Peptides for inducing monocyte cytotoxicity in diagnostics

The present disclosure relates to a new lymphokine molecule termed Monocyte Cytotoxicity Inducing Factor (MCF), to MCF peptides. The disclosure further relates to the development of novel Sezary cell hybridomas which secrete MCF, to the purification of MCF and its constituent polypeptides and to the identification of small peptides with MCF activity. Sezary OKT4+ lymphocytes were fused to CEM.8aza.sup.r.C, an HGPRTase lacking clone of CEM, to generate hybrid cells, certain of which produced soluble mediators of human monocyte cytotoxicity. A single sezary hybrid clone, FtF3, produced a novel monocyte cytotoxicity inducing factor found to be distinct from IFN.gamma. and IFN.alpha.. MCF, purified by dye ligand and ion-exchange chromatography, comprises two polypeptides of 29 and 14.7kD (P29 and P14.7) which can be further purified by preparative SDS/PAGE and hydrophobic chromatography, respectively. Amino acid composition analyses and immunoblotting of two-dimensional gels indicate that these are distinct but possibly related polypeptides. N-terminal analysis of P29 reveals it to be a previously undescribed cytokine mediator of monocyte function. A peptide having the sequence Gly Ala Ala Val Leu Glu Asp Ser Gln, corresponding to the native N-terminal sequence of P29, also exhibits MCF activity equivalent to that of the intact 29kD protein.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the fields of immunology and cancer 
therapy, and is directed to biological compositions and methods for 
inducing human monocytes to a cytoxic state. The invention concerns the 
purification and characterization of soluble factors, and particularly, of 
peptides, which induce human monocyte cytotoxicity and have utility in 
anti-cancer therapy. 
2. Description of the Related Art 
Immune protection of vertebrates is provided by a dual system that 
maintains two basic defenses against foreign invaders. These two defenses, 
termed cellular and humoral immunity, are adaptive and respond 
specifically to most foreign substances, although one response generally 
is favored. While cellular immunity is particularly effective against 
foreign tissue, cancer cells, intracellular viral infections and 
parasites, the humoral immune response defends primarily against the 
extracellular phases of bacterial and viral infections. Therefore, the 
cellular response is directed primarily against invading cells, while the 
humoral response is directed against primarily cell products, such as 
toxins. Moreover, whereas cellular immunity is provided by cells of the 
lymphoid system, humoral immunity is provided by proteins called 
antibodies that circulate through the fluid compartments of the body. 
The dual nature of the immune system is generated from two separate 
populations of morphologically indistinguishable lymphoid cells called 
lymphocytes. While one class of lymphocytes, the T-cell lymphocytes, 
mediates the cellular immune response, the other class of lymphocytes, the 
B-cells, is responsible for the humoral immune response. Thus, when the 
organism is invaded by a foreign substance, for example an altered cell 
(e.g. viral transformed cell or tumor cell), some of the T-cells that 
recognize it are activated and initiate reactions that include binding to 
and eliminating the altered cells. On the other hand, when individual 
B-cells are activated, they differentiate to plasma cells that secrete 
specific antibodies directed against substances secreted by the foreign 
invader. For a review of the foregoing, see Hood et al., (Immunology, 
Second Edition, Benjamin/Cummings Publishing Company, Inc., Menlo Park, 
Calif., 1984). 
While cells of B-lymphocyte lineage have found widespread clinical and 
industrial application in the generation of monoclonal antibodies, cells 
of T-lymphocyte lineage have proved of interest in part due to the 
numerous soluble factors they secrete. In the biological system, T-cell 
factors play an important role in modulating and activating various immune 
functions. Isolation and characterization of various T-cell factors has 
been the goal of many clinical research endeavors attempting to identify 
those factors which might be useful in treating a number of disease 
states, for example, in the treatment of tumor cells and viral infectious 
states. Of particular interest has been the recent characterization of a 
factor termed T-cell growth factor, or interleukin II, which is produced 
and secreted by effector T.sub.A cells. (See U.S. Pat. Nos. 4,401,756; 
4,404,208; 4,407,945; and 4,473,642). When T.sub.C cells are stimulated by 
interleukin II, they undergo an effector phase and are stimulated to 
mature into killer T-cells which are capable of identifying and 
eliminating various target cells. As demonstrated by the above patents, 
interleukin II has become an important pharmaceutical agent in the 
treatment of various disease states. 
Optimism spurred by the preliminary success of interleukin II has lead 
researchers on a quest to identify other immune-mediating factors having 
potential clinical applicability. However, this search has generally been 
hampered by the existence of numerous factors secreted by the same or 
similar cell types. Moreover, confusion often results from the general 
overlapping nature of the factor activities and often times from a lack of 
currently available test systems for identifying individual factor 
activities. Without highly sensitive test systems for identifying 
individual factor activities, the existence of a particular factor cannot 
be readily distinguished from other factor activities. 
Recently, interest has been shown in identifying soluble factors which 
serve to stimulate human monocyte cytotoxicity. Monocytes are a phagocyte 
of the blood which, along with macrophages and polymorphonuclear 
leukocytes, bind and ingest foreign substances often prior to an antibody 
response. "Activated" monocytes have recently been shown to exert an 
antitumor activity. For example, Fischer et al. (Cell. Immunol., 
58:426-435, 1981) disclose that human peripheral blood monocytes can 
reproducibly lyse a variety of tumor cells. More recently, researchers 
have disclosed various factors thought to play a role in monocyte 
activation. For example, Kleinerman et al. (Cancer Res., 45:2058-2064, 
1985), discusses the activation of human blood monocytes by incubation 
with concanavalin A-stimulated lymphokine (macrophage-activating factor 
(MAF)), lipopolysaccharide endotoxin, and human recombinant gamma 
interferon. It was reported that gamma interferon, in the presence of 
endotoxin, was capable of activating monocyte tumoricidal activity. 
Moreover, MAF treatment exhibited a similar effect. 
Other monocyte cytotoxicity promoting factors have been identified as well. 
For example, Le et al. (J. Immunol., 131:2821-2826, 1983) has reported a 
T-cell hybridoma line capable of producing a macrophage activating factor 
with the ability to activate human blood. Monocytes to show enhanced 
cytotoxicity against a human colon adenocarcinoma line. However, this 
activity was found to be neutralized with specific antiserum to purified 
human interferon-gamma. These authors concluded that this MAF was in fact 
interferon-gamma. 
More recently, Jones and Clouse (Immunobiol., 167: Abstract No. 365, 1984) 
reported the use of lymphocytes from patients with Sezary's syndrome in 
the production of a human T-cell hybridoma line which is capable of 
producing a factor which stimulates human monocyte antitumor cytotoxicity. 
In contrast to the factor identified by Le et al., the factor reported in 
the Jones and Clouse publication was not inhibited by antibodies having 
specificity for interferon-gamma. Although the Jones and Clouse reference 
did observe that two molecular weight species having the biological 
activity was observable, the methodology used to identify this particular 
activity was not identified. Moreover, the methodology for identifying and 
isolating Sezary/T-cell hybridomas which secrete the particular factor was 
not disclosed. Clearly, not all Sezary/T-cell hybridomas are capable of 
producing monocyte stimulatory factors (See, e.g., Grillot-Courvalin et 
al., "Helper T-Hybridoma Produced By Fusion With Sezary Cells," in: T-Cell 
Hybridomas, ed. by M. J. Taussig, CRC Press, Inc., Boca Raton, Fla., 
1985). 
It is apparent from the foregoing references that, not only are there 
numerous factors potentially involved in the stimulation of human monocyte 
cytotoxicity, but additionally that these factor activities may be 
indistinguishable in previously available assays for detecting various 
cytotoxic actions. Moreover, as with other cytokines, the purification and 
characterization of monocyte cytotoxicity inducing factors has been 
hampered simply by their naturally low abundance in biological systems. 
Accordingly, the present invention is directed to methods for accomplishing 
the isolation of particular discrete soluble factors which exhibit human 
monocyte cytotoxicity inducing activity. The present disclosure is further 
directed to the purification and characterization of these factors and to 
the preparation of T-cell hybridoma lines which produce these factors in 
vitro and thereby provide a ready source for isolating the factors. In 
that the novel factors of the present invention demonstrate a surprising 
ability to elicit an antitumor response by monocytes in vitro, similar to 
that possessed by interleukin II for lymphoid cells, it is believed that 
these factors will provide an important new addition to the antineoplastic 
armament of medical science. 
SUMMARY OF THE INVENTION 
The present invention is directed to compositions and methods for inducing 
human monocytes to a cytoxic state. The invention generally concerns the 
purification and characterization of soluble factors with human monocyte 
cytotoxicity inducing activity, referred to herein as MCF activity, and 
more particularly, to peptides with MCF activity. The factors and peptides 
of the present invention are contemplated for use in the treatment of 
cancer. 
Substantially purified MCF, is a factor capable of inducing human monocytes 
to a cytotoxic state which comprises two polypeptides having molecular 
weights of about 29,000 Daltons (P29) and of about 14,700 Daltons (P14.7) 
when determined by polyacrylamide gel electrophoresis under the conditions 
as described herein. However, it is known that the migration of a 
polypeptide can vary, sometimes significantly, with different conditions 
of SDS/PAGE (Capaldi et al., Biochem. Biophys. Res. Comm., 76:425, 1977, 
incorporated herein by reference). It will therefore be appreciated that 
under differing electrophoresis conditions, these molecular weights may 
vary. 
The larger polypeptide of the human monocyte cytotoxicity inducing factor 
(P29) exhibits a molecular weight of about 29 kilodaltons on SDS 
polyacrylamide gel electrophoresis (SDS/PAGE) when the factor is obtained 
from cells grown in serum-free media. However, following gel filtration 
chromatography it exhibits a molecular weight of between about 78 and 63 
kilodaltons, i.e., essentially the same as bovine serum albumin, when 
obtained from cells grown in media containing serum. In that bovine serum 
albumin itself has a molecular weight of approximately 68,000 Daltons, it 
is likely the case that this higher molecular weight species of MCF has an 
affinity for serum albumin. This would explain why, when obtained from 
cells grown in the presence of serum albumin, the higher molecular weight 
factor exhibits a molecular weight essentially indistinguishable from that 
of serum albumin. 
The smaller polypeptide of MCF (P14.7) has an apparent molecular weight of 
approximately 11,500 Daltons when subjected to gel filtration 
chromatography under the conditions described herein. Again, as with gel 
electrophoresis, it will be appreciated that gel filtration chromatography 
does not provide an exact molecular weight determination. In this case, 
the monocyte cytotoxicity inducing activity elutes from such columns as a 
broad peak, corresponding to a molecular weight of approximately 11,500 
Daltons. However, this molecular weight may vary with variations in 
conditions, for example, running buffer, exclusion limit, column size and 
the particular gel filtration methodology which is utilized. 
A composition which includes one or both of the above factors may be 
further characterized by physicochemical and biological characteristics. 
For example, the composition and factor(s) retains biological stability 
following treatment at pH 2 for one hour. The term biological stability, 
as used herein, is defined as the retention of substantial biological 
activity following the indicated treatment as defined by the present 
disclosure. The composition similarly retains biological stability 
following treatment at 60 degrees Centigrade for one hour, and further 
retains biological activity in the presence of antiserum to 
interferon-gamma, interferon-alpha, or a combination of antisera to 
interferon-alpha and gamma. MCF activated monocytes retain biological 
activity in the presence of antiserum to Leu-llb plus complement 
indicating that the factor does not appear to activate NK cells, and 
following treatment by the enzymes Rnase, Dnase and trypsin. Biological 
activity is reduced following treatment by the enzyme chymotrypsin. The 
polypeptides of MCF can also be characterized according to their 
isoelectric points, P29 having an isoelectric point of 4.2, and that of 
P14.7 being 6.5. 
Certain aspects of this study concern a continuous cell line which produces 
a factor as defined by the foregoing characteristics. In particular, a 
continuous cell line is provided which produces a factor capable of 
inducing human monocytes to a cytotoxic state, wherein the cell line is 
produced by a process which includes the steps of immortalizing human 
T-cells to produce continuous cell clones; identifying a clone which 
produces the factor; and culturing the clone to provide the continuous 
cell line. 
The first step of immortalizing human T-cells to produce continuous cell 
clones is generally defined as providing human T-cells in a manner whereby 
they may be cultured continuously for an indefinite period. The most 
convenient manner for providing such continuous cell clones is through the 
development of a T-cell hybridoma. T-cell hybridomas are generally well 
known in the art and may be generated by a variety of well known methods. 
In general, such methods include fusing human T-cells with a second cell 
population which is sensitive to growth in a selective media and culturing 
the fused cells in the selective media to produce the continuous cell 
clones. Cells may be fused in numerous ways, for example, through the use 
of polyethylene glycol or Sendai virus. 
In a preferred embodiment, the second cell population is a T-cell lymphoma 
population which has been selected for growth in 8-azaguanine. By 
selecting for T-cell lymphomas capable of growth in 8-azaguanine, a cell 
population sensitive to growth in HAT selective media is obtained. Those 
of skill in the art will recognize that other cell lines having other 
selective criteria may be utilized for fusion with human T-cells to 
provide the continuous cell clones. For example, additional T-cell 
lymphoma subtypes could be used to clone other subclasses of human 
T-lymphocytes. Moreover, drug sensitivities and other selective criteria 
can be generated using other approaches including, 6-thioguanine, oubaine 
or oncogenic transformation. Additionally, inter-species hybrids can be 
generated to allow for chromosomal localization. 
However, fusion is not the only means of achieving immortalized human 
T-cells. For example, certain human T-cells are responsive to T-cell 
growth factor and may be immortalized by continuous culturing in the 
presence of T-cell growth factor (interleukin II). Additionally, certain 
human T-cells, for example, certain neoplastic T-cell lines are capable of 
continuous growth in cell culture as are certain transformed (e.g. virus 
transformed) T-cell lines. While most T-lymphotrophic viruses are toxic, 
it is known that HTLV-1, as well as portions of the EB virus genome, 
commonly utilized in B-lymphocyte transformation, can be used to transform 
and thereby immortalize, T-lymphocytes. All such continuously growing 
T-cells, and methods of providing continuously growing T-cells, are 
included within the scope of the present invention. 
After obtaining the immortalized human T-cells in the form of continuous 
cell clones, a clone is identified which produces the monocyte 
cytotoxicity inducing factor. The crux of the successful practice of the 
present invention relies on the ability to identify clones which produce 
this factor rather than the numerous other immune regulatory and 
stimulatory factors known in the art. It is now believed that many 
hundreds of peptides, whose functions are unknown, are secreted by various 
activated T-cells. (see, e.g., Zurawski et al., Science, 232: 772-775, 
1986). Moreover, depending on the particular T-cell which is immortalized, 
the number of clones positive for factor production may be quite low. 
Therefore, the assay must be not only highly specific for the present 
factor, but must be quite sensitive to the presence of small amounts of 
the factor, in order to successfully practice the present invention. 
Accordingly, the present disclosure is directed to an assay particularly 
adapted to identification of the present factor. 
The final step of the present process is simply culturing the identified 
clone to produce the cell line. Where the immortalized human T-cell is 
achieved through hybridoma development, culturing will include simply 
culturing in an acceptable media. However, where the immortalized cell 
line does not involve cell fusion and instead requires the presence of a 
growth maintaining factor such as T-cell growth factor, culturing will 
require the inclusion of the particular growth factor. 
In that it is believed that the factor of the present invention is secreted 
by a very small proportion of T-cells in general, it is a preferred 
embodiment of the present invention to employ Sezary cells as the T-cells 
to be immortalized. This is because it has been determined that a 
relatively large proportion of Sezary cells do in fact produce the present 
factor. However, in that it appears clear that not all Sezary cells 
produce the factor, the selection step is still required in order to 
identify clones producing the factor. However, if Sezary cells are 
unavailable, and one does not desire to screen the large number of clones 
which must necessarily be screened where T-cells in general are utilized, 
one may desire to use effector T-cells. It has been determined that a 
population of effector T-cells include a larger proportion of factor 
positive cells than do T-cells in general. 
In a very general sense, the method of identifying a clone which produces 
the factor includes the steps of stimulating the clone with a T-cell 
mitogen to release lymphokines; culturing human monocytes together with 
appropriate target cells, for example, human cancer cells, in the presence 
of the released lymphokines; and detecting target cell lysis, wherein such 
lysis is indicative of the presence of human monocyte cytotoxicity 
inducing factor in the released lymphokines. As used herein, and as 
appreciated in the art, lymphokines is a generic term directed to any 
molecule having biological activity for modulating the immune system. A 
T-cell mitogen, as will be appreciated by those of skill in the art, is a 
molecule or a compound having the ability to stimulate the release of 
lymphokines from T-cells. In a preferred aspect of the present invention, 
the T-cell mitogen used is phytohemagglutin, commonly referred to as PHA. 
However, concanavalin A and other T-cell mitogens known to the art may be 
successfully utilized. 
The second step of culturing human monocytes together with appropriate 
target cells in the presence of released lymphokines allows for the 
specific induction of monocyte cytotoxicity inducing factor where such 
factor is present in the released lymphokines. As noted previously, this 
particular step is quite important to the successful practice of the 
present invention and is disclosed in detail in connection with the 
disclosure of a preferred embodiment in a later section. Of course, to 
demonstrate that the present factor is indeed effective against human 
target cells, an appropriate human target cell is preferred. Useful target 
cells have been identified as the human myeloid leukemic cell line, K562 
and also HL60, L5178Y and TU5 cells. This group of tumor targets includes 
both NK-sensitive and NK-resistant cells. However it is believed that 
numerous additional cell types may be employed, for example, melanoma, 
lung carcinoma and bladder cell tumors. 
The final step of detecting target cell lysis is conveniently performed 
through the use of a radioisotope which is maintained extra or 
intracellularly when the target cell is in a non-lysed condition, and 
wherein the radioisotope is released into the surrounding media when the 
target cell is lysed. However, it will be appreciated that additional 
methods known in the art, and disclosed herein, may be used. 
The present invention is additionally directed to a method for generating 
substantially purified monocyte cytotoxicity inducing factor, including 
both the P29 and P14.7 polypeptides, which comprises the steps of: 
(a) stimulating a continuous cell line which produces the monocyte 
cytotoxicity inducing factor with T-cell mitogen to release the factor 
into the culture supernatant; 
(b) subjecting the supernatant, or a fraction thereof, to affinity 
chromatography using a Matrex Gel Red A column; 
(c) assaying the chromatography fractions to identify those fractions which 
retain a human monocyte cytotoxicity inducing factor; 
(d) subjecting said monocyte cytotoxicity inducing factor-containing 
fractions to ion-exchange chromatography; and 
(e) identifying the chromatography fractions which contain human monocyte 
cytotoxicity inducing activity and collecting said fractions. 
Practicing the method in this manner is preferred in that it will provide a 
factor-containing composition which is substantially purified with respect 
to biological activity as defined by the disclosed assay. However, it is 
believed that sufficient purity may be obtained by simply subjecting the 
culture supernatant to chromatography on a Matrex Gel Red A column 
(Amicon), i.e., performing steps (a) to (c), as above. To conduct such a 
purification step, one would pass the supernatant over the column in a low 
salt-containing buffer, such as 20 mM phosphate-buffered-saline (PBS), 
0.15N NaCl, to bind the factor to the Matrex gel, wash the column to 
remove non-binding material, and then elute the bound fraction from the 
column with a buffer having an increasing salt concentration, such as PBS 
with a salt gradient of 0.15--1N NaCl. 
As stated above, to achieve a more purified factor preparation containing 
both the P29 and P14.7 polypeptides, one may combine the Matrex Gel Red A 
column procedure with an ion-exchange chromatography step. To perform 
ion-exchange chromatography in this manner one would preferably include 
the steps of: 
(a) dialyzing the monocyte cytotoxicity inducing factor-containing 
fractions from the Matrex Gel Red A column against a low salt-containing 
buffer; 
(b) passing said dialyzed fractions over an ion exchange column in a low 
salt-containing buffer to bind the factor to the column; 
(c) washing the column to remove non-binding materials; and 
(d) eluting bound material from the column with a high salt-containing 
buffer. 
A preferred ion-exchange column for use in such embodiments is a DEAE 
column. However, other columns are contemplated to be of use, such as a 
Mono Q column (Pharmacia). 
In further embodiments, the present invention provides methods to prepare 
substantially purified P27 or P14.7 polypeptides of human monocyte 
cytotoxicity inducing factor. The preferred starting material for the 
further purification of either P27 or P14.7 is the substantially purified 
MCF obtained following both Matrex Gel Red A and ion-exchange 
chromatography. 
To obtain the MCF P27 polypeptide one would then subject the 
ion-exchange-eluted material to one-dimensional polyacrylamide gel 
electrophoresis, elute fractions from the polyacrylamide gel and identify 
the fractions having monocyte cytotoxicity inducing activity. 
The preferred procedure for obtaining a substantially purified MCF P14.7 
polypeptide involves subjecting the ion-exchange-eluted material to 
hydrophobic chromatography. To purify P14.7 in this manner one would 
include the following steps: 
(a) prepare a sample of ion-exchange-eluted material in a buffer containing 
a high concentration ammonium sulphate solution; 
(b) pass the sample in the high concentration ammonium sulphate over an 
octyl sepharose column to bind the factor to the column; 
(c) wash the column to remove non-binding materials; and 
(d) elute bound material from the column with a buffer containing a low 
concentration ammonium sulphate solution and containing sodium dodecyl 
sulphate. 
In using hydrophobic chromatography to further purify MCF P14.7, it is 
contemplated that the high concentration ammonium sulphate buffer used to 
prepare the sample will have an ammonium sulphate concentration of over 
6M, and more preferably, of about 8M ammonium sulphate. To elute the bound 
P14.7 from the column one would use a buffer with a decreasing ammonium 
sulphate concentration, such a buffer with a gradient of 8-0M ammonium 
sulphate. It is also preferred that the elution buffer contain a more 
hydrophobic component, for example, a detergent such as Tween 20, or more 
preferably, SDS. 
However, other combinations of hydrophobic columns and buffer systems are 
contemplated to be of use in accordance herewith. For example, one could 
use an RP8 column with acetonitrile/pyridine. Appropriate techniques for 
use with other hydrophobic columns, such as phenyl sepharose, will be 
known to those of skill in the art in light of the present disclosure. 
In further and particularly important embodiments, the present invention is 
directed to novel MCF-derived peptides and to synthetic peptides with MCF 
activity. The inventor has determined the N-terminal amino acid sequence 
of MCF P29 to be: 
EQU Gly Ala Ala Val Leu Glu Asp Ser Gln (Seq id no:1) 
The inventor determined that a synthetic peptide comprising this 9 amino 
acid sequence (termed MJ-2) is capable of activating human blood monocytes 
for tumor cytotoxicity. Variants of the 9 residue peptide with certain 
amino acid substitutions have also been shown to exhibit human monocyte 
cytoxicity inducing capabilities. A variant examined to date and found to 
have biological MCF activity was the peptide MJ-1 in which the single 
aspartate residue at position 7 had been replaced by asparagine. A 5-mer 
peptide, MJ-5, having the sequence Leu Glu Asp Ser Gln was also found to 
be critical for biological activity. 
Therefore, in certain embodiments, the present invention is directed to the 
use of proteins or peptides of at least 5 amino acid residues in length, 
and having an apparent molecular weight of less than or equal to 29 
kilodaltons, as determined by SDS polyacrylamide gel electrophoresis, the 
protein or peptide including at its amino terminus a sequence being 
selected from the group consisting of: 
EQU Gly Ala Ala Val Leu Glu Asp Ser Gln (seq id no:1); 
EQU Gly Ala Ala Val Leu Asp Asn Ser Gln (seq id no:2); 
EQU Leu Glu Asp Ser Gln (seq id no:3); 
wherein Gly=Glycine (G); Ala=Alanine (A); Val=Valine (V); Leu=leucine (L); 
Glu=Glutamate (E); Asn=Asparagine (N); Ser=Serine (S); Gln=Glutamine (Q); 
and Asp=Aspartate (D). 
The synthesis of synthetic peptides, such as those outlined above, using an 
automated peptide synthesizer, will be generally known to those of skill 
in the art in light of the present disclosure. 
The peptides of the present invention are contemplated to be os use in a 
variety of different embodiments. For example, in important embodiments, 
it is believed that any one, or a combination of, the above peptides could 
be employed in any of the clinical or diagnostic embodiments described 
herein below. Moreover, the peptide sequences could be used to design 
corresponding oligonucleotide probes for use in the molecular cloning of 
the gene encoding the MCF P29 polypeptide. 
Such cloning techniques are often employed in the art for the preparation 
of a so-called "recombinant" protein, which recombinant proteins may then 
be expressed in, and subsequently obtained from, recombinant host cells. 
"Cloning" the 29kD protein, refers to the process of obtaining a specific 
DNA molecule which encodes this protein, in a form distinct from other 
portions of DNA. To achieve this, one may screen a cDNA or genomic DNA 
library with, for example, an oligonucleotide probe or probes designed 
from a knowledge of the amino acid sequence of portions of the protein, 
such as the N-terminal sequence disclosed herein. Following the cloning of 
an appropriate DNA molecule, it may be inserted into any one of the many 
vectors currently known in the art and transferred to a prokaryotic or 
eukaryotic host cell where it may be used to direct the expression and 
production of the so-called recombinant version of the protein. 
A synthetic peptide, termed MJ-2, having the sequence Gly Ala Ala Val Leu 
Glu Asp Ser Gln (seq id no:1) is shown herein to be capable of inducing 
the cytotoxic activity of human blood monocytes, from all donors tested, 
to a similar extent as the intact protein of 29kD. The use of this 
peptide, or an equivalent thereof, in any or all of the clinical 
embodiments for which MCF could be used is therefore contemplated. Indeed, 
the use of peptides in such embodiments is preferred for several reasons. 
The advantages of using peptides in human therapy are many, and include, 
for example, the cost and relative ease of large scale synthesis as 
opposed to purification from natural sources; the invariant composition of 
the purified peptides obtained from different syntheses; and the 
elimination of the possibility that any other natural factors or compounds 
may be present which may adversely affect the human recipient despite 
their low concentration. Furthermore, peptides have preferable 
pharmacological properties such as the ease with which they can penetrate 
tissues and their low immunogenicity. 
Accordingly, in prefered embodiments, it is contemplated that the use of a 
peptide of substantially less than 29kD in molecular weight will be 
advantageous. The present invention therefore concerns peptides of between 
5 and about 100 amino acid residues in length, having at their amino 
terminus (N-terminus) an amino acid sequence corresponding to any one of 
the sequences set forth in seq id no:1, seq id no:2, seq id no:3, or a 
biologically functional equivalent thereof. Peptides of between about 5 
and about 50 residues in length which terminate with such a sequence, or 
an equivalent thereof, are preferred; and peptides of between 5 and about 
20 residues in length which terminate with such a sequence, or an 
equivalent thereof, are particularly preferred. Even more preferred, are 
peptides of at least 9 amino acid residues in length which peptides have 
at their N-terminus the sequence Gly Ala Ala Val Leu Glu Asp Ser Gln (seq 
id no:1). 
As is generally understood in the art, modifications and changes may be 
made in the structure of a protein or peptide and still obtain a molecule 
having like or otherwise desirable characteristics. For example, certain 
amino acids may be substituted for other amino acids in a protein 
structure without appreciable loss of interactive binding capacity with 
other structures such as, for example, substrate molecules, enzymes or 
receptors. Since it is the interactive capacity and nature of a protein 
that defines that protein's biological functional activity, certain amino 
acid sequence substitutions can be made in a protein sequence (or, of 
course, its underlying DNA coding sequence) and nevertheless obtain a 
protein with like properties. It is thus contemplated by the inventors 
that various changes may be made in the sequence of the peptides disclosed 
herein without appreciable loss of their biological utility or activity. 
In making such changes, the hydropathic index of amino acids may be 
considered. The importance of the hydropathic amino acid index in 
conferring interactive biological function on a protein is generally 
understood in the art (Kyte et al., J. Mol. Biol., 157:105-132, 1982). It 
is known that certain amino acids may be substituted for other amino acids 
having a similar hydropathic index or score and still retain a similar 
biological activity. Each amino acid has been assigned a hydropathic index 
on the basis of their hydrophobicity and charge characteristics, these 
are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine 
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); 
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); 
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); 
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and 
arginine (-4.5). 
It is believed that the relative hydropathic character of the amino acid 
determines the secondary structure of the resultant protein or peptide, 
which in turn defines the interaction of the protein with other molecules 
such as enzymes and receptors. It is known in the art that an amino acid 
may be substituted by another amino acid having a similar hydropathic 
index and still obtain a biological functionally equivalent protein. In 
general, the substitution of amino acids whose hydropathic indices are 
within .+-.2 is considered to be an appropriately conservative change. In 
the present case, it is contemplated that amino acid changes that are 
within .+-.1 will be preferable, and that changes within .+-.0.5 will be 
particularly preferred. 
Substitution of like amino acids can also be made on the basis of 
hydrophilicity, as disclosed in U.S. Pat. No. 4,554,101, incorporated 
herein by reference. In U.S. Pat. No. 4,554,101, the following 
hydrophilicity values are assigned to amino acid residues: arginine 
(+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); 
serine (+0.3); asparagine (+0.2); glutamine ( +0.2); glycine (0); 
(0.+-.1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine 
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine 
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is 
understood that an amino acid can be substituted for another having a 
similar hydrophilicity value and still obtain a biologically equivalent 
protein. In a similar manner to that described above, the substitution of 
amino acids whose hydrophilicity values are within .+-.2 is generally 
considered to be an appropriately conservative change. In regard to the 
present invention, is contemplated that amino acid changes that are within 
.+-.1 will be preferable, and that changes within .+-.0.5 will be 
particularly preferred. 
Amino acid substitutions are generally therefore based on the relative 
similarity of the amino acid side-chain substituents, for example, their 
hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary 
substitutions which take various of the foregoing characteristics into 
consideration are well known to those of skill in the art and include: 
arginine and lysine; glutamate and aspartate; serine and threonine; 
glutamine and asparagine; and valine, leucine and isoleucine. 
It is further contemplated that the peptides of the present invention may 
be modified to render them biologically "protected". As is generally known 
in the art, biologically protected peptides have certain advantages over 
unprotected, i.e., unmodified, peptides when administered to human 
subjects. As disclosed in U.S. Pat. No. 5,028,592, incorporated herein by 
reference, a peptide which is protected, for example, through acylation of 
the amino terminus and/or amidation of the carboxyl terminus often 
exhibits an increase in pharmacological activity. 
Bioactive peptides which contain an acetyl group bound to the N-terminus 
and/or an amido function bound to the C-terminus have been found to 
maintain biological activity, but to be less susceptible to acid 
hydrolysis. This is beleived to be due, in part, to the protecting groups 
playing a role in reducing the susceptibility of the protected peptide to 
enzymatic attack and degradation. Therefore, in further embodiments, the 
invention contemplates the use of pharmaceutical preparations of a 
protected peptide(s) comprising the active peptide in combination with 
pharmaceutically acceptable buffers, diluents, stabilizers and the like. 
For a listing of appropriate techniques and suitable pharmaceutical 
agent/additives compositions, one may wish to refer to Remington's 
Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., incorporated 
herein by reference. 
In that it is believed that the monocyte cytotoxicity inducing factor of 
the present invention, and synthetic peptides with MCF-like activity, will 
be clinically useful in a manner similar to that identified for 
interleukin II and the various interferons, methods are additionally 
disclosed for clinical utilization and treatment of disease states using 
MCF. In addition to clinical applicability with respect to tumor 
treatment, as indicated by demonstrated in vitro efficacy in stimulating 
monocyte antitumor activity, it is believed that pharmaceutical 
compositions of the present invention will be useful in the treatment of 
infectious diseases, particularly those infectious diseases wherein the 
causative organisms reside in mononuclear phagocytes, for example, 
tuberculosis and leishmaniasis. 
Additionally, it is believed that compositions included by the present 
invention will find diagnostic utility. Antiserum specific for MCF would 
be of value in determining blood levels of MCF, as well as documenting the 
ability of patients' mononuclear cells to produce MCF. Since MCF is 
produced by T-lymphocytes, and in particular, neoplastic T-lymphocytes, 
MCF will likely serve as a marker for diagnosis and/or evaluation of 
T-cell malignancies. 
With respect to therapeutic utilization of MCF, one treatment protocol 
would include the ex vivo activation of a patient's mononuclear cells for 
reinfusion into the patients in a manner analogous to LAK cells as 
described by Rosenberg et al. (J. Natl. Cancer Inst., 75:595, 1985, and N. 
Eng. J. Med., 313:1485, 1985). For direct delivery of MCF to tissue 
macrophages, it is contemplated that MCF, or peptides with MCF activity, 
may be given by direct transfusion, encapsulated in liposomes, or could be 
incorporated into a viral transvecting particle for use in transduction in 
gene therpay protocols. Such techniques have recently been found to 
increase the efficacy and significantly prolong the half-life of related 
low molecular weight mediators. Liposome encapsulation can be accomplished 
in a number of manners, for example, as described by Fidler et al. (Cancer 
Res., 36:3608, 1976) incorporated herein by reference. 
Additionally, combination therapy employing MCF or peptides with MCF 
activity in combination with interleukin II, interferon, tumor necrosis 
factor and cytoxan, are contemplated. These additional agents may be 
obtained and employed in a manner known in the art as further disclosed 
herein. 
It is further believed that pharmaceutical compositions which include MCF 
or peptides with MCF activity will find utility in direct infusion 
treatment in a manner similar to that utilized for interferon treatment. 
It is believed that dosage determination, as well as proper infusion 
techniques, is well within the skill of the art as exemplified by 
Goldstein et al. (Cancer Res., 46:4315-4329, 1986), incorporated herein by 
reference. Moreover, in that interferons have found applicability in the 
treatment of various infectious disease states as noted above, it is 
believed that such utility will be applicable to MCF as well, for example, 
when employed as suggested by Nathan et al. (J. Exp. Med., 160:600-605, 
1984), incorporated herein by reference. 
Diagnostic procedures, utilizing antibodies specific for MCF, may be 
employed in a manner similar to the current clinical test for acquired 
immune deficiency syndrome. Most conveniently, this would include a 
standard enzyme linked immunosorbent assay (ELISA), well known to those 
skilled in the art.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Introduction 
Concepts of monocyte activation for cytotoxicity have undergone a change 
from the "all or none" paradigm to the more precise concept of a discrete 
stepwise process. Our present understanding of monocyte activation has 
been addressed by Cohn (Jrnl. Immunol., 121:813-816, 1978). Moreover it is 
generally recognized that activation is regulated by products of 
T-lymphocytes. Soluble mediators of mononuclear phagocyte activation have 
recently been reviewed by Nathan et al., supra. 
From these studies and earlier reports, interferon gamma would appear to 
the most important if not the only regulator of cytotoxicity against 
microorganisms and has recently been identified as a migration inhibitory 
factor. However, other studies have suggested that colony stimulating 
factor (CSF I), and the interferons alpha and beta, may have roles in 
regulating secretory or proliferative functions. The present invention is 
directed to the perhaps surprising discovery that an additional factor, 
termed monocyte cytotoxicity inducing factor (MCF) by the present 
inventors also plays a role. In light of the present finding, it is likely 
that additional factors will be identified through the development of the 
individual assays particularly suited to identification of factor 
activities. 
The MCF disclosed by the present invention is characterized biologically by 
an ability to stimulate monocytes to a cytotoxic antitumor state. While 
the precise function of MCF within the biological system is unknown, it is 
believed that it might play a role in affording specific activation of the 
cellular immune system. MCF appears to cause an increase in the synthesis 
and release of IL 1 by monocytes, which may act to either perpetuate the 
cytolytic state, to directly lyse susceptible tumor targets, or to induce 
cytostasis. MCF has no TNF, m-CSF, or IFN-like anti-viral activity. 
Similarly MCF has no activity when tested in the IL 1, IL 2, or m-CSF 
assays. Moreover, MCF was not directly cytotoxic for the targets K562 or 
L929, indicating that MCF has no lymphotoxin-like activity of its own. The 
factor is characterizable as having two molecular weight forms, one about 
29,000 Daltons, and a lower molecular weight form of between 11,500 and 
18,100 Daltons, depending on the particular molecular weight determination 
method utilized. It is emphasized that these two molecular weight forms 
exhibit indistinguishable biological activity in terms of monocyte 
activation. Therefore, the term MCF is meant to relate to either of these 
two molecular weight forms individually or in combination. 
Sensitivity of MCF to the enzymes and metabolic inhibitors described herein 
are consistent with a conclusion that MCF is a peptide. For example, 
responses to metabolic inhibitors (actinomycin-D, puromycin, and 
cycloheximide), suggest that transcription of message is necessary and 
that messenger RNA for MCF does not persist. Because treatment of 
non-lectin-treated cells with puromycin did lead to production of MCF, and 
because total suppression of MCF could not be achieved with cycloheximide 
and puromycin, these data are consistent with either regulation of 
transcription by a repressor protein or its product, or at the level of 
translation, by stabilization of message. 
MCF is most conveniently isolated through the preparation of a T-cell 
hybridoma employing Sezary cells isolated from an individual having 
Sezary's syndrome. Sezary's syndrome is characterized by a proliferation 
of leukemic helper T-cells, and is discussed in some detail by Broder et 
al. (J. Clin. Invest., 58:1297-1306, 1976). Sezary's cells circulate in 
the peripheral blood and are known to provide T-cell help for 
immunoglobulin production. These cells are identified by their 
characteristic cerebriform nuclei, PAS positive vacuoles, and additionally 
by their surface marker phenotype (OKT3+, OKT4+, OKT8-, OKIal-(+)). Helper 
function has been defined by the ability of Sezary cells to stimulate 
polyclonal IgG production by B lymphocytes and by their production of MIF. 
Although lymphokine production does not necessarily segregate exclusively 
to any one subclass of T cells, Sezary's cells most probably represent a 
homogeneous clonal expression of one particular subclass of human 
T-lymphocytes which can be studied for production of lymphokine mediators 
uncontaminated by other T-cells. 
The use of Sezary cells, as noted above, is not crucial to the practice of 
the present invention in that it is believed that MCF production is a 
feature common to lymphocyte populations in general. However, not all 
cells of a particular T-cell population produce MCF. Thus, where a general 
T-cell population is utilized, only a very small percentage of cells are 
likely to produce MCF. In the case of Sezary's cells, it appears as though 
MCF production is a more generalized phenomenon. Therefore, Sezary's cells 
are preferred in that T-cell hybridomas produced from Sezary's cells 
provide clones wherein there is a much greater likelihood that any one 
particular clone would produce MCF. Conversely, it is likely that when 
general T-cell populations are employed for hybridoma production, a 
substantial number of clones will likely have to be screened before a 
positive clone is identified. However, where Sezary's cells are not 
available, one may employ helper T-cells, in that it appears that helper 
T-cell populations contain a sufficient percentage of positive cells to 
avoid the exhaustive screening which would be necessitated by employing 
general T-cell lymphocyte populations. 
Recently, strong evidence has been presented that interferon-gamma is the 
major mediator of macrophage/monocyte activation, and may be identical 
with the lymphokine macrophage activation factor (MAF). Interferon-gamma 
has also been described as the mediator of inhibition of mononuclear 
phagocyte migration and hence may be migration inhibition factor (MIF). 
However, it is known that activated Factor B of the alternative complement 
pathway (Bb), plasminogen activator (PA), and other products of monocytes 
will inhibit migration. Nathan et al., supra, have recently reviewed the 
range of mediators of mononuclear phagocyte activation. 
The production of lymphokines by malignant T-cells is important in 
understanding host defense in conditions with chronic courses such as 
Sezary's syndrome and may reflect production of such mediators by 
non-malignant counterparts in the normal host. In developing the present 
invention, attempts were made to expand Sezary cells with interleukin II 
(IL-2), but these attempts were unsuccessful. Similar results have been 
reported by other groups, and have been attributed to the lack of IL-2 
receptor expression or proliferation of Sezary cells by non-IL-2 dependent 
means. Clearly, as additional cell lines are screened for MCF production, 
cells will be identified which may be immortalized through the inclusion 
of growth factors in the cell growth medium, thus avoiding the need for 
hybridoma development. 
Previously, hybridization has been utilized to perpetuate subclasses of 
murine T-cells and this technique was applied to Sezary's cells for the 
present invention. Other groups have employed Sezary cell hybridomas to 
study BCGF (B-cell growth factor) production by Sezary's cells. In the 
present invention, six cell lines were obtained by hybridization and all 
were capable of inducing human blood monocytes to become cytotoxic for the 
myeloid leukemia target, K562. For further study, one hybrid, FtF3, was 
selected whose phenotype (OKT3+, 4+, 8-, OKII1-, sIgG-) was identical to 
the parent Sezary's cell. It is of interest that all lines produced a 
lymphokine capable of inducing monocyte cytotoxicity (MCF). This probably 
results from positive selection of fusion partners since similar 
hybridization experience using whole murine T-cell populations have given 
rise to much smaller percentages of clones with MAF-like function. 
Supernatants from PHA-stimulated FtF3 induced human mononuclear 
cytotoxicity but contained no detectable interferon. Antisera to native 
interferon-gamma when added in excess to MCF having no antiviral activity 
produced only a 10-15% decrease in specific lysis of the target K562. 
Moreover, when formal titrations of the anti-IFN-gamma antisera were 
carried out using the constant antibody method previously described as 
being optimal for the detection of cross-reactivity with IFN-gamma, no 
neutralization titer could be determined. In contrast, treatment with the 
antisera to interferon-alpha caused a small increase in specific release. 
Addition of both antisera together produced no change in specific lysis. 
Both antisera were produced against partially purified native human 
interferons. In the case of the antisera to interferon-gamma, some 
cross-reactivity may be detected in that an MCF-like molecule could have 
been present in the preparation used to raise the antisera which was 
previously not recognized. 
The development of a series of monoclonal antibodies which are capable of 
differentially blocking antiviral or MAF activity of interferon-gamma, 
respectively, has been described by Schreiber. In particular, monoclonal 
antibodies directed against the C-terminus of recombinant interferon-gamma 
were reported to block MAF but not the antiviral activity of the 
recombinant interferon-gamma, while the antibodies to the N-terminus of 
recombinant interferon-gamma blocked antiviral but not MAF activity. 
Therefore, these findings could be consistent with an interpretation that 
MCF was an altered interferon species produced by a T-cell 
leukemia-derived hybridoma. However, the fact that MCF has recently been 
determined by the present inventors to be produced by normal effector 
T-cells, appears to rebut this theory. 
These issues were further approached by direct comparison of the 
physicochemical properties of MCF and native interferon-gamma. Unlike 
interferon-gamma, MCF was stable at pH 2 but was partially inactivated at 
pH 8 and was much more stable than interferon-gamma. IFN- was inactivated 
by trypsin, in contrast to the effect of this enzyme on MCF. These 
findings would support the concept that MCF is not an altered 
interferon-gamma which has lost antiviral, but not MAF activity, because 
interferon-gamma lost both MCF and antiviral activity with both low pH and 
heat treatment. 
MCF is a lymphokine distinct from TNF, IL 1, IL 2, m-CSF or IFN.MCF has no 
activity when tested in assays for IL 1, IL2 or M-CSF. MCF had no TNF, 
m-CSF, or IFN-like antiviral biological activity when compared to RTNF, 
purified human m-CSF, IFN gamma or alpha/beta, respectively. In addition, 
MCF had no cross reactivity with m-CSF in the radioimmunoassay for m-CSF. 
Gel filtration chromatography of concentrated supernatants from cells 
raised in the presence of serum, revealed two molecular weight species, 
one with a molecular weight of 64,000 daltons which co-eluted with the 
major protein peak, bovine serum albumin, and a second with a molecular 
weight of 11,500 daltons. Although yields from our Bio-Gel P-100 column 
have been low, these values differ from the molecular weight of 50,000 
reported for native human interferon-gamma when chromatographed under 
similar conditions. When such cells were cultured in serum-free media, the 
higher molecular weight species migrates at about 29 kD upon SDS gel 
electrophoresis. 
MCF was found to bind to Matrex Gel Red A (procion-red agarose; Amicon) and 
eluted with 1-N NaCl. Similar results have been reported for native 
interferon-gamma. MCF eluted from Matrex Gel Red A was electrophoresed 
under reducing conditions on a 15% SDS-PAGE gel. Two molecular weight 
species were identified. The first had a molecular weight of approximately 
29,000 daltons when produced in serum-free media and the second had a 
molecular weight of about 14,700 Daltons. The specific activity was 610 
MCF units/mg protein and 1350 MCF units/mg protein, respectively. Yields 
from this procedure have been excellent with recoveries greater than 90%, 
despite treatment with SDS. 
Electrophoresis under reducing conditions followed by dialysis to remove 
SDS has resulted in each case in a diminution of biological activity 
associated with the higher molecular weight form and greater recovery of 
the lower molecular weight form. 
Further and important aspects of this study concern the delineation of 
N-terminal sequence of P29 and the inventor's discovery that synthetic 
peptides corresponding to this sequence, or variants thereof, exhibit MCF 
activity. The N-terminal amino acid sequence of MCF P29 was found to be: 
Gly Ala Ala Val Leu Glu Asp Ser Gln (seq id no:1). A synthetic 9mer 
peptide having this sequence is capable of activating human blood 
monocytes for tumor cytotoxicity to a similar extent as MCF itself. A 
further peptide with the substituted sequence Gly Ala Ala Val Leu Glu Asn 
Ser Gln (seq id no:2), and a shorter peptide with the sequence Leu Glu Asp 
Ser Gln (seq id no:3) also have MCF activity. 
The peptides of the present invention are contemplated to be os use in the 
variety of clinical and diagnostic embodiments proposed for MCF itself. 
The use of peptides in such embodiments is actually preferred for various 
reasons, such as the low cost and relative ease of large scale 
preparation, and the reliability of the product; the ease with which 
peptides can penetrate tissues; and their low immunogenicity. As mentioned 
above, the peptides may be further modified prior to administration by the 
addition of a stabilising group to their N- or C-termini, for example, by 
acylation or amidation. 
It is contemplated that peptides in accordance with the present invention 
may be synthesized by any of the automated methods currently known in the 
art, for example, as disclosed hereinbelow. Alternatively, they may be 
produced as a fusion protein by employing the techniques of genetic 
engineering and expression in host cells. Such techniques are generally 
known in the art and used to produce proteins or peptides linked to 
various proteins, or parts of proteins, such as, for example, 
.beta.-galactosidase or ubiquitin. The fusion proteins may then be 
purified by any of the standard techniques known in the art. The fusion 
proteins may be used themselves, or the peptides released from the 
remainder of the fusion prior to use. The latter technique is particularly 
employed where the genetic construct has been so designed to include a 
protease sensitive site, to allow cleavage by exposure to a free or 
immobilized protease. 
EXAMPLES 
1. Isolation of Sezary's cells 
Mononuclear cells were isolated from 60 ml whole blood from a patient 
previously known to have Sezary's syndrome. Mononuclear cells were 
isolated by centrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, 
N.J.). 7.5.times.10.sup.7 cells having a phenotype of 87% 0KT 3+, 77% 0KT 
4+, and 12% 0KT B+ were recovered from the F/P centrifugation and applied 
to a 35 ml gradient of Percoll (Pharmacia) according to the method of 
Gemlig-Meyling and Waldman (J. Immunol. Method., 33:1, 1980, incorporated 
herein by reference). Briefly, the gradients were made by mixing 168 ml 
Percoll with 144 ml 2.times. PBS, and centrifuged for 40 min at 
21,000.times.g in a Beckman Model 71 centrifuge with a SS-34 fixed angle 
rotor. The cells were suspended in 5 ml of Hank's balanced salt solution 
(HBSS), layered gently on the gradient, centrifuged at 100.times.g for 20 
min at 20.degree. C. in a swinging bucket rotor. Standard density marker 
beads (Pharmacia) were loaded on a companion gradient. 
Three distinct "bands" were recovered from Percoll. At the interface 
between the Percoll and sample were 1.5.times.10.sup.6 cells, the majority 
of which were polymorphonuclear leukocytes, and were discarded. Band I, 2 
cm below the interface contained 1.9.times.10.sup.7 cells which when 
studied by flow microfluorometry (FMF) were 90.2% 0KT 3+, 80% 0KT 4+, and 
47% 0KT 8+, and were morphologically small lymphocytes. Band II, located 5 
cm below the interface contained 7.8.times.10.sup.6 cells, which were 97% 
0KT 3+, 96% 0KT 4+, and 17% 0KT 8+. Microscopically, these cells were 
larger and had cerebriform nuclei. 38% of the total cells applied to the 
gradient were recovered. 
Cells isolated in Band II were washed and resuspended at 5.times.10.sup.5 
cells/ml in RPMI 1640 with 10% fetal calf serum (FCS). Aliquots of these 
cells were cultured with 10 units/ml final concentration of interleukin II 
from the Gibbon ape cell line NLA-144. These cells failed to divide or 
incorporate 3H-Tdr after 72 hrs of culture thus demonstrating a lack of 
IL-2 responsiveness. 
Preparation of 8-azaguanine Resistant T-lymphocytes 
Since these particular Sezary's cells were not capable of indefinite 
sustained growth in tissue culture, it was decided to prepare a continuous 
T-cell hybridoma line by fusing the Sezary's cells with a T-lymphocyte 
population which has been conditioned to grow continuously in tissue 
culture. To achieve hybrid selectability, it was first necessary to select 
a cell population which was sensitive to HAT selection medium. This was 
accomplished through the preparation of an 8-azaguanine-resistant 
T-lymphocyte population. 
It has been determined that certain commercially available T-lymphocyte 
cell lines are to be preferred over others. CCRF-CEM is a cell line 
obtainable from the ATCC which, due to its apparent genetic stability, is 
to be preferred in generating hybridomas of the present invention. Two 
additional cell lines, MOLT and Jurkat, have been found to be genetically 
unstable upon drug selection and therefore unsuitable. However, the 
techniques of the present invention are applicable to any genetically 
stable T-cell lymphocyte or other cell type which can form a stable fusion 
product with T-cells and which is capable of continuous growth in culture. 
Determination of the foregoing criteria is within the skill of the art and 
the scope of the present disclosure is not limited to the preferred 
embodiment employing CCRF-CEM. 
To generate 8-azaguanine resistant CCRF-CEM, cells were obtained from the 
American Type Culture Collection and grown in RPMI 1640 with 10% FCS. CEM 
was grown in increasing concentrations of 8-azaguanine primarily according 
to the method of Okada (Proc. Natl. Acad. Sci., 78:7717, 1981, 
incorporated herein by reference). However, beginning with a concentration 
of 2 uM, the dose was doubled every 2 days until a dose of 16 uM was 
reached at which time the dose was doubled every 10 days until a dose of 
100 uM was reached. After 6 weeks the cells were recloned and tested for 
their ability to grow in HAT containing media (hypoxanthine, aminopterin, 
and thymidine), and subjected to flow microfluorometry. 
3. Generation of T-cell Hybridomas 
T-cell hybridomas were formed between Sezary cells, isolated as described 
in section 1 above, and the 8- azaguanine-resistant, HAT-sensitive CEM 
T-lymphocyte line (CEM.8aza.sup.r.C), prepared as described in section 2 
above. In particular, 7.5.times.10.sup.6 cells from Band II of the Percoll 
gradient fractionation of patient's mononuclear cells were subjected to 
flow microfluorometry and light microscopic examination. These cells were 
hybridized to an equal number of CEM.8aza.sup.r.C using polyethylene 
glycol (mol wt 1000, Sigma, St. Louis, Mo.) as described by Jones, C. M., 
"T-cell Hybridomas Producing Macrophage Activation Factors," In: T Cell 
Hybridomas, Ed.: M. Taussig, CRC Press., Inc., Boca Raton, Fla., 1985, pp. 
56-68, incorporated herein by reference. The fused cells were cultured for 
24 hrs in RPMI 1640 with 10% FCS prior to addition of HAT-containing 
medium (hypoxanthine 1.times.10.sup.-4 M, aminopterin 4.times.10.sup.-5 M, 
and thymidine 1.6.times.10.sup.-5 M). Colonies were selected after 1 month 
in HAT media and cloned by limiting dilution. 
Individual hybridoma clone colonies which were isolated by this procedure 
were adjusted to a culture density of 5.times.10.sup.5 cells/ml and 
stimulated with 16 ug/ml phytohemagglutinin (PHA; Miles-Yeda, Rehovot, 
Israel) for 24 hours to stimulate lymphokine production. Thus, hybridoma 
clones were screened for positive MCF production by mitogen stimulation 
followed by subjecting the resultant hybridoma supernatants to biological 
screening in the human monocyte cytotoxicity assay, which is discussed in 
detail below. 
The human T-cell hybridoma was also successfully maintained in 5% fetal 
calf serum (FCS), then grown in serum free media for at least 24 hours 
after stimulation by phytohemagglutinin (PHA). Either tissue culture media 
not supplemented by serum, or a serum-free media described by Sachs, L., 
Clin. Exp. Immunol. 33:495, 1978 and incorporated herein by reference, 
will support this growth for a limited time span. This cell growth 
procedure permits analysis of MCF prepared in serum-free conditions. 
In particular, supernatants of stimulated and unstimulated clones were 
incubated 20 hours with the monocyte monolayer. Serial dilutions of the 
supernatant were made to quantitate MCF in each sample. The data in FIG. 1 
show that all six clones produce a factor(s) which induces human monocytes 
to kill K562. Their phenotypes were studied by FMF and two clones, FtF3 
and FtA5, were uniformly 0KT 3+, 4+, and were 0KT 8- and OKMT 1-, and 
IgG-. 
Table 1 demonstrates the results obtained when supernatants from two 
representative MCF-producing hybridomas, FtA5 and FtF3, were subjected to 
the in vitro monocyte cytotoxicity assay. Cytotoxicity was measured by 
adding a fixed input of supernatant (25, 50 and 100 vl.) to a total test 
volume of 0.2 ml. 
TABLE I 
______________________________________ 
Specific Cytotoxicity Induced 
By Hybridoma Supernatants 
Hybrid Input (ul) % Specific Lysis 
______________________________________ 
FtA5 25 10.3 
50 25.1 .+-. 6.9 
100 38.3 .+-. 8.4 
FtF3 25 11.1 
50 26.2 .+-. 10.4 
100 37.7 .+-. 6.8 
______________________________________ 
4. Human Monocyte Cytotoxicity Assay 
As noted above, successful practice of the present invention rests on the 
ability to successfully identify MCF activity in an in vitro assay which 
has been designed to distinguish MCF activity from the numerous other 
lymphokine activities produced by T-cells. The assay described below has 
been derived in part from an assay method reported by Koren and his 
associates (Fischer et al., Cell Immunol., 58:426, 1981). However, the 
assay as described in the Fischer et al. reference was found to be 
unacceptable in that it is not designed to measure lymphokine induced 
cytotoxicity and does not eliminate natural killer cell activity. It, 
therefore, must be modified as follows. 
Human monocyte enriched leukopaks were obtained as a byproduct of the 
platelet donor program at M. D. Anderson Hospital, and were prepared with 
an IBM Model 2997 Cell Separator. Only healthy volunteer donors were used. 
All volunteer donors signed an informed consent, and the protocol was 
approved by the Committee for the Protection of Human Subjects, University 
of Texas Health Science Center. Mononuclear cells were prepared by 
centrifugation over Ficoll/Hypaque. Monocyte monolayers were prepared by 
adherence to 96-well flatbottom plates previously coated with human serum 
as described by Golightly et al. (Blood, 61:390, 1983, incorporated herein 
by reference), and allowed to adhere to the serum coated plates for 15-30 
mins followed by vigorous washing with warm (37.degree. C.) Hank's 
balanced salt solution. This resulted in a confluent monolayer of &gt;95% 
esterase positive cells. The monolayer was incubated overnight to allow 
for decay of residual natural killer activity before addition of 
lymphokine preparations on day 2. The monolayers were incubated with 
lymphokine for 20 hrs, washed, and the .sup.111 In-Ox labeled K562 added 
as targets. 
The .sup.111 In-Ox labeled K562 target cells were prepared by the method of 
Wiltrout et al. (in: Manual of Macrophage Methodology, Ed. H. B. 
Herscowitz et al., Marcel Dekker, Inc., N.Y., pp 337-344, 1981, 
incorporated herein by reference). The effector to target ratio was 30:1. 
Spontaneous release averaged 15% (7-20%) in greater than 50 experiments. 
LPS (Lipopolysaccharide) free RPMI 1640 (M. A. Bioproducts, Walkersville, 
Md.) with 10% heatinactivated AB-negative human serum (FLOW Laboratories, 
Arlington, Va.) was used throughout the assay procedure. After 18 hrs 
incubation of monocyte monolayer with target, the plates were centrifuged 
and supernatant was removed and cpms were counted in a Beckman Biogamma 
2000. Specific release was calculated as described by Wiltrout, supra. 
Units of MCF activity were calculated as described by Lohmann-Matthes 
(Kniep et al., J. Immunol., 127:417, 1981). 20% specific release equals 1 
unit of MCF. Other tumor targets used were HL-60, L5178Y and TU5 (Table 
V). 
The primary distinction between the present assay and the one described by 
Fischer et al., is the finding that it is absolutely crucial that LPS-free 
media be utilized in order to distinguish the MCF activity from 
interfering activities. For the particular activity investigated by 
Fischer et al., the use of LPS-free media was not crucial in that it is 
noted by those authors that similar activities were observed regardless of 
whether LPS-free media was utilized. However, with respect to MCF, when 
LPS-containing media is used, a spontaneous release of label occurs and 
induction of lymphokine (MCF) directed cytotoxicity cannot be measured. 
5. Deposit of Representative Sezary cell Hybridomas with the American Type 
Culture Collection 
By the foregoing procedures, six hybridoma clones were identified whose 
supernatants exhibited MCF activity. Two were chosen for further 
physiochemical and biological characterization. It has been determined 
that those two hybridomas, designated Ft.A5 and Ft. F3, both secrete the 
same MCF biological profile, as determined by the characterization 
criteria disclosed herein. Accordingly, one of these hybridomas, Ft. F3, 
has been deposited with the ATCC and accorded ATCC reference number 
HB9713. 
6. Characterization of MCF 
A. MCF is Antigenically Distinct From Interferon Gamma 
1) Generation of MCF 
MCF was generated by stimulating 5.times.10.sup.5, 1.times.10.sup.6, 
2.5.times.10.sup.6, or 5.times.10.sup.6 FtF3 or FtA5 cells/ml with 2, 4, 
8, 16, or 32 ug/ml PHA (Miles-Yeda, Rehovot, Israel) in RPMI 1640 with 1, 
2.5, 5, or 10% FCS (Hyclone, Sterile Systems, Logan, Utah) for 24, 48, or 
72 hrs. Unstimulated controls were grown with each to which an equal 
amount of PHA was added at the end of incubation. The cells were 
centrifuged, and the supernatants filter-sterilized, and stored at 
-30.degree. C. For characterization and purification, MCF was then 
prepared by stimulating 1.times.10.sup.6 cells/ml in RPMI 1640, 1% FCS 
with 8 ug/ml PHA for 24 hrs, centrifuged, and filter sterilized. This 
routinely gave approximately 40 U/ml activity. Unstimulated controls to 
which PHA was added back were used in all experiments. 
MCF was also generated in serum-free media. FtF3 cells were first grown in 
RPMI 1640 containing 5% FCS. Cells were then washed twice with Hanks 
balanced salt solution (HBSS) and once with RPMI 1640. Cells were 
resuspended at a concentration of 1.times.10.sup.6 /ml or 3.times.10.sup.6 
/ml in either RPMI 1640 containing 10, 5, 1, or 0.1% heat-inactivated, 
AB-negative human serum, RPMI 1640 alone, or a serum-free media described 
by Sachs (Clin. Exp. Immunol., 33:495, 1978, incorporated herein by 
reference). Phytohemagglutinin (PHA) was added to a final concentration of 
0, 0.5, 1.0, 2.0 or 4.0 micrograms/mi. Supernatants were collected at 24 
and 48 hours, filter sterilized, and stored at -70.degree. C. Satisfactory 
growth could only be maintained in RPMI 1640 having at least 5% fetal calf 
serum. However, after FtF3 had been conditioned to grow in media having 5% 
FCS, this cell line was capable of producing MCF under serum-free 
conditions as described herein. The total units of MCF recovered were 
scarcely different among the serum-containing medias or RPMI alone. The 
serum-free media of Sachs resulted in a decrease in MCF production of 5 to 
10 U/ml. In addition, an increase in cell concentration from 
1.times.10.sup.6 cells/ml to 3.times.10.sup.6 cells/ml did not increase 
levels of MCF production which is likely the result of decreased viability 
at higher cell density. 
2) Interferon Assay 
Human interferon activity was measured as inhibition of plaque formation by 
Sindbis virus on WISH cells as described by Baron et al. (Infect. Immun., 
32:449, 1981). Sindbis virus, human alpha, beta, and gamma interferons 
were prepared by Drs. Samuel Baron and Marlyn Langford, University of 
Texas Medical Branch, Galveston, Tex. WISH cells were obtained from Drs. 
Baron and Langford. Using this plaque inhibition assay, interferon was not 
demonstrated in supernatants of FtF3 and FtA5. 
3) Treatment of IFN-gamma and MCF With Antisera to Various Interferons 
Antibody to a partially purified preparation of native human gamma 
interferon (Langford et al., J. Immunol., 126:1620, 1981), a 20 peptide 
N-terminal fragment of recombinant gamma interferon (Johnson et al., J. 
Immunol., 129:2357, 1982) and human alpha interferon (Langford et al., 
supra) were prepared as described in the referenced articles. In initial 
experiments, 40 Units of MCF or 100 U IFN-gamma in 1 ml RPMI 1640, 1% FCS 
were incubated with 100 U of each of the antisera above alone or in 
combination at 4.degree. C. for 30 min, 1 hr, and 4 hr. Following this 
incubation, serial dilutions of the IFN or MCF were made in RPMI 1640, 10% 
FCS and residual macrophage activating factor activity measured in the MCF 
assay. 
27 U/ml of partially purified MCF were diluted 1/2 (13.5 U), 1/4 (6.75 U), 
and 1/8 (3.375 U). Serial half-log dilutions were made of these dilute 
MCF, and the predicted number of units were confirmed by measurement in 
the MCF bioassay. Next, 27 U/ml of partially purified MCF were subject to 
serial half-log dilutions up to a final dilution of 1/8. 100 U, 50 U, or 
25 U of each of the anti-IFN-gamma, antisera was added to each of these 
MCF dilutions and incubated for 1 hr at 25.degree. C. Residual MCF 
activity was calculated in the constant antibody titration as recommended 
by Kawade (J. Ifn. Res., 4:571-584, 1984). Units are expressed as 
described by Lohmann-Matthes (Kniep et al., supra). 
Results from two representative sets of experiments are summarized in Table 
II. Anti-interferon-alpha produced no significant change in specific 
release. On the contrary, the presence of this antisera during the 
activation has consistently produced small increases in specific lysis. 
The antibody to a partially purified preparation of native human 
interferon-gamma (SEA-activated human PBL) produced a decrease of only 15% 
and 10% specific lysis. Antisera to the 20-peptide N-terminal fragment of 
recombinant interferon-gamma or to a combination of interferons alpha and 
gamma failed to neutralize MCF. The antisera themselves produced only 
small changes in spontaneous release of label from the target K562 when 
added in place of the activating agent (spontaneous release=22.6% with 
media and monocytes alone; specific release=-13.2% with anti-IFN-alpha, 
=4.66% with anti-IFN-gamma native, and =8.4% with anti-IFN-gamma 
N-terminus.) More importantly, IFN-gamma in amounts up to 1000 U/ml final 
concentration increased specific lysis only 6.2%. Because IFN-gamma did 
not produce significant activation for cytotoxicity in the present assay, 
direct comparison with MCF could not be performed. 
TABLE II 
______________________________________ 
Treatment of MCF with Antisera 
% Specific Lysis 
Treatment Exp 1 Exp 2 
______________________________________ 
untreated 54.6 .+-. 3.8 
29.8 .+-. 5.4 
anti-IFN-alpha 57.6 .+-. 5.0 
35.8 .+-. 4.2 
anti-IFN-gamma (native) 
40.6 .+-. 3.8 
19.9 .+-. 8.5 
anti-IFN-gamma N-terminus 26.5 .+-. 8.0 
anti-IFN-gamma (native) + 
48.89 .+-. 5.3 
anti-IFN-alpha 
______________________________________ 
In order to confirm that increasing concentrations of antibody relative to 
MCF units would not affect induction of cytotoxicity, the following 
experiments were performed. Serial checkerboard dilutions of 27 U/ml 
partially purified MCF were carried out as described above. In each 
experiment, 100, 50, or 25 U of the anti-IFN-gamma antibodies were added 
separately to MCF dilutions and residual MCF units were measured. The 
results summarized in Table III demonstrate that 100 U anti-IFN-gamma 
native reduced total bioassayable units by 7 U/ml. This is consistent with 
preceding experiments. Addition of 50 U/ml anti-IFN-gamma (native) and all 
inputs of anti-IFN-gamma N-terminus caused an increase in bioassayable 
units. 
TABLE III 
______________________________________ 
Treatment of MCF with Anti-IFN-gamma 
by the Constant-Antibody Technique 
anti-IFN anti-IFN 
Input Ab (U/ml) 
gamma (native) 
gamma-N-terminus 
______________________________________ 
0 27 27 
25 28 39 
50 58 46 
100 20 40 
______________________________________ 
FtF3 and FtA5 MCF were also capable of activating murine peritoneal exudate 
macrophages for cytotoxicity against the target L5178Y. The presence of 
LPS did not appear to augment cytotoxicity induced by these lymphokines. 
B. MCF is Distinct from IL 1, IL 2, TNF and m-CSF 
1) Measurement of IL 1, IL 2, TNF and CSF Biological Activity 
IL 1 activity was measured using the DlO.G4.1. cell line as described by 
Kaye and Janeway (J. Immunol., 133:2291, 1984). IL 2 activity was measured 
as described by Bonnard, using cultured human T-cells (Cell. Immunol., 
51:390, 1980). TNF activity was measured by direct cytotoxicity against 
L929 cells as described by Gately and Mayer (J. Immunol., 116:669, 1976). 
M-CSF was measured by both murine bone marrow colony formation (Waheed and 
Shadduck, Exp. Hematol., 17:61, 1989; Shadduck and Waheed, Ann. N.Y. Acad. 
Sci., 554:156, 1989) and by radioimmunoassay specific for purified human 
M-CSF. 
2) Addition of Lymphokines to MCF Assay 
Purified human IL 1 (alpha plus beta), rIL la, rIL lb, (Cistron 
Biotechnology, Pinebrook, N.J., USA), rIL 2 (Genzyme Corp., Boston, Mass., 
USA), RGM-CSF, or purified m-CSF (Waheed and Shadduck, supra), was added 
to the human monocyte cytotoxicity assay in order to determine the ability 
of lymphokines other than MCF to activate monocytes for tumor 
cytotoxicity. Antibody to a partially purified preparation of native human 
IFN-gamma, a 20 peptide n-terminal fragment of rIFN-gamma, and human 
IFN-alpha were used, and neutralization was carried out using the constant 
antibody method as previously described by Jones et al. (J. Immunol., 
137:571, 1986). Anti-serum to purified M-CSF, capable of neutralizing 0, 
100 and 1,000 units/ml was added to a preparation of human MCF containing 
33 U/ml. These were incubated one hour at room temperature. Resultant 
supernatants were tested in the MCF biological assay. 
Using K562 as a cytotoxicity assay as previously determined, IFN-alpha/beta 
had no, and IFN-gamma only slight activity. Neither IL 1 or IL 2 had any 
activity in the MCF assay. Anti-sera to m-CSF failed to neutralize MCF 
activity. The CSF's had effects different from MCF. They caused monocyte 
cell division, altering the effector to target ratio and causing cells to 
assume a rounded morphology. MCFs by contrast did not cause cell division, 
but induced cytotoxicity and caused the cells to assume a macrophage-like 
morphology. The details of the effects of cytokines or lymphokines other 
than MCF, in the MCF assay, are shown in Table IV. 
3) MCF Activity is Not Dependent Upon Natural Killer Cells (NK) 
In order to remove human NK cells from whole human peripheral blood 
mononuclear cells, anti-Leu-11B was used (Becton Dickinson, Mountain View, 
Calif., USA) (Itch et al., J. Immunol., 134:802, 1985). Human monocyte 
monolayers were treated with anti-Leu-11B and subjected to either 
activation with MCF for measurement of cytotoxicity, or staining with 
trypan blue for viability. Human monocyte monolayers treated with 
anti-Leu-11B plus complement and activated with MCF did not diminish MCF 
mediated cytolysis, nor was viability at the monocyte monolayers decreased 
when compared to control monolayers. Monocyte monolayers were activated 
with either crude MCF (23.8 U/ml) or MCF prepared by Matrex Gel Red A 
chromatography (83.3 U/ml). Cytotoxicity was measured using K562, HL60, 
L5178Y or TU5. Specific release of MCF was comparable with both 
NK-sensitive and NK-resistant (TU5 and L5178Y) cells. (Table V) 
TABLE IV 
__________________________________________________________________________ 
Effect of other cytokines in the MCF assay 
__________________________________________________________________________ 
Cytokine 
U/ml % specific lysis 
MCF U/ml 
Specific antisera treatment of 
__________________________________________________________________________ 
MCF 
IFN-.alpha. 
1000 -8.9 .+-. 8.8 
18.1 .+-. 2.6 
.alpha.IFN-.alpha.input 
Exp. 1 
Exp. 2 
100 -2.1 .+-. 12.4 % specific lysis 
10 -5.4 .+-. 9.2 0 U/ml 
54.6 .+-. 3.8 
29.8 .+-. 5.4 
1 4.5 .+-. 3.0 100 57.6 .+-. 5.0 
35.8 .+-. 4.2 
.alpha.IFN-.gamma. input 
Native 
N-terminus 
U/ml U/ml 
IFN-.gamma. 
1000 6.2 .+-. 4.4 
40.0 .+-. 2.9 
0 27 27 
100 3.4 .+-. 3.2 25 28 39 
10 -3.5 .+-. 1.1 50 58 46 
1 - 2.2 .+-. 1.9 
100 20 40 
eIL1.alpha. 
10 -0.7 .+-. 7.6 
24.4 .+-. 6.5 
1 -3.6 .+-. 5.6 
0.1 -4.1 .+-. 4.1 
rIL1.beta. 
10 -1.8 .+-. 9.6 
24.4 .+-. 6.5 
1 -4.9 .+-. 4.6 
0.1 -8.7 .+-. 2.8 
pIl1 (.alpha. + .beta.) 
100 -10.2 .+-. 4.0 
18.1 .+-. 2.6 
10 -5.2 .+-. 2.7 
1 -5.4 .+-. 7.9 
0.1 -10.9 .+-. 3.3 
rIL2 1000 2.7 .+-. 6.2 
20.0 .+-. 2.0 
100 -3.2 .+-. 3.5 
10 -4.6 .+-. 4.3 
1 -10.2 .+-. 17.7 
GM-CSF 200 -0.6 .+-. 4.9 
20.0 .+-. 4.4 
100 5.7 .+-. 3.1 
50 -2.5 .+-. 15.9 
am-CSF input 
MCF activity 
U/ml U/ml 
m-CSF 1730 -3.9 .+-. 4.7 
50.0 .+-. 3.9 
0 33.3 
173 14.5 .+-. 4.6 
100 40.0 
17.3 26.0 .+-. 6.5 
1000 50.0 
__________________________________________________________________________ 
Various cytokines were added to the MCF bioassay. Specific antisera were 
added to MCFcontaining supernatants to determine whether any MCF activity 
could be neutralized by antisera to other cytokines. 
TABLE V 
______________________________________ 
MCF-induced cytotoxicity against tumor targets 
Target MCF (units/ml) 
______________________________________ 
crude MCF: 
K562 23.8 .+-. 9.1 
HL-60 23.5 .+-. 1.5 
partially purified MCF: 
K562 83.3 .+-. 12.3 
L5178Y 78.4 .+-. 8.4 
TU5 100.5 .+-. 26.8 
______________________________________ 
Monocyte monolayers were activated with either crude MCF or MCF prepared 
over Matrex Gel Red A for 24 h before the addition of targets. 
Units were calculated as the reciprocal of the dilution giving 20% 
specific lysis. 
These are the results of 3 experiments run in quadruplicate. 
TNF could not be demonstrated in supernatants collected from human 
monocytes incubated with 20 U/ml MCF for 20 h, washed and incubated for 24 
h (dilution of 1:10). However, IL 1 was present at concentrations of up to 
100 U/ml in MCF-activated monocyte supernatant. 
C. Response of MCF to Enzyme Treatment 
In order to treat MCF supernatants with enzymes, 10 ml of the supernatant 
(28.5 units/ml) were treated with 1 mg/ml trypsin at pH 7.4, 1 mg/ml 
chymotrypsin at pH 7.4, 0.5 mg/ml DNase at pH 7.4, or 40 units/ml RNase at 
pH 5 for 1 hour at 25.degree. C. The result of treating MCF with 
chymotrypsin was that there was a reduction in the biological activity of 
MCF by 54.4%. Trypsin, RNAse and DNAse had no significant effect. The 
experiments were repeated using insolubilized enzymes to minimize the 
possibility that enzymes could be carried over into the bioassay and could 
explain the results. However, the results were entirely comparable to 
those using soluble enzymes. Chymotrypsin reduced activity from 28.5 U/ml 
to 12.5 units, whereas trypsin RNAse and DNAse treatment showed no 
significant differences after one hour of treatment, as measured by 
bioassay for MCF and determined by students T test. 
D. Effects of Tunicamycin and Other Agents on MCF 
Urea treatment was accomplished by adding 3 grams of solid urea to 10 ml 
MCF (28.5 U/ml) (5M urea final). Extraction of MCF with 
butanol-diisopropyl ether was performed. FtF3 cells were cultured with 0, 
1, 2.5 or 5.0 ug/ml tunicamycin. Urea decreased biological activity in the 
MCF containing supernatants by 35.7%. MCF supernatants were extracted with 
butanol-diisopropyl ether; the aqueous phase contained 36.7% less MCF than 
the control preparation. Biosynthesis in the presence of either 
2-mercaptoethanol or tunicamycin resulted in no significant change in 
biological activity of MCF. 
E. Effects of Metabolic Inhibitors on MCF 
FtF3 cells at a concentration of 1.times.10.sup.6 /ml in RPMI 1640/10% FCS 
were incubated 4 hours with either actinomycin D, cycloheximide, or 
puromycin (all at 0 to 100 ug/ml, Sigma). Tests for viability after 
treatment were performed by trypan blue exclusion and supernatants were 
collected by centrifugation. In a second set of experiments FtF3 cells at 
the same concentration but using 0.1% human serum were incubated for 5 
hours with either cycloheximide or puromycin. Uptake experiments were 
performed by adjusting FtF3 cells to 1.times.10.sup.6 /ml in media after 
treatment with inhibitor, and adding either 5,6-.sup.3 H-uridine (ICN 
Radiochemicals, Irvine, Calif., USA, 49,Ci/mmol) to actinomycin D treated 
cells or .sup.3 H L-amino acid mixture (25.5 mci/mg) to 
cycloheximide-treated cells at 1 uCi to 1.times.10.sup.5 cells. Cells were 
subsequently lysed and counted by liquid scintillation. 
When MCF was grown in the presence of the metabolic inhibitors, actinomycin 
D totally suppressed production of MCF in a dose-dependent manner 
accompanied by a corresponding fall in .sup.3 H-uridine uptake (FIG. 2). 
Cycloheximide suppressed but did not abolish MCF production (FIG. 3). At 
intermediate doses (25 and 50 ug/ml) some escape from suppression was 
noted. Puromycin, like cycloheximide, suppressed but did not totally 
abolish MCF production under these conditions (FIG. 4). A corresponding 
pattern was observed using tritiated amino acids and examining the pattern 
of incorporation in TCA insoluble material. 
Actinomycin D had no effect, and cycloheximide had only a slight 
stimulatory effect, on MCF production of nonstimulated (non-PHA treated) 
cells. Puromycin at doses of 10-50 ug/ml appeared to stimulate production 
of MCF by FtF3 cells not activated by lectin. Cycloheximide and puromycin 
therefore provide reversible inhibition. When inhibitor was present for 
the entire incubation period, lectin-induced MCF production is not 
suppressed by cycloheximide but puromycin was suppressive in a 
dose-dependent manner. 
F. MCF Behavior in other Bioassays 
The biological activity of MCF was checked in other bioassays to probe the 
issue of multiple biological activities. Supernatants from FtF3 containing 
25 U/ml of MCF activity were substituted for IL 1 and IL 2 in their 
respective bioassays. MCF demonstrated no IL 1 or IL 2 activity. MCF had 
no TNF, M-CSF, or IFN-like antiviral biological activity when compared to 
RTNF, purified human m-CSF, IFN-gamma or alpha/beta respectively. 
G. Physicochemical Characterization of MCF 
1) pH Stability 
pH stability of MCF was compared to IFN-gamma by dialyzing 40 U MCF or 100 
U IFN-gamma in Spectropor tubing (molecular weight cutoff of 
5.times.10.sup.3 Daltons) against 0.1M glycine-HCl, pH 2.0, 0.1M TRIS-HCl, 
pH 5.0, 0.15M PBS, pH 7.4, 0.15M PBS, pH 8.0, or O.lM TRIS, pH 10.0 for 4 
hrs at 25.degree. C. The samples were then dialyzed against Dulbecco's 
PBS, pH 7.4, to restore neutrality prior to bioassay. IFN-gamma was found 
to be totally inactivated at pH 2. However, as demonstrated by Table VI, 
MCF was found to be stable at pH 2. However, partial inactivation of MCF 
has consistently been noted at pH 8.0. 
TABLE VI 
______________________________________ 
pH Stability of MCF 
pH % Specific Lysis 
______________________________________ 
2 41.7 .+-. 8.6 
5 41.1 .+-. 6.4 
7.4 36.8 .+-. 3.2 
8 19.6 .+-. 4.6 
10 45.4 .+-. 5.4 
______________________________________ 
2) Heat Stability of MCF 
To test heat stability of MCF relative to IFN-gamma, 40 units of MCF and 
100 U IFN-gamma in 1 ml RPMI 1640/1% FCS were heated for 2 hrs in a 
constant temperature bath to study heat denaturization. As demonstrated by 
Table VII, MCF was stable at temperatures up 60.degree. C., but was 
totally inactivated at 100.degree. C. IFN-gamma was not stable at 
temperatures higher than 4.degree. C. for periods of 2 hrs or longer. 
TABLE VII 
______________________________________ 
Heat Stability of MCF 
Temperature % Specific Lysis 
______________________________________ 
4.degree. 53.3 .+-. 4.3 
37.degree. 48.6 .+-. 4.9 
60.degree. 48.0 .+-. 3.4 
100.degree. 0.0 
______________________________________ 
7. Preliminary Purification of MCF 
A. Gel Filtration of MCF 
Gel filtration experiments were performed to give an indication of 
approximate molecular weight. 180 ml of MCF from PHA stimulated FtF3 
supernatant (7.2.times.10.sup.3 U MCF) was concentrated 20-fold by 
pressure dialysis over an Anicon YM-10 membrane, applied to a 2.5.times.60 
cm. column of Bio-Gel P100, equilibrated with PBS and eluted at a flow 
rate of 1.5 ml/min. 7.5 ml fractions were collected and were assayed 
undiluted, 1/2, and 1/5 for MCF activity. The column was calibrated with 
aldolase (158K), ovalbumin (45K), chymotrypsin (25K), and ribonuclease A 
(13.7K) (Pharmacia). 
The data in FIG. 5 demonstrated that two peaks of biological activity were 
obtained. The first peak co-eluted with the major protein present, bovine 
serum albumin (from the fetal calf serum present in the culture media). 
The second peak of monocyte cytotoxicity inducing activity eluted in a 
region with an apparent molecular weight of approximately 11,500 Daltons. 
B. Precipitation with Ammonium Sulfate 
Initially, ammonium sulfate precipitation was attempted as a method for 
purification and concentration. MCF appeared to precipitate in the 30-50% 
range but resulted in greater than an 85% loss in biological activity. 
Therefore, ammonium sulfate precipitation was not pursued further as a 
means for purification. 
C. Binding of MCF to Matrex Gel Resins 
Two ml of each of the Matrex Gel Resins (Amicon) was washed with 5M Urea 
and then washed with 20 ml PBS in 1.5M NaCl, pH 7.4. Four ml of FtF3 
supernatant (160 U MCF in RPMI 1640, 1%FCS) was passed twice over each of 
the Matrex Gel Resins (Blue A, Red A, Orange A, Green A, and Blue B), 
washed with 2 volumes of starting buffer, and eluted stepwise with 1 
volume each 0.5M NaCl. 1.OM NaCl, and 2 volumes 1.OM NaCl/ 50% ethylene 
glycol. The starting material, wash, and each fraction was then dialyzed 
at 4.degree. C. for 24 hrs (Spectrapor tubing, 5.times.10.sup.3 D 
molecular weight cut off) against 3 changes of PBS, pH 7.4, and then 4 hrs 
against distilled H.sub.2 O. The samples were placed in a glass tray and 
covered with Sephadex G-10 to reduce volume to approximately 1 ml. Each 
sample was then assayed for units MCF activity. 
MCF was bound and could not be eluted from Matrex Gels Orange A, Green A, 
and Blue B. However as summarized in FIG. 6, MCF bound to both Blue A, and 
Red A. Red A bound 31.3% of the starting material and eluted with 1.OM 
NaCl. Blue A bound only 10.5% of the starting and was not studied further. 
8. Further Purification of MCF & Constituent Polypeptides 
A. Matrex Gel Red A Chromatography 
Ft. F3 serum-free supernatant was diluted 1:1 with distilled H.sub.2 O 
before application to a Matrex Gel Red A column (50.times.2.5 cm), 
equilibrated with 20 mM phosphate-buffered saline (PBS)/0.15N NaCl, pH 
7.4. The column was eluted with a 0-1N NaCl step gradient. Fractions were 
tested for biological activity, and protein was quantitated by comparison 
with ovalbumin silver-stained standards in a 15% SDS-PAGE gel. Fractions 
containing biological activity were pooled and dialyzed against distilled 
H.sub.2 O until the conductivity fell below that of the following starting 
buffer. This purification step resulted in a recovery of 90% of the 
biological activity in less than 10% of the starting protein. 
B. Ion-Exchange Chromatography 
The dialyzed fractions from the Matrex Gel Red A chromatography step were 
applied to a 1.5- .times.25-cm diethylaminoethyl (DEAE) cellulose column 
equilibrated with 0.02M Tris, pH 8.4, and eluted with a 0-0.5 N Nacl/0.02M 
Tris gradient at a flow rate of 2 ml/min. Biological activity and protein 
were measured in the same manner as for the dye ligand column. The peak 
biologically active fractions, discarding the trailing fractions, were 
pooled, dialyzed against distilled H.sub.2 O, and lyophilized to dryness. 
Only a single 29kD protein band prove these pooled fractions can be 
visualized on a silver-strained gel (FIG. 7). However, when companion gels 
were cut into 1 cm segments and the proteins were eluted from the gel, 
biological activity was found to be present in fractions corresponding to 
molecular weights of both 29kD and 14.7kD. 
These DEAE-recovered fractions were used as the starting materials for the 
further purification of the individual polypeptides P29 and P14.7 
C. Polyacrylamide Gel Electrophoresis (SDS-PAGE) to Prepare MCF P29 
A sample of the MCF-containing lyophilized material obtained following DEAE 
chromatography was subjected to preparative SDS PAGE. The sample was mixed 
with sample preparation buffer (0.0625 ml TRIS-HCl, 2% SDS, 5% 2ME, 10% 
sucrose, and 0.002% bromphenol blue), heated 3 min at 100.degree. C. and 
centrifuged at high speed (1 min in an Adams microfuge). 100 .mu.l 
aliquots were loaded into each of 4 lanes of a 2 mm thick preparative SDS 
slab gel (BRL vertical gel apparatus, Gaithersburg, Md.) using a 15% 
polyacrylamide gel, prepared according to Laemmli (Nature, 227:680, 1970), 
and run at 90 V through the stacking gel and 200 V through the running 
gel. The gel was cut into 1 cm slices, crushed, eluted with 2 washes of 5 
ml of PBS/0.1% SDS pH 7.4, for 12 hours at 4.degree. C., and the 
biological activity of the eluted material was determined. Pharmacia low 
molecular weight standards prepared in the same buffer and run in 
companion lanes were used to determine molecular weight: ovalbumin (43kD), 
.alpha.-chymotrypsinogen (25.7kD), oval-a-lactoglobulin (18.4kD), lysozyme 
(14.3kD), bovine trypsin inhibitor (6.2kD), and insulin A or B chain, 
(2.3kD and 3.4kD). A companion gel was divided and stained with 1) 
Coomasie Brilliant Blue and 2) by a silver nitrate method (Oakley et al., 
Anal. Biochem., 105:361, 1980). 
Preparative SDS PAGE and elution in this manner was found to be 
particularly suitable for the preparation of P29 polypeptide for further 
analysis. However, as mentioned above, elution of P29 preparative PAGE 
gels in the region of 14.7 kd demonstrated that MCF biological activity 
was present despite the inability to visualize protein employing either 
silver staining of the primary gel or colloidal gold (Aurodye) staining of 
the resultant Western blots. This tends to indicate that P14.7 may not be 
present in a high enough concentration to allow detection, perhaps partly 
because some loss of protein occurs during the blotting and washing 
procedures themselves. Alternatively blotting to nitrocellulose may have 
altered its antigenic reactivity during the processes of denaturation and 
rebinding to a foreign surface. 
D. Hydrophobic Chromatography to Prepare MCF P14.7 
A further method was developed to yield a reliable source of pure P14.7. A 
sample of the MCF-containing lyophilized material obtained following DEAE 
chromatography was resuspended in 2 ml 0.01M phosphate buffer, pH 6.8, and 
brought to 8M ammonium sulfate. This sample was then applied to a 0.5- 
.times.12-cm octyl sepharose column equilibrated with 0.01M phosphate 
buffer, pH 6.8, and 8M ammonium sulfate. Protein was eluted with a 
declining gradient of ammonium sulfate (8-0M) and an increasing SDS 
(0-0.5%) gradient. Column fractions were analyzed by SDS-PAGE. 
Hydrophobic chromatography in this manner generated two peaks, one of which 
was not retained by the resin and a second which bound tightly. Gel 
analysis of the second peak revealed a single protein band at 14.7 kd 
(FIG. 8). Nearly identical results were obtained using a Lichrosorb RP8 
column with an acetonitrile/pyridine mobile phase. 
Characterization and Analysis of MCF Polypeptides P29 & P14.7 
A. Relationship of P29 and P14.7 
In order to determine if the 14.7-kd species could be derived from the 
29-kd species, three types of experiments were performed. Firstly, P29 was 
eluted from 15% PAGE gel and subjected to treatment with endoglycosidase F 
as follows. Five hundred micrograms of protein in 0.1 ml-0.05 N NaCl, 1% 
SDS, pH 6.8 incubation buffer, was denatured at 100.degree. C. for 3 min. 
The SDS was precipitated with 1.0% nonionic detergent NP-40, and the 
reaction carried out by the addition of 5 and 10 U of endoglycosidase F 
(Boehringer Mannheim) followed by an overnight incubation at 37.degree. C. 
It was found that P14.7 could not be generated from P29 by simple 
deglycosylation using endoglycosidase F treatment. 
Secondly, P29 was subjected to performic acid oxidation. DEAE-prepared P29 
protein was dissolved in 250.mu.l cold performic acid (100 .mu.l 30% 
H.sub.2 O.sub.2 plus 900.mu.l concentrated formic acid incubated 1 h at 
room temperature) and held 4 h at 4.degree. C. This was diluted to 3.0 ml 
with distilled H.sub.2 O.sub.2 frozen in dry ice and ethanol, and 
lyophilized. Products from both procedures were recovered by repetitive 
lyophilization and analyzed by SDS-PAGE. Performic acid treatment in this 
manner did not result in regeneration of P147 from P29. Neither did 
treatment with excess (40.times.) .beta.-mercaptoethanol. 
Lastly, P29 and P14.7 were compared immunologically. Antisera to P29 and 
P14.7 were prepared as follows. Two New Zealand white rabbits were 
immunized with 600 .mu.g of purified P29 MCF in complete Freund's adjuvant 
at multiple sites. Rabbits were boosted at 2-week intervals with 100 .mu.g 
P29 MCF in incomplete Freund's adjuvant. A third rabbit was immunized in 
the same manner with purified P14.7 MCF prepared from octyl sepharose. 
Specificity of both antisera was tested by dot immuno-blotting using human 
serum, purified human IL-1, recombinant human IL-1.alpha. and IL-1.beta. 
(Centikor), purified human tumor necrosis factor alpha (TNF-.alpha.; gift 
of Dr. Paul Massaferro, Bissendorf Peptides, Dallas, Tex.), purified human 
CSF-1 (gift of Dr. Richard Shadduck, University of Pittsburgh), 
recombinant GM-CSF (gift of Dr. Saroj Vadhan-raj, Department of Clinical 
Immunology, M.D. Anderson Hospital), and purified human IFN-.alpha., 
IFN-.beta., and IFN-.gamma. (gifts of Dr. Samuel Barron, University of 
Texas Medical Branch, Galveston, Tex.). 
On testing the specificities of the antisera raised to P29 and P14.7, 
neither was found to react with whole human serum, IL-1.alpha., 
IL-1.beta., TNF-.alpha., CSF-1, IFN-.alpha., IFN-.beta., or IFN-.gamma.. 
To compare the P27 and P14.7 polypeptides directly, a sample (100 ml) of 
crude Ft. F3 supernatant was dialyzed, lyophilized, electrophoresed in two 
dimensions (as described by O'Farrell, J. Biol. Chem., 250:4007, 1975), 
and subjected to immunoblotting. 
The immunoblots were developed according to the method of Bennett and 
Yeoman (J. Immunol. Methods, 61:201, 1983) using alkaline 
phosphatase-labeled goat anti-rabbit IgG (Kirkegaard and Perry, Bethesda, 
Md.) as a secondary antibody. It was found that antiserum to P29 
recognized only P29 in such immunoblots of crude hybridoma supernatant 2D 
gels. 
B. Amino Acid Composition 
P29 prepared by dye ligand and ion exchange chromatography and SDS gel 
electrophoresis in one dimension, was cut and eluted from a single 15% 
gel, lyophilized, and re-electrophoresed for 2D analysis. Protein blotting 
of the second dimension as well as silver staining of the primary gel 
revealed a major spot at 29 kd having an isoelectric point of 4.2. This 
spot was cut out of the second dimension, eluted, lyophilized to dryness, 
and subjected to amino acid composition analysis. 
Amino acid analysis was carried out on 250 pmoles of P29 utilizing 
overnight acid hydrolysis followed by analysis on an LKB model 4151 alpha 
plus amino acid analyzer. Peaks were compared to authentic standards (LKB) 
(Levy et al., PNAS, 76:6186, 1981). The results of the amino acid 
composition are summarized below: 
TABLE VIII 
______________________________________ 
Amino Acid Composition Analysis of P29 MCF 
Mole percent Mole percent 
______________________________________ 
Asp 8.06 Ile 4.06 
Thr 3.89 Leu 7.39 
Ser 9.05 NorL -- 
Gln 12.40 Tyr 3.06 
Gly 20.06 Phe 3.25 
Ala 7.51 His 2.83 
Cys -- Lys 5.72 
Val 5.08 Arg 3.94 
Met 0.99 Pro 2.72 
______________________________________ 
380 moles of P14.7, purified by hydrophobic chromatography, was similarly 
subjected to amino acid analysis giving the following results: 
TABLE IX 
______________________________________ 
Amino Acid Composition Analysis of P14.7 MCF 
Mole percent Mole percent 
______________________________________ 
Asp 11.2 Ile 5.4 
Thr 4.8 Leu 7.6 
Ser 5.1 NorL -- 
Gln 9.2 Tyr 2.2 
Gly 19.25 Phe 3.8 
Ala 9.2 His 1.2 
Cys -- Lys 5.1 
Val 6.8 Arg 4.0 
Met 1.1 Pro 3.9 
______________________________________ 
It can be seen that the amino acid composition analyses of both species are 
similar but not identical. Both proteins are rich in glycine. However, 
this occurs as a contaminant in such analysis and these results should 
therefore be confirmed by primary sequencing. 
C. N-terminal Sequence Analysis 
The purified MCF P29 and P14.7 polypeptides were prepared, as described 
above, and subjected to automated Edman degradation in Beckman Model 890 
Sequenator. The N-terminus of P29, as determined from three separate 
preparations, reads in the three-letter amino acid code--Gly Ala Ala Val 
Leu Glu Asp Ser Gln, whereas that of P14.7 is blocked (results are from 
determinations using 250, 320, and 380 nmol of each protein). 
The N-terminal sequence of P29 was compared with those in the PIR (Protein 
Identification Resource), National Institute of General Medical Sciences, 
and the EMBL Data Banks and was found to be unique. 
To summarize, the data presented above suggest that the MCF P29 and P14.7 
polypeptides may be distinct, but related, proteins important in the 
induction of monocyte cytotoxicity. Furthermore, an N-terminal analysis of 
P29 reveals it to be a previously undescribed cytokine. 
10. MCF Activity of Synthetic Peptides 
A 9-mer synthetic peptide corresponding to the authentic N-terminal amino 
acid sequence of MCF P29 was prepared using a Water's Mode; 438 
Synthesizer. The peptides were cleaved from the resin and purified by 
hydrophobic HPLC over an Aqua Pore C4 column using a TFA/acetonitrile 
gradient. Coupling was monitored by the Ninhydrin method (Kent, The 
chemical synthesis of peptides and proteins annual review of biochemical 
chemistry, 15:951, 1988). The peptide was purified by rechromatographing 
on a C4 reverse phase column and repeated lyophilization. The fidelity of 
synthesis was determined by N-terminal sequence analysis using an Applied 
Biosystems Model 477A sequencer. This peptide, having the sequence Gly Ala 
Ala Val Leu Glu Asp Ser Gln (seq id no:1), was termed MJ-2. 
The potential monocyte cytotoxicity inducing activity of the purified MJ-2 
peptide was tested, as before, using assays based upon the specific lysis 
of the human tumor target K562 by activated human peripheral blood 
monocytes. The purified MJ-2 peptide was found to be capable of activating 
human blood monocytes for cytotoxicity at a concentration of approximately 
10.sup.-9 M (FIG. 9). This level of activity is particularly noteworthy as 
this peptide, of only 9 amino acids, exhibits the same high level activity 
as the intact protein of 29kD. Furthermore, the peptide was found to be 
capable of activating the monocytes obtained from eight different human 
donors. The standard deviation between assays using monocytes from 
different human subjects was found to be relatively low in comparison to 
the high variability which is usually observed in assays of human immune 
function. 
Further truncated or substituted synthetic peptides were also synthesized, 
purified, and analyzed for monocyte cytotoxicity inducing activity. 
A purified peptide termed MJ-1, having the sequence Gly Ala Ala Val Leu Glu 
Asn Ser Gln (seq id no:2) i.e., containing an asparagine at position 7 
instead of an aspartic acid residue, was also found to be capable of 
inducing human monocyte cytotoxicity. When tested in an identical manner 
to MJ-2 in parallel assays using cells from the same human donors, the 
activity of the substituted peptide, MJ-1, was determined to be 10.sup.-6 
M (FIG. 10). This data indicates that the substitution of this particular 
single amino acid residue results in an approximate 3 log fold decrease in 
the biological activity of the peptide. 
A truncated five-mer peptide, MJ3, having the sequence Gly Ala Ala Val Leu, 
did not reliably exhibit biological activity when tested in parallel 
assays (FIG. 11). No dose response could be obtained, although in one 
experiment 25% specific release of label was observed at an input of 
10.sup.2 mg of protein. However, the 5-mer peptide MJ-5 with the sequence 
Leu Glu Asp Ser Gln (seq id no:3) was critical for biological activity. 
11. Treatment Protocols 
Due to precautions which are necessarily attendant to every new 
pharmaceutical, due both to consideration of patient safety and federal 
new drug regulations, the MCF and MCF peptides of the present invention 
have not been tested as yet in a clinical setting in human subjects. 
However, the in vitro activity of MCF in stimulating monocytes to kill 
tumor cells, along with the recent clinical success of interleukin II, is 
believed to demonstrate the utility of the present invention in this 
regard. The following embodiments are therefore prophetic and represent 
the best mode contemplated by the present inventor of carrying out the 
practice of the invention in various clinical settings. 
A. Antitumor Therapy 
1) Direct Infusion 
It is believed that MCF or MCF-derived peptides or synthetic peptides will 
prove to be useful in the treatment of various tumors, and in particular, 
tumors of the blood forming organs such as leukemias, or solid tumors 
which have been described as infiltrated by macrophages, by way of direct 
intravenous infusion of pharmaceutical compositions which include MCF. 
Such compositions would include effective doses of either MCF alone, or in 
combination with other therapeutic agents such as interleukin II, 
interferon, tumor necrosis factor or cytoxan. Interleukin II may be 
obtained as disclosed by numerous U.S. patents, including for example, 
U.S. Pat. No. 4,407,945 and 4,401,756, incorporated herein by reference. 
Cytoxan (cyclophosphamide) is a commercially available antineoplastic 
agent. Interferon is also commercially available as disclosed here@in, and 
its clinical use has been reviewed and described in detail in numerous 
publications, including, for example, in Goldstein et al., Can. Res., 
46:4325-4329, 1986, incorporated herein by reference. Moreover, Goldstein 
discloses in detail the suggested and reported dose regimens for 
interferon antitumor therapy. Preparation of Tumor Necrosis Factor and its 
use is known in the art as exemplified by U.S. Pat. Nos. 4,457,916; 
4,529,594; and 4,447,355 and as further disclosed by Carswell et al., 
Proc. Natl. Acad. Sci, USA, 72:3666, 1975; Ruff et al., J. Immunol., 
125:1671, 1980; Matthews et al., Br. J. 46(9):4357, 1980, all of the 
foregoing references being incorporated by reference. Therefore, it is 
considered that use and dosages of MCF treatment, alone or in combination 
with these agents, is well within the skill of the art in light of the 
present specification. 
MCF, MCF-derived peptides or synthetic peptides could be given daily by 
continuous infusion or given on alternative days with interleukin-2 or 
interferon being given on the other day. Such a treatment would be 
possible since the cytotoxic effect of MCF seems to last for about 
approximately 24 hours. Alternatively a large initial dose of Cytoxan 
could be given which should deplete suppressor T-lymphocytes followed by 
continuous infusion of MCF. Doses of MCF would of course have to be 
determined by experimental methods which are well known to skilled 
immunologists. However, dosages will likely be at least an order of 
magnitude lower than dosages of interferon gamma. Interferons are usually 
given as an IM dose of 3 million units thrice weekly although one would 
have to take into account whether total body water is being saturated. 
With a new agent of any type one would have to initiate a phase I trial 
first to establish levels at which unacceptable toxicity is reached. 
2) Adoptive Immunotherapy 
Adoptive immunotherapy is a new approach to treating metastatic cancer in 
which immune cells with antitumor reactivity are transferred to the 
tumor-bearing patient. Much of this work has been pioneered by Dr. Steven 
Rosenberg and is discussed in more detail in Rosenberg et al., Adv. Cancer 
Res., 25:323, 1977 and Rosenberg, Cancer Treat. Rep., 68:233, 1984, both 
incorporated by reference. In particular, interleukin II, also referred to 
as T-cell growth factor, has been shown to be a useful adjuvant to 
adoptive immunotherapy, wherein it is used to stimulate killer T-cell 
development (see, e.g., Rosenberg, J. Natl. Cancer Inst., 75:595, 1985, 
incorporated herein by reference). Moreover, adoptive therapy utilizing 
interleukin II has demonstrated applicability in the treatment of a 
variety of advanced metastatic cancers in humans (Rosenberg et al., N. 
Eng. J. Med., 313:1485, 1985, incorporated herein by reference). 
Accordingly, it is submitted that the MCF of the present invention, or 
peptides therefrom, can be utilized in an adoptive immunotherapy protocol 
in a manner similar to interleukin II. In particular, it is believed that 
the following proposed protocol will serve as a sufficient basis to teach 
those skilled in the art of adoptive immunotherapy to utilize MCF in this 
manner. 
Monocytes will be harvested by cell separation using, for example, an IBM 
cell separator using accepted techniques. These cells would then be 
incubated with approximately 4 units/ml of MCF or MCF-derived peptides 
overnight followed by slow continuous infusion of the induced cells into 
the patients. Such therapy could initially be given 2 to 3 times a week. 
However, because of the long life span of monocytes it could perhaps be 
given at more infrequent intervals stretched over a much longer period to 
time to insure that infiltration into the tumor occurs. 
3) Gene Therapy 
It is envisioned that the molecular cloning of the 29kD MCF protein will 
open up new avenues of clinical investigation. In particular, it is 
believed that recombinant MCF may prove to be of use in gene therapy 
protocols for human treatment. The basis of this treatment would be to 
enhance human-defence mechanisms by administering human macrophages or 
monocytes into which had been inserted a functioning MCF gene. 
Importantly, in such treatment, the "recombinant" monocytes or macrophages 
would be able to migrate to sites of tumors and interact with both tumor 
cells and host lymphocytes. This may lead to an expanded role for adoptive 
immunity in the treatment of cancer and provide a safe and effective 
method for delivering immunostimulatory cells host sites. 
The first step towards such treatment strategies would be the stable 
transvection of a human monocyte cell line, such as, for example, U937, to 
generate MCF-expressing recombinant cells. Technology regarding 
transvection of human monocyte cell lines is established in the art, for 
example, as used by Mace and colleagues in transvecting U937 cells with a 
plasmid bearing the IFN.beta. gene (Mace et al., J. Immunol., 
147(10):3559, 1991, incorporated herein by reference). 
Following this, it is envisioned that one would next proceed to transvect 
human peripheral blood monocytes expanded in vitro. Expression of 
cytotoxicity by transvected monocytes may be measured using the assays 
disclosed herein. One would naturally follow standard and controlled 
techniques throughout these processes. For example, one would minimize the 
possibility of subsequently inducing monocyte tumors by monitoring the 
monocytes for 48 hours in the absence of growth factors for evidence of 
spontaneous growth which may have resulted from insertional mutagenesis. 
Prior to any clinical trials, one would of course analyze the effects of 
such recombinant cells in an animal model. In vivo monocyte tumor 
cytotoxicity may be monitored by employing a mouse model, such as, for 
example, the B16 melanoma model. Inbred mice would be inoculated by tail 
vein with B16 melanoma cells, and monocytes transformed with recombinant 
MCF or a control gene. The transformed monocytes could be administered 
simultaneously with, or subsequent to, the tumor cells. The mice would 
then be sacrificed 14 days later and pulmonary metastases counted under a 
dissecting microscope. The results should be statistically analyzed using 
students T test. Due to the use of human cells, it is contemplated that 
one would wish to employ nude mice in place of the more commonly used 
c57B1/6. 
Following the animal studies, one could proceed to analyze the recombinant 
MCF-bearing monocytes or macrophages in human gene therapy regimens. As 
monocytes are able to migrate to sites of tumor infiltration, they should 
be particularly useful as vehicles for the delivery of recombinant 
cytokines, such as MCF, to the tumor bed. Patients with melanoma or 
non-small cell lung cancer who have failed to respond to conventional 
therapies are considered to be suitable candidates for inclusion in a 
clinical trial. The various elements of conducting such a trial, including 
patient treatment and monitoring, will be known to those of skill in the 
art in light of the present disclosure. 
B. Diagnostic Utility 
MCF will additionally be of value as a clinical diagnostic aid. For 
example, the anti-MCF antibodies described herein will provide an ability 
to determine MCF blood levels, thereby assisting in maintaining 
therapeutic blood levels and perhaps in the diagnosis of T-cell 
malignancies which may be accompanied by high serum MCF levels. Moreover, 
MCF antiserum may be of use in evaluating T-cell function in normal 
individuals. Similar uses have been described for the so-called 
melanoma-associated antigen. 
The development of antibodies to a particular antigen whether polyclonal or 
monoclonal, are well known in the art and can readily be achieved by 
skilled immunologists. This is the case even where the particular molecule 
is not antigenic in and of itself, through either the attachment of an 
immunostimulating ligand such as keyhole limpet hemocyanin, or by finding 
a species wherein the molecule is antigenic. 
As demonstrated herein, polyclonal anti-MCF-antibodies can be prepared by 
immunizing rabbits. A hetero-antisera of this kind can be quantitated by 
immunodot assay, western blotting, ELISA and various other 
immunodiagnostic techniques may be performed with such a hetero-antiserum. 
Monoclonal antibodies may be developed by a number of accepted techniques, 
for example, as disclosed by U.S. Pat. Nos. 4,172,124 and 4,271,145, both 
to Koprowski et al., incorporated herein by reference. 
For in vitro diagnostic work, for example, in an immunoassay to quantitate 
serum MCF levels, the MCF antibody will be used most preferably in an 
ELISA assay which employs the antibody together with an immuno detection 
reagent capable of detecting quantitatively specific immune complex 
formation. 
However, in general, immunodiagnostic kits would include reagents 
appropriate for either detecting patient-generated anti-MCF antibodies 
(e.g. circulating antibodies) or detecting MCF, for example, 
tumor-generated MCF, in biological fluids or tissues from patients. As 
used herein, a biological fluid or tissue includes any fluid or tissue 
obtained from a patient, including, for example, urine, serum, plasma, and 
biopsy samples. In the case of MCF antibody-detection kits, such kits 
would include antigenically pure, and preferably titrated, MCF together 
with an immuno detection reagent. By antigenically pure MCF is meant an 
MCF preparation which does not substantially cross-react with non-MCF 
directed antibodies. Sufficient antigenic purification could be achieved 
through immunopurification by adsorption with normal sera or 
chromatography over anti-MCF antibodies as is known in the art. 
In the case of MCF antigen detection kits, such kits would typically 
include antigenically pure, preferably titrated, anti-MCF antibody. By 
antigenically pure is meant antibody which will not substantially 
cross-react with antigens other than MCF. As with MCF antigen 
purification, polyclonal antibody purification could be achieved by immuno 
chromatography. However, a preferred antibody would be a monoclonal 
antibody. 
In either case, immunodetection kits would include an immunodetection 
reagent for detecting and/or quantifying the occurrence of specific 
immunoreactions involving MCF. Typically such reagents include, for 
example, a radioactive or enzyme-linked ligand. Such ligands are typically 
associated with either the antibody, antigen or a second antigen or 
antibody. As noted, a preferred immunodetection system are the various 
systems based on the ELISA assay. For a further description of the ELISA 
assay and the various immunodetection reagents, please refer to U.S. Pat. 
Nos. 4,454,233 and 4,446,232, both incorporated herein by reference. It is 
believed that these patents provide sufficient disclosure to enable the 
use of antibody to MCF in a clinical immunoassay. 
The present invention has been disclosed in terms of specific embodiments 
which are believed by the inventor to be the best modes for carrying out 
the invention. However, in light of the disclosure hereby provided, those 
of skill in the various arts will recognize that modifications can be made 
without departing from the intended scope of the invention. For example, 
although the present invention is disclosed in terms of a Sezary cell 
hybridoma for MCF production, it is clear that other types of T-cells may 
be employed. Additionally, numerous embodiments are likely possible for 
isolation of the factor. Moreover, as biological characterization of the 
factor progresses, it is likely that more refined and simpler assays will 
be developed for MCF identification. For example, once an antibody to MCF 
has been developed, such antibody can be used directly to assay for MCF 
production by the various cell populations. These and all other 
modifications and embodiments are intended to be within the spirit and 
scope of the present invention and appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GlyAlaAla ValLeuGluAspSerGln 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GlyAlaAlaValL euGluAsnSerGln 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LeuGluAspSerGln 
15