which is contaminated with the 
inducing agent, or by induction of peripheral blood lymphocytes, which is 
economically impractical, poorly reproducible, and produces product 
contaminated with homologous cellular and plasma proteins; (c) to obtain 
DNA encoding such tumor necrosis factor and to express the DNA in 
recombinant culture; (d) to synthesize such factor in recombinant culture 
in the mature form; (e) to modify the coding sequence or structure of such 
factor; (f) to formulate such factor into therapeutic dosage forms and to 
administer same to animals for the treatment of tumors; and (g) to produce 
diagnostic reagents relating to such factor. 
SUMMARY 
A cytotoxic factor has been purified to homogeneity, characterized and 
expressed in recombinant culture. This factor is designated tumor necrosis 
factor (TNF) for convenience and is defined below. It is provided in 
substantially homogeneous form from cell culture at a specific activity of 
greater than about 10 million units/mg protein, and ordinarily about 100 
million units/mg. 
Human tumor necrosis factor synthesized in recombinant culture is 
characterized by the presence of non-human cell components, including 
proteins, in amounts and of a character which are physiologically 
acceptable for administration to patients in concert with the tumor 
necrosis factor. These components ordinarily will be of yeast, procaryotic 
or non-human higher eukaryotic origin and present in innocuous contaminant 
quantities, on the order of less than about 1 percent by weight. Further, 
recombinant cell culture enables the production of tumor necrosis factor 
absolutely free of homologous proteins. Homologous proteins are those 
which are normally associated with the tumor necrosis factor as it is 
found in nature, e.g. in cells, cell exudates or body fluids. For example, 
a homologous protein for human tumor necrosis factor is human serum 
albumin. Heterologous proteins are the converse, i.e. they are not 
naturally associated or found in combination with the tumor necrosis 
factor in question. 
DNA is provided that encodes tumor necrosis factor and which, when 
expressed in recombinant or transformed culture, yields copious quantities 
of tumor necrosis factor. This DNA is novel because cDNA obtained by 
reverse transcription of mRNA from an induced monocytic cell line contains 
no introns and is free of any flanking regions encoding other proteins of 
the organism from which the mRNA originated. 
Chromosomal DNA encoding TNF is obtained by probing genomic DNA libraries 
with cDNA. Chromosomal DNA is free of flanking regions encoding other 
proteins but may contain introns. 
The isolated tumor necrosis factor DNA is readily modified by substitution, 
deletion or insertion of nucleotides, thereby resulting in novel DNA 
sequences encoding tumor necrosis factor or its derivatives. These 
modified sequences are used to produce mutant tumor necrosis factor and to 
directly express mature tumor necrosis factor. The modified sequences also 
are useful in enhancing the efficiency of tumor necrosis factor expression 
in chosen host-vector systems, e.g. by modification to conform to a host 
cell codon preference. 
These novel DNA sequences or fragments thereof are labelled and used in 
hybridization assays for genetic material encoding tumor necrosis factor. 
In processes for the synthesis of tumor necrosis factor, DNA which encodes 
tumor necrosis factor is ligated into a replicable (reproducible) vector, 
the vector used to transform host cells, the host cells cultured and tumor 
necrosis factor recovered from the culture. This general process is used 
to construct tumor necrosis factor having the characteristics of monocyte 
tumor necrosis factor or to construct novel derivatives of tumor necrosis 
factor, depending upon vector construction and the host cell chosen for 
transformation. The tumor necrosis factor species which are capable of 
synthesis herein include mature (valyl amino-terminal) tumor necrosis 
factor, pretumor necrosis factor ("preTNF", defined herein), and 
derivatives of TNF including (a) fusion proteins wherein TNF or any 
fragment thereof (including mature tumor necrosis factor) is linked to 
other proteins or polypeptides by a peptide bond at the amino and/or 
carboxyl terminal amino acids of TNF or its fragments, (b) TNF fragments, 
including mature tumor necrosis factor or fragments of preTNF in which any 
preprotein amino acid is the amino-terminal amino acid of the fragment, 
(c) mutants of TNF or its fragments (including mature tumor necrosis 
factor) wherein one or more amino acid residues are substituted, inserted 
or deleted, and/or (d) methionyl or modified methionyl (such as formyl 
methionyl or other blocked methionyl species) amino-terminal addition 
derivatives of the foregoing proteins, fragments or mutants. 
Ordinarily, if a mammalian cell is transformed with (a) a vector containing 
the entire tumor necrosis factor structural gene (including a 5' start 
codon), or (b) the gene for mature tumor necrosis factor or a tumor 
necrosis factor derivative operably ligated to a eukaryotic secretory 
leader (which may also include the tumor necrosis factor secretory leader 
presequence), and the cell cultured, then mature tumor necrosis factor is 
recovered from the culture. 
Similarly, if DNA which encodes TNF is operably ligated in a vector to a 
secretory leader which is properly processed by the host cell to be 
transformed (usually the organism from which the leader sequence was 
obtained), the host transformed with the vector and cultured, then the 
tumor necrosis factor is synthesized without amino-terminal methionyl or 
blocked methionyl. In particular, E. coli transformed with vectors in 
which DNA encoding mature tumor necrosis factor is ligated 5' to DNA 
encoding the STII enterotoxin signal polypeptide will properly process a 
high percentage of the hybrid preprotein to mature tumor necrosis factor. 
Secretory leaders and host cells may be selected that also result in 
proper transport of mature protein into cell periplasm. 
Also within the scope of this invention are derivatives of tumor necrosis 
factor other than variations in amino acid sequence or glycosylation. Such 
derivatives are characterized by covalent or aggregative association with 
chemical moieties. The derivatives generally fall into three classes: 
salts, side chain and terminal residue covalent modifications, and 
adsorption complexes. 
If DNA encoding mature tumor necrosis factor is operably ligated into a 
vector, the vector used to transform a host cell and the cell cultured, 
mature tumor necrosis factor is found in the cell cytoplasm. Accordingly, 
it is unnecessary to devise secretion systems in order to obtain mature 
tumor necrosis factor. This was surprising because, ordinarily, direct 
expression yields methionylated protein. Further, the protein is stable 
and soluble in recombinant cell culture, i.e., it is neither 
proteolytically cleaved by intracellular proteases nor deposited as 
refractile bodies. Accordingly, novel fermentations are provided that 
comprise lower eukaryotic or prokaryotic cells having unmethionylated 
mature tumor necrosis factor located within the cytoplasm of such cells. 
While tumor necrosis factor may be prepared by culturing animal cell lines, 
e.g. a monocytic cell line induced by growth in the presence of 
4-beta-phorbol-12-myristate-13-acetate (PMA) or immortal cell lines such 
as hybridomas or EBV transformed cells (U.S. Pat. No. 4,464,465), it is 
preferable to synthesize tumor necrosis factor in recombinant cell culture 
as described further below. 
Once tumor necrosis factor is prepared by fermentation it generally is 
purified by recovering the supernatant culture fluid or lysed cell 
culture, removing solids, adsorbing tumor necrosis factor from the 
supernatant admixture (containing tumor necrosis factor and other 
proteins) onto a hydrophobic substance, eluting tumor necrosis factor from 
the substance, adsorbing tumor necrosis factor onto a tertiary amino anion 
exchange resin, eluting tumor necrosis factor from the resin, adsorbing 
tumor necrosis factor onto an anion exchange resin (preferably quaternary 
amino-substituted) having substantially uniform particle size, and eluting 
tumor necrosis factor from the resin. Optionally, the tumor necrosis 
factor compositions are concentrated and purified by chromatofocusing at 
any point in the purification procedure, for example by isoelectric 
focusing or passage through a sieving gel such as Sephadex G-25. 
The purified tumor necrosis factor from recombinant or induced cell culture 
is combined for therapeutic use with physiologically innocuous stabilizers 
and excipients and prepared in dosage form as by lyophilization in dosage 
vials or storage in stabilized aqueous preparations. Alternatively, tumor 
necrosis factor is incorporated into a polymer matrix for implantation 
into tumors or surgical sites from which tumors have been excised, thereby 
effecting a timed-release of the tumor necrosis factor in a localized high 
gradient concentration. 
The compositions herein are obtained free of contaminant cytotoxic factors 
such as lymphotoxin, interferons or other cytotoxic proteins referred to 
in the literature. However, in therapeutic applications tumor necrosis 
factor is advantageously combined with predetermined amounts of 
lymphotoxin and/or interferon. Compositions containing tumor necrosis 
factor and an interferon such as gamma interferon are particularly useful 
since they have been found to exert a synergistic cytotoxic activity. 
Tumor necrosis factor compositions are administered to animals, 
particularly patients bearing malignant tumors, in therapeutically 
effective doses. Suitable dosages will be apparent to the artisan in the 
therapeutic context, as is further described infra.

DETAILED DESCRIPTION 
Tumor necrosis factor is defined for the purposes herein as a polypeptide 
other than lymphotoxin capable of preferential cytotoxic activity and 
having a region showing functional amino acid homology with the mature 
tumor necrosis factor amino acid sequence set forth in FIG. 10, a fragment 
thereof, or a derivative of such polypeptide or fragment. 
Preferential cytotoxic activity is defined as the preferential destruction 
or growth inhibition of tumor cells when compared to normal cells under 
the same conditions. Preferential cytotoxic activity is detected by the 
effect of the polypeptide on tumor cells in vivo or in vitro as compared 
with normal cells or tissue. Cell lysis is generally the diagnostic 
indicia in vitrn, while tumor necrosis is determined in in vivo 
experiments. However, cytotoxic activity may be manifest as cytostasis or 
antiproliferative activity. Suitable assay systems are well known. For 
example, the cell-lytic assay used to determine the specific activity of 
tumor necrosis factor described below is acceptable, as is the assay 
described in B. Aggarwal et al., in "Thymic Hormones and Lymphokines", 
1983, ed. A. Goldstein, Spring Symposium on Health Sciences, George 
Washington University Medical Center (the A549 cell line referred to in 
this literature is available from the ATCC as CCL185). 
The specific activity of TNF is cast in terms of target cell lysis, rather 
than cytostasis. One unit of tumor necrosis factor is defined as the 
amount required for 50 percent lysis of the target cells plated in each 
well in accord with Example 1. However, this is not meant to exclude other 
assays for measuring specific activity, e.g. methods based on target cell 
growth rate. 
PreTNF is a species of tumor necrosis factor included within the foregoing 
definition of tumor necrosis factor. It is characterized by the presence 
in the molecule of a signal (or leader) polypeptide which serves to 
post-translationally direct the protein to a site inside or outside of the 
cell. Generally, the signal polypeptide (which will not have tumor 
necrotic activity in its own right) is proteolytically cleaved from a 
residual protein having tumor necrosis factor activity as part of the 
secretory process in which the protein is transported into the host cell 
periplasm or culture medium. The signal peptide may be microbial or 
mammalian (including the native, 76 residue presequence), but it 
preferably is a signal which is homologous to the host cell. 
Native tumor necrosis factor from normal biological sources has a 
calculated molecular weight of about 17,000 on sodium dodecyl sulfate 
polyacrylamide gel electrophoresis (SDS-PAGE) as described infra, an 
isoelectric point of about 5.3, and susceptibility to trypsin hydrolysis 
at multiple sites. Native tumor necrosis factor which has been purified by 
reverse phase HPLC is trypsin hydrolyzed into at least nine fragments 
under the conditions described infra. The precise number of fragments into 
which tumor necrosis factor is hydrolyzed by trypsin will depend upon such 
factors as trypsin activity, tumor necrosis factor concentration and 
incubation parameters, including the ionic strength, pH, temperature and 
time of incubation. 
Tumor necrosis factor does not appear to be a glycoprotein, as it is not 
retained on lectin affinity columns and analysis of the deduced amino acid 
sequence contains no likely glycosylation sites. Also, material produced 
in recombinant E. coli culture (which does not have the ability to 
glycosylate) co-migrates with natural TNF on SDS-PAGE. 
The degree of amino acid sequence homology which brings a polypeptide 
within the scope of the definition of tumor necrosis factor herein will 
vary depending upon whether the homology between the candidate protein and 
tumor necrosis factor falls within or without the tumor necrosis factor 
regions responsible for cytotoxic activity; domains which are critical for 
cytotoxic activity should exhibit a high degree of homology in order to 
fall within the definition, while sequences not involved in maintaining 
tumor necrosis factor conformation or in effecting receptor binding may 
show comparatively low homology. In addition, critical domains may exhibit 
cytolytic activity and yet remain homologous as defined herein if residues 
containing functionally similar amino acid side chains are substituted. 
Functionally similar refers to dominant characteristics of the side chains 
such as basic, neutral or acid, or the presence or absence of steric bulk. 
However, tumor necrosis factor as defined herein specifically excludes any 
proteins identified as lymphotoxin in copending U.S. Ser. No. 616,503, 
incorporated herein by reference. 
Generally a polypeptide defined as tumor necrosis factor will contain 
regions substantially homologous with the FIG. 10 protein or fragments 
thereof over a continuous block of about from 20 to 100 amino acid 
residues in particular the blocks encompassed by residues 35-66 and 
110-133. 
A most significant factor in establishing the identity of a polypeptide as 
tumor necrosis factor is the ability of antisera which are capable of 
substantially neutralizing the cytolytic activity of mature tumor necrosis 
factor as set forth in FIG. 10 to also substantially neutralize the 
cytolytic activity of the polypeptide in question. However it will be 
recognized that immunological identity and cytotoxic identity are not 
necessarily coextensive. A neutralizing antibody for the tumor necrosis 
factor of FIG. 10 may not bind a candidate protein because the 
neutralizing antibody happens to not be directed to specifically bind a 
site on tumor necrosis factor that is critical to its cytotoxic activity. 
Instead, the antibody may bind an innocuous region and exert its 
neutralizing effect by steric hinderance. Therefore a candidate protein 
mutated in this innocuous region might no longer bind the neutralizing 
antibody, but it would nonetheless be tumor necrosis factor in terms of 
substantial homology and biological activity. 
It is important to observe that characteristics such as molecular weight, 
isoelectric point and the like for the native or wild type mature human 
tumor necrosis factor of FIG. 10 obtained from peripheral lymphocyte or 
established cell line cultures are descriptive only for the native species 
of tumor necrosis factor. Tumor necrosis factor as contemplated by the 
foregoing definition will include other species which will not exhibit all 
of the characteristics of native tumor necrosis factor. While tumor 
necrosis factor as defined herein includes native tumor necrosis factor, 
other related cytotoxic polypeptides will fall within the definition. For 
example, TNF derivatives like the insertion mutants, deletion mutants, or 
fusion proteins described above will bring tumor necrosis factor outside 
of the molecular weight established for native human tumor necrosis factor 
(fusion proteins with mature tumor necrosis factor or preTNF itself as 
well as insertion mutants will have a greater molecular weight than 
native, mature tumor necrosis factor, while deletion mutants of native, 
mature tumor necrosis factor will have a lower molecular weight). 
Similarly, tumor necrosis factor may be engineered in order to reduce or 
eliminate susceptibility to hydrolysis by trypsin or other proteases. 
Finally, post-translational processing of human preTNF in cell lines 
derived from nonprimate mammals may produce microheterogeneity in the 
amino terminal region, so that valine will no longer be the amino terminal 
amino acid. 
The amino acid sequence for human tumor necrosis factor deduced from its 
cDNA is described in FIG. 10. Mature or native tumor necrosis factor is 
represented by amino acid residues 1 to 157. A 76 residue signal sequence 
which is believed to be removed during normal processing of the translated 
transcript to produce the mature protein. Trypsin hydrolysis sites are 
denoted by arrows. 
Note that the language "capable" of cytotoxic activity or in vivo tumor 
necrosis means that the term tumor necrosis factor includes polypeptides 
which can be converted, as by enzymatic hydrolysis, from an inactive state 
analogous to a zymogen to a polypeptide fragment which exhibits the 
desired biological activity. Typically, inactive precursors will be fusion 
proteins in which mature tumor necrosis factor is linked by a peptide bond 
at its carboxyl terminus to a human protein or fragment thereof. The 
sequence at this peptide bond or nearby is selected so as to be 
susceptible to proteolytic hydrolysis to release tumor necrosis factor, 
either in vivo or, as part of a manufacturing protocol, in vitro. The 
tumor necrosis factor that is so generated then will exhibit the 
definitionally-required cytotoxic activity. 
While tumor necrosis factor ordinarily is meant to mean human tumor 
necrosis factor, tumor necrosis factor from sources such as murine, 
porcine, equine or bovine is included within the definition of tumor 
necrosis factor so long as it otherwise meets the standards described 
above for homologous regions and cytotoxic activity. TNF is not species 
specific, e.g., human TNF is active on mouse tumors. Therefore, TNF from 
one species can be used in therapy of another. 
Tumor necrosis factor also includes multimeric forms. Tumor necrosis factor 
spontaneously aggregates into multimers, usually dimers or higher 
multimers. Multimers are cytotoxic and accordingly are suitable for use in 
in vivo therapy. While it is desirable to express and recover tumor 
necrosis factor as a substantially homogeneous multimer or monomer, tumor 
necrosis factor may be used therapeutically as a mixture of different 
multimers. 
Derivatives of tumor necrosis factor are included within the scope of the 
term tumor necrosis factor. Derivatives include amino acid sequence 
mutants, glycosylation variants and covalent or aggregative conjugates 
with other chemical moieties. Covalent derivatives are prepared by linkage 
of functionalities to groups which are found in the TNF amino acid side 
chains or at the N- or C-termini, by means known in the art. These 
derivatives may, for example, include: aliphatic esters or amides of the 
carboxyl terminus or residues containing carboxyl side chains, e.g., 
asp10; 0-acyl derivatives of hydroxyl group-containing residues such as 
ser52, ser3, ser4 or ser5; N-acyl derivatives of the amino terminal amino 
acid or aminogroup containing residues, e.g. lysine or arginine; and 
derivatives of cys101 and cys69. The acyl group is selected from the group 
of alkylmoieties. (including C3 to C1O normal alkyl), thereby forming 
alkanoyl species, and carbocyclic or heterocyclic compounds, thereby 
forming aroyl species. The reactive groups preferably are difunctional 
compounds known per se for use in cross-linking proteins to insoluble 
matrices through reactive side groups. Preferred derivatization sites are 
at cysteine and histidine residues. 
Covalent or aggregative derivatives are useful as reagents in immunoassay 
or for affinity purification procedures. For example, tumor necrosis 
factor is insolubilized by covalent bonding to cyanogen bromide-activated 
Sepharose by me:hods known per se or adsorbed to polyolefin surfaces (with 
or without glutaraldehyde cross-linking) for use in the assay or 
purification of anti-TNF antibodies or cell surface receptors. Tumor 
necrosis factor also is labelled with a detectable group, e.g., 
radioiodinated by the chloramine T procedure, covalently bound to rare 
earth chelates or conjugated to another fluorescent moiety for use in 
diagnostic assays, especially for diagnosis of TNF levels in biological 
samples by competitive-type immunoassays. Such derivatives may fall 
outside of the TNF definition above because it is not necessary that they 
show cytotoxic activity, only cross-reactivity with anti-TNF. 
Mutant tumor necrosis factor derivatives include the predetermined, i.e. 
site specific, mutations of TNF or its fragments. The objective of 
mutagenesis is to construct DNA that encodes tumor necrosis factor as 
defined above, i.e., tumor necrosis factor which exhibits cytotoxic 
activity towards tumor cells in vitro or causes tumor necrosis in vivo, 
and which retains residual homology with the FIG. 10 sequence, but which 
also exhibits improved properties and activity. Mutant tumor necrosis 
factor is defined as a polypeptide otherwise falling within the homology 
definition for tumor necrosis factor set forth herein but which has an 
amino acid sequence different from that of tumor necrosis factor whether 
by way of deletion, substitution or insertion. For example, the lysine or 
arginine residues of tumor necrosis factor (arginine 2, 6, 82, 44 and 131 
and lysine 98, 90, and 65,) may be mutated to histidine or another amino 
acid residue which does not render the protein proteolytically labile. 
Similarly, cysteine 101 could be replaced by other residues and 
cross-linked chemically in order to confer oxidative stability. It is not 
necessary that mutants meet the activity requirements for tumor necrosis 
factor, for even biologically inactive mutants will be useful upon 
labelling or immobilization as reagents in immunoassays. However, in this 
case the mutants will retain at least one epitopic site which is 
cross-reactive with antibody to tumor necrosis factor. 
The regions of the tumor necrosis molecule within residues 35 to 66 and 110 
to 133 inclusive show substantial homology (50 percent) with lymphotoxin. 
The hydrophobic carboxy-termini (tumor necrosis factor residues 150 to 
157) of the two molecules also are significantly conserved. Since both 
proteins demonstrate cytotoxic activity or in vivo tumor necrosis these 
regions are believed to be important in the shared activity of lymphotoxin 
and tumor necrosis factor. As such, the residues in these regions are 
preferred for mutagenesis having the objective of directly affecting the 
activity of tumor necrosis factor on a given cell. The relatively 
unconserved region at tumor necrosis factor residues 67-109 may function 
to correctly position the two surrounding relatively homologous regions in 
a conformation essential for cytotoxic activity. Such positioning, which 
could be achieved by a Cys69-Cys101 disulfide bond in tumor necrosis 
factor, might be similarly accomplished by the corresponding region of 
lymphotoxin and could account for differences in specificity and activity 
between the two molecules. In this connection, since these residues are 
postulated to represent the active core of tumor necrosis factor they may 
be synthesized chemically or by deletion mutagenesis as truncated, 
short-length polypeptides having tumor necrosis factor activity. 
While the mutation site is predetermined, it is unnecessary that the 
mutation per se be predetermined. For example, in order to optimize the 
performance of the position 131 mutants random mutagenesis may be 
conducted at the codon for arginine 131 and the expressed tumor necrosis 
factor mutants screened for the optimal combination of cytotoxic activity 
and protease resistance. Techniques for making substitution mutations at 
predetermined sites in DNA having a known sequence is well known, for 
example M13 primer mutagenesis. 
Tumor necrosis factor mutagenesis is conducted by making amino acid 
insertions, usually on the order of about from 1 to 10 amino acid 
residues, or deletions of about from 1 to 30 residues. Substitutions, 
deletions, insertions or any subcombination may be combined to arrive at a 
final construct. Insertions include amino or carboxyl-terminal fusions, 
e.g. a hydrophobic extension added to the carboxyl terminus. Preferably, 
however, only substitution mutagenesis is conducted. Obviously, the 
mutations in the encoding DNA must not place the sequence out of reading 
frame and preferably will not create complementary regions that could 
produce secondary mRNA structure. 
Not all mutations in the DNA which encode the tumor necrosis factor will be 
expressed in the final secreted product. For example, a major class of DNA 
substitution mutations are those in which a different secretory leader or 
signal has been substituted for the native human secretory leader, either 
by deletions within the leader sequence or by substitutions, wherein most 
or all of the native leader is exchanged for a leader more likely to be 
recognized by the intended host. For example, in constructing a 
procaryotic expression vector the human secretory leader is deleted in 
favor of the bacterial alkaline phosphatase or heat stable enterotoxin II 
leaders, and for yeast the leader is substituted in favor of the yeast 
invertase, alpha factor or acid phosphatase leaders. However, the human 
secretory leader may be recognized by hosts other than human cell lines, 
most likely in cell culture of higher eukaryotic cells. When the secretory 
leader is "recognized" by the host, the fusion protein consisting of tumor 
necrosis factor and the leader ordinarily is cleaved at the leader-tumor 
necrosis factor peptide bond in the events that lead to secretion of the 
tumor necrosis factor. Thus, even though a mutant preTNF DNA is used to 
transform the host, and mutant preTNF is synthesized as intermediate, the 
resulting tumor necrosis factor ordinarily is native, mature tumor 
necrosis factor. 
Another major class of DNA mutants that are not expressed as tumor necrosis 
factor derivatives are nucleotide substitutions made to enhance 
expression, primarily to avoid amino terminal loops in the transcribed 
mRNA (see copending U.S. Ser. No. 303,687, incorporated by reference) or 
to provide codons that are more readily transcribed by the selected host, 
e.g. the well-known E. coli preference codons for E. coli expression. 
Substantially homogeneous tumor necrosis factor means tumor necrosis factor 
which is substantially free of other proteins native to the source from 
which the tumor necrosis factor was isolated. This means that homogeneous 
tumor necrosis factor is substantially free of blood plasma proteins such 
as albumin, fibrinogen, serine proteases, .alpha.-globulins, non-tumor 
necrosis factor cytotoxic polypeptides such as lymphotoxin or interferons, 
or other proteins of the cell or organism which serves as the synthetic 
origin of the tumor necrosis factor, including whole cells and particulate 
cell debris. However, homogeneous tumor necrosis factor may include such 
substances as the stabilizers and excipients described below, 
predetermined amounts of proteins from the cell or organism that serves as 
the synthetic origin, proteins from other than the tumor necrosis factor 
source cells or organisms, and synthetic polypeptides such as 
poly-L-lysine. Recombinant tumor necrosis factor which is expressed in an 
allogeneic, e.g. bacterial, host cell of course will be expressed 
completely free of gene source proteins. 
Tumor necrosis factor is preferably synthesized in cultures of recombinant 
organisms. Neither peripheral blood lymphocytes (PBLs) nor cell lines are 
desirable. It is difficult in practice to obtain PBLs of one class which 
are free of contamination by cells of other classes, e.g. to obtain 
macrophages free of B or T cells. Such contamination will render the 
separation procedure applied to the products of such cells more difficult 
because of other potential cytotoxic factors and proteins released by the 
contaminant cells. Furthermore, tumor necrosis factor obtained from 
nonrecombinant culture is expensive and will consist solely of native 
tumor necrosis factor, such cultures thereby lacking in the flexibility of 
recombinant culture to improve upon the characteristics of tumor necrosis 
factor. 
DNA which encodes tumor necrosis factor is obtained by chemical synthesis, 
by screening reverse transcripts of mRNA from PBL or cell line cultures, 
or by screening genomic libraries from any cell. Suitable cell line 
cultures comprise monocytic cell lines such as promyelocytic cell lines 
designated "HL-60" in the art (one of which is available from the ATCC as 
CCL 240) or the histiocytic lymphoma cell line U 937 (ATCC CRL 1593). 
These and other cell lines are induced to express and secrete tumor 
necrosis factor by exposure of the cells to chemical or physical agents 
known in the art, generally tumorigenic or mitogenic agents. Tumor 
necrosis factor can be induced effectively in certain monocytic cell lines 
only with PMA; otherwise conventional agents such lipopolysaccharide, 
staphylococcal enterotoxin B, or thymosin .alpha.-1 were not as effective 
as PMA at inducing tumor necrosis factor in these cell lines. Since a 
variable amount of screening is required to locate a cell line expressing 
tumor necrosis factor (and therefore containing the desired mRNA) it may 
be more efficient to simply synthesize the gene. Synthesis is advantageous 
because unique restriction sites can be introduced (thereby facilitating 
the use of the gene in vectors containing restriction sites otherwise 
present in the native sequence) and steps can be taken to enhance 
translational efficiency, as discussed below. 
This DNA is covalently labelled with a detectable substance such as a 
fluorescent group, a radioactive atom or a chemiluminescent group by 
methods known per se. It is then used in conventional hybridization 
assays. Such assays are employed in identifying TNF vectors and 
transformants as described in the Examples infra, or for in vitro 
diagnosis such as detection of TNF mRNA in blood cells. 
The mRNA for TNF, surprisingly, is relatively rare even in induced HL-60 
cells, perhaps due to instability in the messenger resulting from unknown 
causes. Furthermore, the time course of appearance of TNF mRNA after cell 
induction is important. TNF mRNA appears in the cells for only a brief 
period at about 4 hours post-induction. In this respect its appearance is 
distinct from that of lymphotoxin, which appears at about 12 hours 
post-induction. This makes the cDNA easy to overlook were one not apprised 
as to what to look for. However, once its presence is appreciated and 
completely complementary DNA made available, as is enabled by the 
disclosures herein, it is routine to screen cDNA libraries of induced 
HL-60 or PBLs for tumor necrosis factor cDNA using probes having sequences 
of such DNA. The two HL-60 phage libraries screened in the Examples 
contain a relatively consistent number of positive plaques, so it is clear 
that routine hybridization assays will identify phage containing the 
desired cDNA. 
Tumor necrosis factor-synthesizing cells of an HL-60 cell line initially 
are cultured in conventional fashion until reaching a density of about 
8-12.times.10.sup.5 cells/ml. The cells are removed from the culture, 
washed, transferred to serum-free medium and grown in medium containing 
PMA. The culture then is continued until the desired concentration of 
tumor necrosis factor has accumulated in the culture medium, ordinarily 
about 400 tumor necrosis factor units/ml. Thereafter the culture 
supernatant is preferably clarified by centrifugation or other means of 
separating cell debris from the soluble components. Centrifugation should 
be carried out at low speed so as to move only suspended particles. The 
supernatant is then purified as described infra. 
Alternatively, and preferably, tumor necrosis factor is synthesized in host 
cells transformed with vectors containing DNA encoding tumor necrosis 
factor. A vector is a replicable DNA construct. Vectors are used herein 
either to amplify DNA encoding tumor necrosis factor and/or to express DNA 
which encodes tumor necrosis factor. An expression vector is a replicable 
DNA construct in which a DNA sequence encoding tumor necrosis factor is 
operably linked to suitable control sequences capable of effecting the 
expression of tumor necrosis factor in a suitable host. Such control 
sequences include a transcriptional promoter, an optional operator 
sequence to control transcription, a sequence encoding suitable mRNA 
ribosomal binding sites, and sequences which control termination of 
transcription and translation. 
Vectors comprise plasmids, viruses (including phage), and integratable DNA 
fragments (i.e., integratable into the host genome by recombination). Once 
it has transformed a suitable host, the vector replicates and functions 
independently of the host genome, or may, in some instances, integrate 
into the genome itself. In the present specification, "vector" is generic 
to "plasmid", but plasmids are the most commonly used form of vector at 
present. However, all other forms of vectors which serve an equivalent 
function and which are, or become, known in the art are suitable for use 
herein. Suitable vectors will contain replicon and control sequences which 
are derived from species compatible with the intended expression host. 
Transformed host cells are cells which have been transformed or 
transfected with tumor necrosis factor vectors constructed using 
recombinant DNA techniques. Transformed host cells ordinarily express 
tumor necrosis factor. The expressed tumor necrosis factor will be 
deposited intracellularly or secreted into either the periplasmic space or 
the culture supernatant, depending upon the host cell selected. 
DNA regions are operably linked when they are functionally related to each 
other. For example, DNA for a presequence or secretory leader is operably 
linked to DNA for a polypeptide if it is expressed as a preprotein which 
participates in the secretion of the polypeptide; a promoter is operably 
linked to a coding sequence if it controls the transcription of the 
sequence; or a ribosome binding site is operably linked to a coding 
sequence if it is positioned so as to permit translation. Generally, 
operably linked means contiguous and, in the case of secretory leaders, 
contiguous and in reading phase. 
Suitable host cells are prokaryotes, yeast or higher eukaryotic cells. 
Prokaryotes include gram negative or gram positive organisms, for example 
E. coli or Bacilli. Higher eukaryotic cells include established cell lines 
of mammalian origin as described below. A preferred host cell is the phage 
resistant E. coli W3110 (ATCC 27,325) strain described in the Examples, 
although other prokaryotes such as E. coli B, E. coli X1776(ATCC 31,537), 
E. coli 294 (ATCC 31,446), pseudomonas species, or Serratia Marcesans are 
suitable. 
Prokaryotic host-vector systems are preferred for the expression of tumor 
necrosis factor. While the tumor necrosis factor molecule contains two 
cysteine residues, thereby implying a modest potential requirement for 
post-translational processing to form a potential disulfide bond, E. coli 
for example expresses biologically active tumor necrosis factor A plethora 
of suitable microbial vectors are available. Generally, a microbial vector 
will contain an origin of replication recognized by the intended host, a 
promoter which will function in the host and a phenotypic selection gene, 
for example a gene encoding proteins conferring antibiotic resistance or 
supplying an auxotrophic requirement. Similar constructs will be 
manufactured for other hosts. E. coli is typically transformed using 
pBR322, a plasmid derived from an E. coli species (Bolivar, et al., 1977, 
"Gene" 2: 95). pBR322 contains genes for ampicillin and tetracycline 
resistance and thus provides easy means for identifying transformed cells. 
Vectors must contain a promoter which is recognized by the host organism. 
This is generally a promoter homologous to the intended host. Promoters 
most commonly used in recombinant DNA construction include the 
.beta.-lactamase (penicillinase) and lactose promoter systems (Chang et 
al., 1978, "Nature", 275: 615; and Goeddel et al., 1979, "Nature" 281: 
544), a tryptophan (trp) promoter system (Goeddel et al., 1980, "Nucleic 
Acids Res." 8:4057 and EPO App. Publ. No. 36,776) and the tac promoter [H. 
De Boer et al., "Proc. Nat'l. Acad. Sci. U.S.A." 80: 21-25 (1983)]. While 
these are the most commonly used, other known microbial promoters are 
suitable. Details concerning their nucleotide sequences have been 
published, enabling a skilled worker operably to ligate them to DNA 
encoding tumor necrosis factor in plasmid vectors (Siebenlist et al., 
1980, "Cell" 20: 269) and the DNA encoding tumor necrosis factor. At the 
present time the preferred vector is a pBR322 derivative containing the E. 
coli alkaline phosphatase promoter with the trp Shine-Dalgarno sequence. 
The promoter and Shine-Dalgarno sequence are operably linked to the DNA 
encoding the TNF, i.e., they are positioned so as to promote transcription 
of TNF mRNA from the DNA. 
In addition to prokaryates, eukaryotic microbes such as yeast cultures are 
transformed with tumor necrosis factor-encoding vectors. Saccharomyces 
cerevisiae, or common baker's yeast is the most commonly used among lower 
eukaryotic host microorganisms, although a number of other strains are 
commonly available. Yeast vectors generally.. will contain an origin of 
replication from the 2 micron yeast plasmid or an autonomously replicating 
sequence (ARS), a promoter, TNF, sequences for polyadenylation and 
transcription termination and a selection gene. A suitable plasmid for 
tumor necrosis factor expression in yeast is YRp7, (Stinchcomb et al., 
1979, "Nature", 282: 39; Kingsman et al., 1979, "Gene", 7: 141; Tschemper 
et al., 1980, "Gene", 10: 157). This plasmid already contains the trp1 
gene which provides a selection marker for a mutant strain of yeast 
lacking the ability to grow in tryptophan, for example ATCC No. 44076 or 
PEP4-1(Jones, 1977, "Genetics", 85: 12). The presence of the trp1 lesion 
in the yeast host cell genome then provides an effective environment for 
detecting transformation by growth in the absence of tryptophan. 
Suitable promoting sequences in yeast vectors include the promoters for 
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., 1980, "J. 
Biol. Chem.", 255: 2073) or other glycolytic enzymes (Hess et al., 1968, 
"J. Adv. Enzyme Reg.", 7: 149; and Holland et al., 1978, "Biochemistry", 
17: 4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, 
hexokinase, pyruvate decarboxylase, phosphofructokinase, 
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, 
triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. 
Suitable vectors and promoters for use in yeast expression are further 
described in R. Hitzeman et al., EPO Publn. No. 73,657. 
Other promoters, which have the additional advantage of transcription 
controlled by growth conditions, are the promoter regions for alcohol 
dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes 
associated with nitrogen metabolism, and the aforementioned 
metallothionein and gyceraldehyde-3- phosphate dehydrogenase, as well as 
enzymes responsible for maltose and galactose utilization. In constructing 
suitable expression plasmids, the termination sequences associated with 
these genes are also ligated into the expression vector 3' of the tumor 
necrosis factor coding sequences to provide polyadenylation of the mRNA 
and termination. 
In addition to microorganisms, cultures of cells derived from multicellular 
organisms may also be used as hosts. This, however, is not preferred 
because of the excellent results obtained thus far with TNF expressing 
microbes. In principal, any higher eukaryotic cell culture is workable, 
whether from vertebrate or invertebrate culture. However, interest has 
been greatest in vertebrate cells, and propagation of vertebrate cells in 
culture (tissue culture) has become a routine procedure in recent years 
[Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)]. 
Examples of useful host cell lines are VERO and HeLa cells, Chinese 
hamster ovary (CHO) cell lines, and WI38, BHK, COS-7 and MDCK cell lines. 
Expression vectors for such cells ordinarily include (if necessary) an 
origin of replication, a promoter located upstream from the gene to be 
expressed, along with a ribosome binding site, RNA splice site (if 
intron-containing genomic DNA is used), a polyadenylation site, and a 
transcriptional termination sequence. 
The transcriptional and translational control sequences in expression 
vectors to be used in transforming vertebrate cells are often provided by 
viral sources. For example, commonly used promoters are derived from 
polyoma, Adenovirus 2, and most preferably Simian Virus 40 (SV40). The 
early and late promoters are particularly useful because both are obtained 
easily from the virus as a fragment which also contains the SV40 viral 
origin of replication (Fiers et al., 1978, "Nature", 273: 113). Smaller or 
larger SV40 fragments may also be used, provided the approximately 250 bp 
sequence extending from the Hind III site toward the Bg1 I site located in 
the viral origin of replication is included. Further, it is also possible, 
and often desirable, to utilize human genomic promoter, control and/or 
signal sequences normally associated with tumor necrosis factor, provided 
such control sequences are compatible with the host cell systems. 
An origin of replication may be provided either by construction of the 
vector to include an exogenous origin, such as may be derived from SV40 or 
other viral (e.g. Polyoma, Adenovirus, VSV, or BPV) source, or may be 
provided by the host cell chromosomal replication mechanism. If the vector 
is integrated into the host cell chromosome, the latter is often 
sufficient. 
In selecting a preferred host mammalian cell for transfection by vectors 
which comprise DNA sequences encoding both tumor necrosis factor and 
dihydrofolate reductase (DHFR), it is appropriate to select the host 
according to the type of DHFR protein employed. If wild type DHFR protein 
is employed, it is preferable to select a host cell which is deficient in 
DHFR thus permitting the use of the DHFR coding sequence as a marker for 
successful transfection in selective medium which lacks hypoxanthine, 
glycine, and thymidine. An appropriate host cell in this case is the 
Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared 
and propagated as described by Urlaub and Chasin, 1980, "Proc. Natl. Acad. 
Sci."(U.S.A.) 77: 4216. 
On the other hand, if DNA encoding DHFR protein with low binding affinity 
for methotrexate (MTX) is used as the controlling sequence, it is not 
necessary to use DHFR resistant cells. Because the mutant DHFR is 
resistant to MTX, MTX containing media can be used as a means of selection 
provided that the host cells are themselves MTX sensitive. Most eukaryotic 
cells which are capable of absorbing MTX appear to be methotrexate 
sensitive. One such useful cell line is a CHO line, CHO-K1 (ATCC No. CCL 
61). 
Tumor necrosis factor initially is recovered from cultures. Transformed 
nonsecreting cells are lysed by sonication or other acceptable method and 
debris separated by centrifugation, while the supernatants from secreting 
cells (such as induced cell lines) are simply separated from the cells by 
centrifugation. Then any one or more of the following steps may be used, 
or other methods entirely may be substituted. The following method was 
used to purify tumor necrosis factor to a degree sufficient for 
sequencing. This is not necessarily coextensive with the purification 
required for a therapeutic product. 
As an initial purification step, tumor necrosis factor is adsorbed onto a 
hydrophobic substance from the lysed culture or supernatant culture 
medium. The hydrophobic substance preferably is a nongelatinous 
hydrophobic surface such as a silicate or polyolefin, although alkyl 
Sepharose also is suitable. The prefered embodiment is controlled pore 
glass. A ratio of about 1 volume of controlled pore glass is mixed with 50 
volumes of supernatant and the adsorption allowed to proceed at about 
4.degree. C. without agitation over a period of about 30 minutes to 2 
hours, preferably about 1 hour, under slightly alkaline conditions. The 
adsorbent generally should thereafter be washed with a suitable buffer to 
remove entrapped contaminant proteins. 
The adsorbed tumor necrosis factor is eluted from the hydrophobic substance 
by altering the solvation properties of the surrounding medium. The 
elution can be accomplished by passing a solution buffered at 
approximately pH 7 to 8.5, preferably around 8, containing 1M salt and an 
effective amount of an aqueous solution of a water miscible organic 
polyol, such as, for example, ethylene glycol or glycerin, ordinarily 
ethylene glycol in the range of 10-30 percent v/v, preferably around 20 
percent v/v. Of course the optimum conditions will depend upon the polyol 
which is used. The tumor necrosis factor-containing elution fractions are 
detected by in vitro assay as described below or by other suitable assay. 
The purification and yield of this step from monocytic cell culture, as 
well as subsequent steps, are shown below in Table I. 
Further purification is obtained by adsorption of tumor necrosis factor 
onto a tertiary or quaternary amino anion exchange resin. The preferred 
resins for this purpose are hydrophilic matrix resins such as cross-linked 
polystyrene, dextran or cellulose substituted with alkyl tertiary or 
quaternary amino groups. Commercial products of this type are available as 
DEAE cellulose, QAE Sephadex or under the trade name Mono Q (in each 
instance where ethyl is the alkyl substituent in each of these products). 
Best results are achieved with the fast protein liquid chromatography 
system described by J. Richey, Oct. 1982, "American Laboratory" using the 
macroporous substantially uniform particles of Ugelstad et al., 1983, 
"Nature" 303: 95-96. This system has enabled the purification of tumor 
necrosis factor to a high level. 
Purification to substantial homogeneity is achieved only upon further 
separation on SDS PAG electrophoresis or C4-reverse phase high pressure 
liquid chromatography (HPLC) as described in the Examples below. This 
product, however, is not desirable for therapy because it has lost 
substantial activity upon exposure to SDA or HPLC organic solvent. Protein 
concentration was determined by the method of M. Bradford, 1976, "Anal. 
Biochem." 72:248-254. During the final stages of purification, the protein 
concentration was estimated by amino acid composition and also by amino 
acid sequence. 
Tumor necrosis factor is prepared for administration by mixing tumor 
necrosis factor having the desired degree of purity with physiologically 
acceptable carriers, i.e., carriers which are nontoxic to recipients at 
the dosages and concentrations employed. Ordinarily, this will entail 
combining the tumor necrosis factor with buffers, antioxidants such as 
ascorbic acid, low molecular weight (less than about 10 residues) 
polypeptides, proteins, amino acids, carbohydrates including glucose or 
dextrins, chelating agents such as EDTA, and other stabilizers and 
excipients. The carrier should be formulated to stabilize the tumor 
necrosis factor as a dimer and/or, preferably, a trimer. This is 
accomplished by avoiding salts or detergents in concentrations that 
dissociate tumor necrosis factor into monomers. Alternatively, conditions 
that aggregate tumor necrosis factor into higher multimers should be 
avoided. Generally a nonionic surfactant such as Tween 20 is employed to 
exhibit excessive aggregation during purification as well as 
lyophilization or aqueous storage. Tumor necrosis factor to be used for 
therapeutic administration must be sterile. This is readily accomplished 
by filtration through sterile filtration membranes. Tumor necrosis factor 
ordinarily will be stored in lyophilized form. 
TABLE I 
__________________________________________________________________________ 
Purification of Human Tumor Necrosis Factor from HL-60 Cell Culture 
Medium 
Relative 
Final 
Total 
Cytolytic 
Specific 
Purification 
Volumes 
Protein 
Activity 
Activity Recovery 
Step (ml) (mg) 
(units) 
(units/mg) 
Purification 
(Percent) 
__________________________________________________________________________ 
Starting 58,000 
1,964 
14.2 .times. 10.sup.6 
0.007 .times. 10.sup.6 
-- -- 
Material 
Controlled 
1,080 
88.9 
11.1 .times. 10.sup.6 
0.12 .times. 10.sup.6 
17 78.5 
Pore Glass 
Chromatography 
DEAE-Cellulose 
285 9.05 
8.9 .times. 10.sup.6 
0.98 .times. 10.sup.6 
140 62.7 
Chromatography 
Mono Q-Fast 
75 0.44 
6.9 .times. 10.sup.6 
15.68 .times. 10.sup.6 
2,240 48.6 
Protein Liquid 
Chromatography 
Preparative 
6 0.028 
2.71 .times. 10.sup.6 * 
96.79 .times. 10.sup.6 * 
13,387* 
19.1* 
SDS PAG 
Electrophoresis 
or 
C4-Reverse 
phase-HPLC 
__________________________________________________________________________ 
*Corrected for partial destruction of tumor necrosis factor activity 
caused by SDS, or by TFA and propanol. 
Tumor necrosis factor optionally is combined with other antineoplastic 
agents such as chemotherapeutic antibiotics (actinomycin-D, adriamycin, 
aclacinomycin A), or with agents to augment or stimulate the immune 
response, for example immunoglobulins such as gamma globulin, including 
immunoglobulins having affinity for the cell surface antigens of 
neoplasms. In addition, since interferons act synergistically with tumor 
necrosis factor in cell lysis assays, alpha, beta or gamma interferon is 
desirably combined with tumor necrosis factor compositions or tumor 
necrosis factor and lymphotoxin-containing compositions. A typical 
formulation comprises tumor necrosis factor and gamma interferon in a unit 
activity proportion of about from 0.1:1 to 200:1, ordinarily 10 to 1, and 
may contain lymphotoxin in place of a proportion of tumor necrosis factor. 
These proportions, of course, are subject to modification as required by 
therapeutic experience. 
Tumor necrosis factor compositions are administered to tumor-bearing 
animals. The route of administration is in accord with known methods, e.g. 
the intravenous, intraperitoneal, intramuscular, intralesional infusion or 
injection of sterile tumor necrosis factor solutions, or by timed release 
systems as noted below. Tumor necrosis factor is administered 
intralesionally, i.e., by direct injection into solid tumors. In the case 
of disseminated tumors such as leukemia, administration is preferably 
intravenous or into the lymphatic system. Tumors of the abdominal organs 
such as ovarian cancer are advantageously treated by intraperitoneal 
infusion using peritoneal dialysis hardware and peritoneal-compatible 
solutions. Ordinarily, however, tumor necrosis factor is administered 
continuously by infusion although bolus injection is acceptable. 
Tumor necrosis factor desirably is administered from an implantable 
timed-release article. Examples of suitable system for proteins having the 
molecular weight of tumor necrosis factor dimers or trimers include 
copolymers of L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et 
al., 1983, "Biopolymers" 22 (1): 547-556), poly 
(2-hydroxyethyl-methacrylate) (R. Langer et al., 1981, "J. Biomed. Mater. 
Res." 15: 167-277 and R. Langer, 1982, "Chem. Tech." 12: 98-105) or 
ethylene vinyl acetate (R Langer et al.,Id.). This article is implanted at 
surgical sites from which tumors have been excised. Alternatively, tumor 
necrosis factor is encapsulated in semipermeable microcapsules or 
liposomes for injection into the tumor. This mode of administration is 
particularly useful for surgically inexcisable tumors, e.g. brain tumors. 
The amount of tumor necrosis factor that is administered will depend, for 
example, upon the route of administration, the tumor in question and the 
condition of the patient. Intralesional injections will require less tumor 
necrosis factor on a body weight basis than will intravenous infusion, 
while some tumor types, e.g., solid tumors appear to be more resistant to 
tumor necrosis factor than others, e.g. leukemic. Accordingly, it will be 
necessary for the therapist to titer the dosage and modify the route of 
administration as required to obtain optimal cytotoxic activity towards 
the target tumor, as can be determined for example by biopsy of the tumor 
or diagnostic assays for putative cancer markers such as carcinoembryonic 
antigen, in view of any recombinant toxicity encountered at elevated 
dosage. Ordinarily, tumor necrosis factor dosages in mice up to about 120 
micrograms/kg body weight/day by intravenous administration have been 
found to be substantially nontoxic and efficacious in vivo. 
Tumor necrosis factor is not believed to be species specific in its 
cytotoxic activity, so that tumor necrosis factor other than human tumor 
necrosis factor, e.g. from bovine or porcine sources, might be employed in 
the therapy of human tumors. However, it is desired to use tumor necrosis 
factor from the species being treated in order to avoid the potential 
generation of autoantibodies. 
In order to simplify the Examples certain frequently occurring methods will 
be referenced by shorthand phrases. 
Plasmids are designated by a low case p preceded and/or followed by capital 
letters and/or numbers. The starting plasmids herein are commercially 
available, are publically available on an unrestricted basis, or can be 
constructed from such available plasmids in accord with published 
procedures. In addition, other equivalent plasmids are known in the art 
and will be apparent to the ordinary artisan. 
"Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme 
that acts only at certain locations in the DNA. Such enzymes are called 
restriction enzymes, and the sites for which each is specific is called a 
restriction site. "Partial" digestion refers to incomplete digestion by a 
restriction enzyme, i.e., conditions are chosen that result in cleavage of 
some but not all of the sites for a given restriction endonuclease in a 
DNA substrate. The various restriction enzymes used herein are 
commercially available and their reaction conditions, cofactors and other 
requirements as established by the enzyme suppliers were used. Restriction 
enzymes commonly are designated by abbreviations composed of a capital 
letter followed by other letters and then, generally, a number 
representing the microorganism from which each restriction enzyme 
originally was obtained. In general, about 1 .mu.g of plasmid or DNA 
fragment is used with about 1 unit of enzyme in about 20 .mu.l of buffer 
solution. Appropriate buffers and substrate amounts for particular 
restriction enzymes are specified by the manufacturer. Incubation times of 
about 1 hour at 37.degree. C. are ordinarily used, but may vary in 
accordance with the supplier's instructions. After incubation, protein is 
removed by extraction with phenol and chloroform, and the digested nucleic 
acid is recovered from the aqueous fraction by precipitation with ethanol. 
Digestion with a restriction enzyme infrequently is followed with 
bacterial alkaline phosphatase hydrolysis of the terminal 5' phosphates to 
prevent the two restriction cleaved ends of a DNA fragment from 
"circularizing" or forming a closed loop that would impede insertion of 
another DNA fragment at the restriction site. Unless otherwise stated, 
digestion of plasmids is not followed by 5' terminal dephosphorylation. 
Procedures and reagents for dephosphorylation are conventional (T. 
Maniatis et al., 1982, Molecular Cloning pp. 133-134). 
"Recovery" or "isolation" of a given fragment of DNA from a restriction 
digest means separation of the digest on polyacrylamide gel 
electrophoresis, identification of the fragment of interest by comparison 
of its mobility versus that of marker DNA fragments of known molecular 
weight, removal of the gel section containing the desired fragment, and 
separation of the gel from DNA. This procedure is known generally. For 
example, see R. Lawn et al., 1981, "Nucleic Acids Res." 9: 6103-6114, and 
D. Goeddel et al., 1980, "Nucleic Acids Res." 8: 4057. 
"Southern Analysis" is a method by which the presence of DNA sequences in a 
digest or DNA-containing composition is confirmed by hybridization to a 
known, labelled oligonucleotide or DNA fragment. For the purposes herein, 
unless otherwise provided, Southern analysis shall mean separation of 
digests on 1 percent agarose, denaturation and transfer to nitrocellulose 
by the method of E. Southern, 1975, "J. Mol. Biol." 98: 503-517, and 
hybridization as described by T. Maniatis et al., 1978, "Cell" 15: 
687-701. 
"Transformation" means introducing DNA into an organism so that the DNA is 
replicable, either as an extrachromosomal element or chromosomal 
integrant. Unless otherwise provided, the method used herein for 
transformation of E. coli is the CaCl.sub.2 method of Mandel et al., 1970, 
"J. Mol. Biol." 53: 154. 
"Ligation" refers to the process of forming phosphodiester bonds between 
two double stranded nucleic acid fragments (T. Maniatis et al., Id., p. 
146). Unless otherwise provided, ligation may be accomplished using known 
buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 
.mu.g of approximately equimolar amounts of the DNA fragments to be 
ligated. 
"Preparation" of DNA from transformants means isolating plasmid DNA from 
microbial culture. Unless otherwise provided, the alkaline/SDS method of 
Maniatis et al., Id. p. 90., may be used. 
"Oligonucleotides" are short length single or double stranded 
polydeoxynucleotides which are chemically synthesized by known methods and 
then purified on polyacrylamide gels. 
All literature citations are expressly incorporated by reference. 
EXAMPLE 1 
Assays 
The specific activity of tumor necrosis factor was determined by a 
previously described modified cell lytic assay (B. Spofford, 1974, "J. 
Immun." 112: 2111). Mouse L-929 fibroblast cells (ATCC CCL-929) were grown 
in 96 well flat bottomed trays (3040; Falcon Plastics, Oxnard, CA) at 
30,000 cells (vol 0.1 ml) per well in the presence of 1 .mu.g/ml of 
actinomycin D and a serially diluted test sample (0.125 ml). The cells 
were incubated in a humidified atmosphere at 37.degree. C. with 5 percent 
CO.sub.2. The test sample was removed after 18 h, the plates were washed 
and cell lysis was detected by staining the plates with 0.5 percent 
solution of crystal violet in methanol:water (1:4)(v/v). The endpoint on 
the microtiter plates was determined by a Microelisa autoreader (Dynatech) 
set for absorption at 450 nm and transmission at 570 nm. The cells exposed 
to culture medium alone were set at 0 percent lysis and those exposed to 
3M guanidine hydrochloride solution provided an end point for 100 percent 
lysis. One unit of tumor necrosis factor is defined as the amount of tumor 
necrosis factor (when assayed in the 0.125 ml volume) which is required 
for 50 percent cell lysis. 
Tumor necrosis factor also was tested in an in vivo tumor necrosis assay. 
Briefly, this assay was carried out by growing Meth A Sarcoma cells 
(5.times.10.sup.5 cells) in CB6F.sub.1 female mice 
(BALB/c.times.C57BL/6)F.sub.1 for 7-10 days and then injecting 
intratumorly with a sample of tumor necrosis factor. After 24 hrs, mice 
were sacrificed by cervical dislocation, tumors were removed and the 
necrosis was scored histologically as previously described in E. Carswell 
et al., 1975, "Proc. Nat. Acad. Sci." 72: 3666-3670. 
EXAMPLE 2 
Use of PBLs or a Monocytic Cell Line to Synthesize Tumor Necrosis Factor 
An HL-60 human promyelocytic cell line seed culture having a cell density 
of 1.times.10.sup.5 cells/ml was grown in 2-liter roller bottles (890 
cm.sup.2) using 500 ml of RPMI 1640 medium (Irvine Scientific, Santa Ana, 
CA) containing 10 mM HEPES, 0.05 mM .beta.-mercaptoethanol, 100 units/ml 
penicillin, 100 .mu.g/ml streptomycin and 10 percent fetal calf serum. 
After three days at 37.degree. C., when the culture reached a cell density 
of 8-12.times.10.sup.5 cells/ml, cells were harvested by centrifugation at 
1000 g for 10 min., washed twice with serum free RPMI 1640 medium and 
transferred into the same medium as described above without serum at a 
cell density of 15-20.times.10.sup.5 cells/ml. Cells were grown in 2-liter 
roller bottles in the presence of 10 ng/ml PMA. After 16-24 h, the cells 
were removed by filtration through a 3 .mu.m Sealkleen filter (Pall 
Trinity Micro Corp. Cortland, NY). The clear filtrate was assayed for 
tumor necrosis factor activity and used for subsequent purification and 
characterization. This procedure produced about 400 units of tumor 
necrosis factor units/ml of supernatant culture medium. 
Human peripheral blood monocytes were also used for the production of tumor 
necrosis factor. Plateletpheresis residues were obtained from American Red 
Cross, Boston, MA, and used within 24 h of collection. The initial 
separation of monocytes from erythrocytes was achieved by centrifugation 
on Ficoll-Hypaque gradients at 1000 g for 30 minutes. Cells collected at 
the interface were washed three times with phosphate buffered saline. 
Monocytes derived from each donor were grown separately in 2-liter roller 
bottles in serum free RPMI 1640 medium at a cell density of 
2.5.times.10.sup.6 cells/ml. 1 .mu.g/ml each of Staphlococcal enterotoxin 
B (SEB) and recombinant thymosin .alpha.-1 were added to the culture and 
the cells incubated in a humidified atmosphere at 37.degree. C. with 10 
percent CO.sub.2. After 24-72 h, depending upon the donor, the cell 
supernatants were harvested and processed in a similar manner to those 
derived from the HL-60 cell line. Tumor necrosis factor yields from PBL 
cultures varied widely, depending upon the inducing agents employed. 
Adding PMA to the induction system described above increased the cytolytic 
activity of cell supernatants. However, the cell supernatants contained 
both tumor necrosis factor and lymphotoxin (determination of lymphotoxin 
or tumor necrosis factor in mixtures of tumor necrosis factor and 
lymphotoxin was made by conducting the cell lytic assay with test samples 
preincubated with rabbit neutralizing antibody to tumor necrosis factor or 
lymphotoxin and determining residual activity in the L-929 cell lytic 
assay). 
EXAMPLE 3 
Controlled Pore Glass Bead Chromatography 
Tumor necrosis factor activity from cell culture was batch absorbed to 
controlled pore glass beads (Catalogue No. CPG 00350, Electro-Nucleonics, 
Fairfield, NJ) equilibrated with 10 mM sodium phosphate buffer, pH 8.0, by 
constant stirring at 4.degree. C. One hundred ml of glass beads were used 
per 5 liters of medium. After stirring for one hour, beads were allowed to 
settle and the supernatant was decanted off. The beads were then poured 
into a 5.times.50 cm column at room temperature and washed with 10 mM 
sodium phosphate buffer, pH 8.0, containing 1M NaCl. The tumor necrosis 
factor activity was eluted from glass beads with 20 percent ethylene 
glycol in 10 mM sodium phosphate buffer, pH 8.0, containing 1M NaCl. The 
elution profile of the HL-60 supernatant from the column is shown in FIG. 
1. 
EXAMPLE 4 
DEAE Cellulose Chromatography 
The eluate from Example 3 was directly applied to a DEAE cellulose 53 
(Whatman) column (2.5.times.20 cm) equilibrated with 10 mM sodium 
phosphate buffer at pH 8.0 and 0.01 percent Tween 20, at a flow rate of 
approximately 500 ml/h. After the flow rate of the column was adjusted to 
100 ml/h, 4.2.times.10.sup.6 units of tumor necrosis factor in 1,080 ml of 
sample was loaded at 4.degree. C., the column was washed with 
equilibration buffer and eluted with step-up gradients of 75 mM 150 mM and 
500 mM sodium chloride in 10 mM phosphate buffer (pH 8.0). The eluate was 
monitored for absorbance at 280 nm and tumor necrosis factor activity as a 
function of elution fractions. The results are shown in FIG. 2. 
EXAMPLE 5 
Fast Protein Liquid Chromatography 
The tumor necrosis factor active fraction from Example 4 was concentrated 
and dialyzed against 20 mM Tris HCl, pH 8.0, containing 0.01 percent Tween 
20 and 1 mM sodium azide (buffer A) in an Amicon stir cell using a YM-10 
membrane or other dialysis membrane with a molecular weight cut-off below 
that of TNF. The membrane was washed twice with buffer A. A quaternary 
ammonium-group substituted Sepharose bead column (9.8 .mu.M beads in a 
5.times.0.5 cm column; sold as Mono Q resin, Pharmacia) in a fast protein 
liquid chromatography (FPLC) unit (Pharmacia) equipped with a gradient 
programmer (GP-250) and two pumps (P-500), was preequilibrated with 
dialysis buffers via a superloop at a flow rate of 1 ml/min as further 
described in J. Richey, "American Laboratory" October 1982, page 1. The 
pooled washes and dialysis concentrate were loaded on the column, the 
column was washed with buffer A and then eluted with a linear gradient of 
40-75 mM sodium chloride in buffer A. Linear gradients were programmed as 
follows: 0-5 min equilibration buffer; 5.1-15 min, 25 mM NaCl; 15.1-25 
min, 40 mM NaCl; 25-60 min, 40-75 mM NaCl linear gradient; 60-65 min, 75 
mM NaCl; 65.1-70 min, 100 mM NaCl; 70-80 min, 100-1000 mM NaCl linear 
gradients; 80-90 min, 100 mM NaCl; 90.1-110 min, equilibration buffer. The 
effluent was taken as 2 ml per fractions and monitored for absorbance at 
280 nm, conductivity, and for tumor necrosis factor activity. The results 
are shown in FIG. 3. 
EXAMPLE 6 
Chromatofocusing 
Chromatofocusing was performed using a Pharmacia Mono P column 
(20.times.0.5 cm) in an FPLC system as in Example 5. The biologically 
active (tumor necrosis factor) fraction eluted in fraction numbers 37 to 
45 from Example 5 was concentrated and dialyzed on an Amicon stir cell 
with a YM-10 membrane against the column equilibration buffer, i.e., 0.025 
M bis-Tris HCl, pH 6.7. The sample was loaded onto the Mono P column at 
room temperature via a superloop at a flow rate of 1 ml/min. The column 
was washed with equilibration buffer until the absorbance at 280 nm 
returned to baseline and then eluted with a linear pH gradient established 
by washing the column with 7.5 percent polybuffer 74 at pH 4.7 
(Pharmacia). One ml fractions were collected and the absorbance at 280 nm 
and pH of the effluent recorded. The results are shown in FIG. 4. As can 
be seen from FIG. 4, the isoelectric point of tumor necrosis factor was 
about 5.3. 
EXAMPLE 7 
Preparative SDS-Polyacrylamide Gel Electrophoresis 
Fifteen percent polyacrylamide gels (11.times.16 cm) with a thickness of 
1.5-3.0 mm were prepared according to a modification of the procedure of 
U. Laemmli, 1970, "Nature" 227: 680-685. Both resolving and stacking gels 
contained 0.1 percent SDS and 0.05 percent Tween 20. Other buffers and 
concentration of cross linking reagent were the same as for analytical 
SDS-PAGE gels. Tumor necrosis factor active fractions from Examples 5 or 6 
step were pooled, concentrated and dialyzed against 6.25 mM Tris HCl, pH 
7.0, containing 0.005 percent SDS on an Amicon stir cell using a YM-10 
membrane. After removal of the dialyzed concentrate, the membrane was 
washed three times with a small volume of sample buffer (0.2 percent SDS, 
0.02 percent Tween 20, 30 percent glycerol, 0.03M Tris HCl, pH 6.8, 0.005 
percent tracking dye). The dialyzed concentrate and washes were pooled 
(total volume 1-4 ml), mercaptoethanol optionally added to establish SDS 
PAGE reducing conditions, and the sample loaded into a large well cast in 
the stacking gel. Small wells adjacent to the sample well were used for 
prestained molecular weight markers phosphorylase-a (94K), bovine serum 
albumin (67K), ovalbumin (43K), carbonic anhydrase (30K), soybean trypsin 
inhibitor (20K) and Lysozyme (14.4K). The gels were run in a Biorad 
vertical electrophoresis system cooled to 12.degree. C., at a constant 
current of 20 mA per mm of gel thickness, until the tracking dye reached 
the bottom of the gel. 
Following electrophoresis, one of the glass plates was removed from the 
gel, and the positions of the molecular weight markers were noted. The 
lane containing the applied tumor necrosis factor sample was then cut into 
0.25 cm sections in accord with the molecular weights of the marker 
proteins. These gel slices were then placed in polypropylene tubes 
containing 1-2 ml of 10 mM ammonium bicarbonate and 0.01 percent Tween 20, 
pH 8, and allowed to elute for 16 h at 4.degree. C. The eluates were then 
assayed for tumor necrosis factor activity and the results shown in FIG. 
5. The tumor necrosis factor molecular weight on SDS gel was about 17,000, 
whether under reducing or nonreducing conditions, thus indicating a single 
chain molecule. 
The protein was recovered from the eluate of gel slices free of salts and 
low molecular weight substances by the following treatment: Small columns 
were prepared containing 0.2 ml Sep-pak C18 resin, which had been 
pre-washed with acetonitrile, 1-propanol, 1 percent trifluoroacetic acid 
(TFA), and distilled water and then equilibrated with 10 mM ammonium 
bicarbonate containing 0.01 percent Tween-20, pH 8.0. The gel eluate was 
loaded onto the column and the effluent collected. The resin was then 
washed with approximately 5 ml each of distilled water and 0.1 percent 
TFA, to remove free amiono acids and buffer salts. Tumor necrosis factor 
was eluted from the resin with 1 ml of 50 percent 1-propanol in 0.1 
percent TFA. Further elutions with 1 ml each of 50 percent 1-propanol in 1 
percent TFA, and 99 percent 1-propanol in 1 percent TFA were also 
performed, but the protein was usually eluted with the first buffer. 
Approximately 80 percent of the tumor necrosis factor bioactivity was 
inactivated in this step. While the tumor necrosis factor so obtained can 
be employed for sequence analysis it is preferred that the HPLC effluent 
described below in Example 8 be used for this purpose. 
EXAMPLE 8 
High Pressure Liquid Chromatography 
The molecular weight of native intact tumor necrosis factor was determined 
by high pressure gel permeation chromatography. The latter was carried out 
at room temperature using a TSK G2000 SW gel HPLC column (Alltech 
Associates, Deerfield, IL)(7.5.times.60 mm). A one ml sample of purified 
tumor necrosis factor from Example 5 containing approximately 1 .mu.g of 
protein and 15,600 units of activity was isocratically eluted from the gel 
column at a flow rate of 0.5 ml/min, with 0.2M sodium phosphate buffer, pH 
7.0. The column was calibrated with bovine serum albumin (MW 66,000), 
ovalbumin (MW 45,000), bovine carbonic anhydrase B (MW 29,000), and 
lysozyme (MW 14,300). One ml fractions were obtained and assayed for tumor 
necrosis factor activity. The fractions showing tumor necrosis factor 
activity eluted consistent with a molecular weight of 45,000.+-.6,000 
(FIG. 6). 
EXAMPLE 9 
Reverse-phase HPLC 
Tumor necrosis factor also was purified by reverse-phase HPLC using C4 
Synchropak columns on Water's Associates, Inc. chromatograph system as 
described previously (W. Kohr et al, 1982, Anal. Biochem, 122: 348-359). 
Protein peaks were detected at 210 nm and at 280 nm after elution with a 
linear gradient of 1 to 23 percent v/v 1-propanol in 0.1 percent aqueous 
TFA in the first 15 minutes and 23-30 percent v/v 1-propanol in 0.1 
percent TFA for the next 15 minutes at a flow rate of one ml per minute. 
The peaks were assayed for cytolytic activity. The organic solvents 
employed in eluting tumor necrosis factor from the C4 column reduced tumor 
necrosis factor activity about 80 percent. Tumor necrosis factor purified 
by this method was dried under vacuum, and then processed for amino acid 
analysis and sequencing. The results are recorded in FIG. 7. FIG. 7 shows 
that the tumor necrosis factor obtained in the effluent from Example 5 
contained biologically inactive protein contaminants eluting at about 16 
and 19 minutes retention time. The bioactive effluent from C4-RP-HPLC was 
substantially homogeneous, by the criteria of amino terminal sequence. 
EXAMPLE 10 
Determination of Partial Amino Acid Sequence for Tumor Necrosis Factor 
Tumor necrosis factor was trypsin digested as follows: Homogeneous tumor 
necrosis factor from Example 9 was dissolved, dried down and redisolved in 
100 mM of ammonium bicarbonate buffer pH 8.0 containing 5 percent w/w TPCK 
trypsin (Worthington Biochemicals), 1 mM CaCl.sub.2 and 0.01 percent 
Tween-20 at an enzyme to substrate ratio of 1:20, incubated for 6 hours at 
37.degree. C., an additional 5 percent by w/w of trypsin added and the 
hydrolysis mixture further incubated for 12 hours at 37.degree. C. The 
reaction mixture was applied onto C4 HPLC as described above in order to 
separate the peptide fragments. The results are shown in FIG. 8. A total 
of 9 fragments were observed (fragments 2 and 2' eluted together at the 
peak designated T2 in FIG. 8). An additional tenth fragment is believed to 
not be retained by the column. Amino acid sequences for intact tumor 
necrosis factor from Examples 8 and 9 and the trypsin hydrolysis fragments 
obtained in this Example were determined by automated sequential Edman 
degradation using a modified Beckman sequencer model 890B equipped with 
cold traps. Polybrene (1.25 mg) was used as a carrier in the cup. Based on 
the amino acid composition of the intact molecule, the molecular weight of 
intact tumor necrosis factor is 17,100. This figure was consistent with 
the SDS-PAGE data and constitutes confirmation of the absence of 
glycosylation. 
EXAMPLE 11 
Synergistic Action of Tumor Necrosis Factor and Gamma Interferon 
Cells of the murine melanoma B16, (Mason Research, Worcester, MA.) a cell 
line of C57B1/6 origin, were seeded in a microtiter plate at 5,000 
cells/well and incubated for 4 hours at 37.degree. C. in a 5 percent 
CO.sub.2 -humidified incubator before the addition of the lymphokines. 
Tumor necrosis factor obtained from Example 1 was purified to substantial 
homogeneity by HPLC and quantitated by its activity in the above-described 
bioassay for cytolysis of L929 cells. Similarly, purified recombinant 
murine gamma interferon (P. Gray et al., 1983, "Proc. Natl. Acad. Sci. 
U.S.A." 80: 5842-5846) was assayed by its antiviral activity against 
EMC-infected L cells (D. Goeddel et al., 1980, "Nature" (London) 287: 
411-416). Murine gamma interferon and human tumor necrosis factor were 
separately diluted to the dilutions shown in FIG. 9. Gamma interferon was 
added first to the designated wells, and diluted tumor necrosis factor was 
added immediately thereafter, to a final volume of 0.2 ml/well. At the end 
of 72 hours of incubation, the cells were stained with 0.5 percent crystal 
violet in 20 percent methanol. The results are shown in FIG. 9. B16 is 
relatively resistant to either tumor necrosis factor or IFN-.gamma. alone; 
at 1,000 units/ml of tumor necrosis factor no visually detectable 
cytolysis was observed. However, addition of very small amounts of gamma 
interferon (as little as 5 units/ml) resulted in cytolysis. 
EXAMPLE 12 
Messenger RNA Isolation 
Total RNA from HL-60 cell cultures (4 hours after PMA induction) or 
peripheral blood monocytes cultured as described in Example 2 was 
extracted essentially as reported by Ward et al., 1972, "J. Virol." 9: 61. 
Cells were pelleted by centrifugation and then resuspended in 10 mM NaCl, 
10 mM Tris-HCl pH 7.5, 1.5 mM MgCl.sub.2. Cells were lysed by the addition 
of NP-40 (1 percent final concentration), and nuclei were pelleted by 
centrifugation. The supernatant contained the total RNA which was further 
purified by multiple phenol and chloroform extractions. The aqueous phase 
was made 0.2 M in NaCl and then total RNA was precipitated by the addition 
of two volumes of ethanol. A typical yield from 1 gram of cultured cells 
was about 6 milligrams of total RNA. Polyadenylated mRNA (about 100 .mu.g) 
was obtained on oligo (dT) cellulose by the method of H. Aviv et al., 
1972, "Proc. Natl. Acad. Sci. U.S.A." 69: 1408-1412. 
EXAMPLE 13 
cDNA Library 
7.5 .mu.g of poly(A).sup.+ mRNA from Example 12 was converted to double 
stranded cDNA by the successive action of reverse transcriptase, DNA 
polymerase Klenow fragment, and S1 nuclease (P. Gray et al., 1982, 
"Nature" 295: 503-508; M. Wickers et al., 1978, "J. Biol. Chem." 253: 
2483-2495). About 80 ng of cDNA having greater than 600 bp in length were 
isolated from a polyacrylamide gel. 
The synthetic DNA adaptor sequence 
##STR1## 
was ligated to the cDNA to create EcoRI cohesive termini. As is 
conventional in the art, the adaptor was synthesized chemically as two 
separate strands, the 5' end of one of the strands was phosphorylated with 
polynucleotide kinase and the two strands annealed. The cDNA (20 ng) was 
then re-isolated from a polyacrylamide gel, inserted by ligation into 
EcoRI-digested .lambda.gt-10, packaged into phage particles and propagated 
in E. coli strain C600 hfl (Huynh et al., 1984, Practical Approaches in 
Biochemistry, IRL Press Ltd., Oxford England) or other known strain 
suitable for lamda phage propagation. A cDNA library of about 200,000 
independent clones was obtained. 
EXAMPLE 14 
Preparation of a Deoxyoliqonucleotide Probe for Tumor Necrosis Factor cDNA 
A 42 nucleotide DNA hybridization probe, based upon the preliminary amino 
acid sequence of tumor necrosis factor tryptic peptide TD-6 
(E-T-P-E-G-A-E-A-K-P-W-Y-E-K-) was designed on the basis of published 
codon usage frequencies (R. Grantham et al., 1981 "Nucleic Acids Res." 9: 
43-74), and the codon bias of human IFN-.gamma. (P. Gray et al., 1982, 
"Nature" 295: 503-508), and human lymphotoxin The preliminary sequence was 
in error (the final K should have been P). Nonetheless, this sequence led 
to a successful probe. The synthetic probe had the sequence 5' 
dGAAACCCCTGAAGGGGCTGAAGCCAAGCCCTGGTATGAAAAG 3' and was synthesized by the 
method of R. Crea et al., 1980, "Nucleic Acids Res." 8: 2331-2348. The 
probe was phosphorylated with (.gamma.-.sup.32 P) ATP and T4 
polynucleotide kinase as described previously (Goeddel et al., 1979, 
"Nature" 281: 544). 
EXAMPLE 15 
Identification of a cDNA Clone Containing Tumor Necrosis Factor Coding 
Sequences 
About 200,000 recombinant phage from the .lambda.gt 10 cDNA library were 
screened by DNA hybridization using the .sup.32 P-labelled 42-mer from 
Example 14 under conditions of low stringency in accordance with A. 
Ullrich et al., 1984, "EMBO J." 3: 361-364 (or alternatively P. Gray et 
al., 1983, "Proc. Natl. Acad. Sci. U.S.A." 80: 5842-5846, S. Anderson et 
al., 1983, "Proc. Natl. Acad. Sci. U.S.A." 80: 6836-6842 and M. Jaye et 
al., 1983, "Nucleic Acids Res." 11: 2325-2335). Nine distinct clones 
hybridized with the probe and were plaque purified. Then .sup.32 
P-labelled cDNA was prepared using mRNA from uninduced HL-60 cells. DNA 
from seven of these nine phage clones failed to hybridize with this 
"uninduced" probe and were therefore judged to be candidates for tumor 
necrosis factor cDNA sequences. The cDNA clone containing the largest 
insert was designated .lambda.42-4. This insert was sequenced by the 
dideoxy chain termination method (A. Smith, 1980, "Methods in Enzymology" 
65: 560-580 and F. Sanger et al., 1977, "Proc. Natl. Acad. Sci. U.S.A." 
74: 5463-5467) after subcloning into the vector M13mp8 (J. Messing et al., 
1981, "Nucleic Acids Res." 9: 309-321). 
The cDNA sequence obtained for .lambda.42-4 contained the entire coding 
region for mature tumor necrosis factor plus a portion of its signal 
peptide. The correct orientation and reading frame of the DNA was deduced 
by comparison with the amino acid sequence of tryptic peptide T4 of tumor 
necrosis factor. The amino terminal valine residue of tumor necrosis 
factor determined by protein sequencing is indicated as amino acid 1, and 
is followed by 156 additional amino acids before a stop codon in reading 
phase is encountered. The calculated molecular weight is 17,356 daltons. 
EXAMPLE 16 
Identification of a cDNA Clone Containing Complete PreTNF Coding Sequences 
The cDNA clone .lambda.42-4 contains the entire coding region of mature TNF 
but lacks a complete signal peptide coding sequence as evidenced by its 
lack of an initiation codon. To obtain the missing sequence information, 
the hexadecanucleotide primer dTGGATGTTCGTCCTCC was chemically 
synthesized. This primer was annealled to mRNA from Example 12 and then 
cDNA was synthesized using the method of Example 13. A new library of 
about 200,000 cDNA clones was prepared in .lambda.gt10 following the 
method described in Example 13. This library was screened by hybridization 
analysis using as a probe the .lambda.42-4 cDNA insert which had been 
.sup.32 P-labelled. Sixteen positive clones were obtained, the longest 
(.lambda.16-4) of which contained a cDNA insert extending 337 bp further 
5' than the .lambda.42-4 insert. The composite sequence of the TNF cDNA 
inserts of .lambda.16-4 (nucleotides 1-870) and .lambda.42-4 (nucleotides 
337-1643) contained the complete TNF cDNA (nucleotides 1-1643) 
EXAMPLE 17 
Construction of an Expression Vector for Direct Expression of Tumor 
Necrosis Factor 
The procedure used to express the cDNA sequence for tumor necrosis factor 
obtained in Example 15 is set forth in FIG. 11. Phage .lambda.42-4 from 
Example 15, containing the entire mature tumor necrosis factor coding 
sequence and a portion of the putative tumor necrosis factor secretory 
leader, was digested with EcoRI and an approximately 800 bp fragment 
containing the tumor necrosis factor coding region was recovered. This 
fragment was digested with Ava I and Hind III and a 578 bp fragment 
(designated "C" in FIG. 11) recovered. This fragment encodes tumor 
necrosis factor amino acids 8-157. 
Two synthetic deoxyoligonucleotides (designated fragment "B" in FIG. 11) 
were prepared (see the construction of the adaptor sequence in Example 13) 
which incorporated an Xba I cohesive terminus at the 5' end, an Ava I 
cohesive terminus at the 3' end, a met initiation codon and codons for the 
first seven amino terminal amino acids of tumor necrosis factor. The 
codons for these amino acids were chosen on the basis of E. coli 
preference. The AATT sequence upstream from the start codon was selected 
to properly space the start codon from the trp ribosome binding sequence 
and, in combination with the amino acid codons, to eliminate a potential 
messenger RNA loop. 
Segments B and C are then joined in a three-fold ligation with a pBR322 
derivative containing the trp promoter sequence with the Shine-Dalgarno 
sequence of the trp leader peptide (European Patent Application Publn. No. 
36776). The derivative is obtained or designed to contain unique Xba I and 
Hind III sites between the trp promoter and the Tet.sup.R gene. Either 
p20KLT (copending U.S.S.N. 616,503, incorporated by reference) or ptrpETA 
(Gray et al., 1984, "Proc. Natl. Acad. Sci. U.S.A." 81: 2645-2649) are 
suitable starting vectors of this type, although others may be constructed 
from pBR322, the trp promoter and any required synthetic linkers. Both 
pBR322 and plasmids containing the trp promoter are publicly available. 
The pBR322 portion of the vector chosen may have the AvaI-PvuII segment 
from bp 1424 to 2065 deleted (designated "XAP" in the plasmid name). Any 
of the foregoing plasmids are simultaneously digested with Xba I and Hind 
III and the large vector fragment recovered. This fragment, and fragments 
B and C are ligated with T4 DNA ligase and the ligation mixture used to 
transform E. coli 294 (ATCC 31446). Ampicillin resistant colonies were 
selected, plasmid DNA recovered and characterized by restriction 
endonuclease mapping and DNA sequencing. pTrpXAPTNF was obtained which 
contained inserts B and C. 
EXAMPLE 18 
Tumor Necrosis Factor Expression in E. coli 
E. coli ATCC 31446 which had been transformed with pTNFtrp was grown in M9 
medium containing 20 .mu.g/ml ampicillin and the culture grown to an 
A.sub.550 =0.3. Indole acetic acid was added to give a final concentration 
of 20 .mu.g/ml and the culture grown to A.sub.550 =1. 10 ml of cells were 
concentrated and resuspended in phosphate buffered saline. The cells were 
sonicated and diluted for tumor necrosis factor determination in the 
Example 1 assay. Approximately 10.sup.5 units of activity were obtained 
per ml of culture. This activity was neutralized by preincubation with 
rabbit antisera from rabbits immunized against human tumor necrosis 
factor. 
EXAMPLE 19 
Tumor Necrosis Factor Expression in E. coli 
This method is preferred over that of Example 18. 
Preferably, the host for use with the above vectors is a non-revertable 
tonA E. coli strain. Such strains are bacteriophage resistant and 
therefore far more suitable for large scale culture than wild-type 
strains. Following is a description of a suitable method for generating 
such a strain. The method is further described in copending U.S. patent 
application Ser. No. 673,955. E. coli W3110 is transduced with 
.lambda.::Tn10, a lambda bacteriophage containing the transposable element 
Tn10, to generate a Tn10 hop pool of E. coli W3110. (N. Klecker et al., 
1977 "J. Mol. Biol." 116: 125). 
The E. coli W3110::Tn10 hop pool is grown in L broth at 37.degree. C. to a 
cell density of about 1.times.10.sup.9 /ml 0.5 ml of the culture is 
centrifuged and the pellet is resuspended in 0.2 mls of a .lambda.phi80 
(or T1) lysate containing 7.0.times.10.sup.9 pfuo. The phage is allowed to 
adsorb for 30 minutes at 37.degree. C. The suspension is then spread on 
EMB plates supplemented with tetracycline (15 .mu.g/ml). After an 
overnight incubation at 37.degree. C., the light pink colonies are pooled 
in 3 ml of L broth, grown overnight at 37.degree. C., washed twice, and 
resuspended in L broth. This culture is infected with bacteriophage Pl kc, 
and the phage lysate recovered (J. Miller, 1972, Experiments in Molecular 
Biology, Cold Spring Harbor Laboratory, p 304). 
E. coli AT982 (no. 4546, E. coli Genetic Stock Center, New Haven, Conn.) is 
transduced to tetracycline resistance by this Pl kc lysate. Transductants 
are selected on L broth plates supplemented with tetracycline (15 
.mu.g/ml) and (40 .mu.g/ml) dap (diaminopimelic acid). The resulting 
transductants are screened for tetracycline resistance and the 
regeneration of the dap gene (dap.sup.+, tet.sup.R) dap.sup.+, tet.sup.R 
transductants ar then tested for .lambda.phi80 (or T1) resistance. 
P1 kc lysates are then made on several dap.sup.+, tet.sup.R, .lambda.phi80 
(or T1) resistant strains. The lysates are used to transduce E. coli W3110 
to tetracycline resistance. The transductants are screened and selected 
for .lambda.phi80 (or T1) resistance. 
Tetracycline sensitive isolates are selected from the W3110 
fhuA::Tn10-.lambda.phi80R transductants (S. Naloy et al., 1981 "J. Bact." 
145: 1110). These isolates are checked for phage .lambda.phi80 resistance 
and tetracycline sensitivity after single colony purification. 
DNA is isolated from several tetracycline sensitive .lambda.phi80 phage 
resistant mutants and digested with SstII. The SstII digested DNA is 
characterized by the Southern blot procedure using radioactively labelled 
and SstII digested .lambda.::Tn10 DNA as a probe to determine if the Tn10 
has excised (R. Davis et al., 1980, Advanced Bacterial Genetics, Cold 
Spring Harbor Laboratory). One of the tetracycline sensitive isolates is 
shown to have lost two of the Tn10 hybridization bands as compared to the 
hybridization between DNA from the .lambda.::Tn10 and the parental W3110 
fhuA::Tn10.mu.phi80R. A third hybridization band has an altered mobility 
indicating that a deletion caused by the imprecise excision of Tn10 has 
occurred. 
SDS-gel electrophoresis of outer membrane preparations from the strain with 
an imprecise Tn10 excision reveal that the band assumed to be the fhuA 
protein has an altered electrophoretic mobility as compared to the 
wild-type fhuA protein. The resulting protein is non-functional as a 
.lambda.phi80 phage receptor protein. A second independent strain which 
also has undergone imprecise excision of Tn10 shows no fhuA protein on the 
SDA gel. 
Neither of these strains demonstrate reversion to tetracycline resistance 
or to .lambda.phi80 susceptability indicating that there is an imprecise 
excision of all or part of the Tn10 transposon together with either a 
partial or complete deletion of the fhuA gene. Preferably, one of such 
W3110 strains (NL106) is used as a host for the TNF-encoding vehicles 
described elsewhere herein. 
NL106 was transformed with ptrpXAPTNF and inoculated into 10 liters of a pH 
7.4 medium having the following formula: 
______________________________________ 
Component gms/L 
______________________________________ 
(NH.sub.4).sub.2 SO.sub.4 
5.0 
K.sub.2 HPO.sub.4 6.0 
NaH.sub.2 PO.sub.4 3.0 
Na Citrate 1.0 
L-Tryptophan 0.2 
NZ Amine AS 4.0 
Yeast Extract 4.0 
MgSO.sub.4 1.2 
Glucose 25.0 
Trace Element solution 0.5 ml 
(Fe, Zn, Co, Mo, Cu, B and 
Mn ions) 
Tetracycline 1.0 mg 
______________________________________ 
Glucose was fed to the culture at a rate of 1 gm/minute when the A.sub.550 
of the culture reached about 20. The fermentation was conducted at 
37.degree. C. until an A.sub.550 of 136 was reached (about 20 hours). The 
culture is centrifuged to form a cell paste and the paste then extracted 
for 30 min. at pH 8.0 and room temperature with a buffer containing 50 mM 
tris, 10 mM EDTA, 1000 mM NaCl, 2000 mM urea and 0.1 percent 
beta-mercaptoethanol. The extract was diluted and assayed as described in 
Example 1. 1.times.10.sup.8 units of tumor necrosis factor activity in 
this assay was established as equivalent to 1 mg of tumor necrosis factor. 
Up to about 2 grams of tumor necrosis factor were obtained per liter of 
culture. Amino terminal sequencing demonstrated that about from 75 to 86 
percent by weight was valyl amino-terminal (mature) tumor necrosis factor, 
the remainder being met-TNF. Furthermore, in addition to high expression 
levels the protein was not present in refractile bodies, nor did it 
otherwise appear to be toxic to the cells as evidenced by the extremely 
high cell densities that were obtained. 
EXAMPLE 20 
Construction and Expression of a Mutant Tumor Necrosis Factor Gene 
In this contemplated example, Examples 17-18 were repeated except that the 
oligonucleotide fragment B was synthesized with a histidine codon CAT in 
place of the arginine 6 codon CGT. Mutant tumor necrosis was expressed. 
EXAMPLE 21 
Construction and Expression of Another Mutant Tumor Necrosis Factor Gene 
In this example, the procedure of Examples 17-18 was repeated with an 
oligonucleotide fragment B encoding leucine (CTT) in place of the residue 
2 arginine codon. About 1200 mg of mature TNF activity were obtained per 
liter of culture in initial trials. Unprocessed TNF was not detectable in 
the culture. 
EXAMPLE 22 
Construction of a Vector Encoding a Tumor Necrosis Factor Fusion with a 
Secretory Signal Sequence 
The sequence of the E. coli heat stable enterotoxin gene STII is depicted 
in FIG. 12. In this Example a fragment containing the STII secretory 
signal and Shine-Dalgarno sequence was ligated downstream of the E. coli 
alkaline phosphotase promoter. The STII signal is followed in the 3' 
direction with a synthetic oligonucleotide which supplies codons for the 
initial seven amino-terminal tumor necrosis factor amino acids and the 
remainder of the coding sequence for tumor necrosis factor. All of the 
foregoing were assembled in a pBR322 vector. 
pWM501 (Picken et al., 1983, "Infection and Immunity" 42(1): 269-275) 
contains the STII gene depicted in FIG. 12. pWM501 was digested with XbaI 
and NsiI and the approximately 90 bp fragment isolated. This fragment also 
could be synthesized organically by methods known per se (fragment A). 
A pBR322-Trp plasmid as described in Example 17 (p20kLT) was digested with 
XbaI and HindIII, and the large vector fragment recovered (fragment B). 
This fragment contains an E. coli origin of replication and a gene 
conferring the phenotype of ampicillin resistance. 
A synthetic oligonucleotide was synthesized as two strands and annealed to 
yield the following structure (the restriction site sticky ends and amino 
acids coded for by the oligonucleotide are also indicated). 
##STR2## 
This is designated fragment C. 
pTNFtrp from Example 18 was digested with AvaI and HindIII. The 578 bp 
AvaI-HindIII fragment (fragment D) was recovered. It contains all of the 
TNF coding sequence except for the first seven amino acids. 
A DNA sequence comprising an E. coli alkaline phosphatase (AP) promoter 
linked to a heterologous Shine-Dalgarno (S.D.) sequence (trp) and having 
EcoRI and XbaI termini was constructed as follows. A DNA fragment 
containing a portion of the AP promoter was isolated from the plasmid 
pHI-1 (H. Inouye et al., 1981, "J. Bacteriol." 146: 668-675), although any 
appropriate sources containing AP promoter DNA also could be used. pHI-1 
was digested with HpaI to open the plasmid, a synthetic EcoRI linker 
##STR3## 
ligated to the plasmid and the linkered plasmid digested with an excess of 
EcoRI to cleave all EcoRI site and with a deficiency of RsaI activity in 
order to only partially cleave all of the RsaI sites (the EcoRI and RsaI 
steps also could be done sequentially rather than simultaneously). A 420 
bp fragment containing the AP promoter was recovered from the EcoRI-RsaI 
partial digest. 
A trp S.D. sequence was obtained as follows. A plasmid or organism 
containing the trp promoter (pIFN-beta 2, D. Leung et al., 1984, 
"Biotechnology" 2: 458-464) was digested with XbaI and RsaI and the 30 bp 
fragment recovered which contains the trp S.D. sequence. This fragment was 
ligated to the 420 bp AP promoter fragment to yield a 450 bp EcoRI-XbaI 
fragment E. Fragment E has the nucleotide sequence 
##STR4## 
Fragments A, B, C and D were ligated in a four-part ligation and the 
ligation mixture used to transform E. coli 294. Transformants were 
identified by growth on LB plates containing ampicillin. Plasmid 
trpSTIITNF was isolated from a transformant colony. This plasmid was 
digested with XbaI and EcoRI to remove the trp promoter, then ligated to 
the 450 bp long EcoRI-XbaI fragment E containing the E. coli alkaline 
phosphatase promoter. The resulting plasmid is called pAPSTIITNF. 
EXAMPLE 23 
Expression and Processing of a Tumor Necrosis Factor Fusion with a 
Secretory Signal Sequence 
E. coli NL106 was transfected with pAPSTIITNF and innoculated into 10 
liters of a pH 7.0 medium having the following formula: 
______________________________________ 
Component gms/L 
______________________________________ 
(NH.sub.4).sub.2 SO.sub.4 
5.0 
K.sub.2 HPO.sub.4 2.6 
NaH.sub.2 PO.sub.4 1.3 
Na Citrate 1.0 
KCI 1.5 
NZ Amine AS 5.0 
Yeast Extract 2.0 
MgSO.sub.4 1.2 
Glucose 25.0 
Trace Element solution 0.5 ml 
(Fe, Zn, Co, Mo, Cu, B and 
Mn ions) 
Ampicillin 20.0 mg 
______________________________________ 
The culture was conducted in the same fashion as described above in Example 
19, except that an A.sub.550 of 140 was reached. The culture at this point 
contained about 400 mg of tumor necrosis factor/liter, about 70-80 percent 
by weight of which was properly processed to the mature protein as 
estimated from electrophoresis gels. Approximately the same activity of 
tumor necrosis factor was recovered upon whole cell extraction by the 
method used in Example 19 as was recovered by osmotic shock of the cells.