Note: Descriptions are shown in the official language in which they were submitted.
~ 094l20L~ 2 ~ ~ 7 3 7 9 PCT~S94/0~39
h~l~O~ OF CANCER T~F~M~.~T
RELATED APPLICATION DATA
This application is a continuation-in-part application
of copending Application Serial No. 07/891,718, filed
~une 1, 1992, which is a continuation-in-part application of
Application Serial No. PCT/US91/00342, which is a
continuation-in-part application of Application Serial
No. 07/466,577, filed on January 17, 1990, which is a
continuation-in-part application of Application Serial
No. 07/416,530, filed Ocotber 3, 1989.
FIELD OF THE INVENTION
The invention generally relates to the treatment of
cancer, and, more specifically, to the treatment of solid
tumors, including their metastases, without radiation,
surgery or st~n~rd chemotherapeutic agents.
BACKGROUND
Therapy for cancer has largely involved the use of
radiation, surgery and chemotherapeutic agents. However,
results with these measures, while beneficial in some
tumors, has had only marginal or no effect in many others.
Furthermore, these approaches have often unacceptable
toxicity.
Both radiation and surgery suffer from the same
theoretical drawback. It has been recognized that, given
that a single clonogenic malignant cell can give rise to
sufficient progeny to kill the host, the entire population
of neoplastic cells must be eradicated. See generally,
Goodman and Gilman The Pharmacoloqical Basis of Therapeutics
(Pergamon Press, 8th Edition) (pp. 1202-1204). This concept 30 of "total cell kill~ implies that total excision of a tumor
is necessary for a surgical approach, and complete
WO94/20L~ PCT~S94/0~39
2~.573~9
destruction of all cancer cells is needed in a radiation
approach, if one is to achieve a cure. In practice this is
rarely possible; indeed, where there are metastases, it is
impossible.
The term ~chemotherapy" simply means the treatment of
disease with chemical substances. The father of
chemotherapy, Paul Ehrlich, imagined the perfect
chemotherapeutic as a "magic bullet;" such a compound would
kill invading organisms without harming the host. This
target specificity is sought in all types of
chemotherapeutics, including anticancer agents.
However, specificity has been the major problem with
anticancer agents. In the case of anticancer agents, the
drug needs to distinguish between host cells that are
cancerous and host cells that are not cancerous. The vast
bulk of anticancer drugs are indiscriminate at this level.
Typically anticancer agents have negative hematological
effects (e.g., cessation of mitosis and disintegration of
formed elements in marrow and lymphoid tissues), and
immunosuppressive action (e.g., depressed cell counts), as
well as a severe impact on epithelial tissues (e.g.,
intestinal mucosa), reproductive tissues (e.g., impairment
of spermatogenesis), and the nervous system. P. Calabresi
and B.A. Chabner, In: Goodman and Gilman The Pharmacoloqical
Basis of Therapeutics (Pergamon Press, 8th Edition) (pp.
1209-1216).
Success with chemotherapeutics as anticancer agents has
also been hampered by the phPno~non of multiple drug
resistance, resistance to a wide range of structurally
unrelated cytotoxic anticancer compounds. J.H. Gerlach et
al., Cancer Surveys, 5:25-46 (1986). The underlying cause
of progressive drug resistance may be due to a small
population of drug-resistant cells within the tumor (e.g.,
mutant cells) at the time of diagnosis. J.H. Goldie and
~ 094/20L~ 21 ~ 7 3 7 9 PCT~S94tO~39
Andrew J. Cold-m-an~ Cancer Research, 44:3643-36S3 (1984).
Treating such a tumor with a single drug first results in a
remission, where the tumor shrinks in size as a result of
the killing of the predomin~nt drug-sensitive cells. With 5 the drug-sensitive cells gone, the remaining drug-resistant
cells continue to multiply and eventually dominate the cell
population of the tumor.
Treatment at the outset with a combination of drugs was
proposed as a solution, given the small probability that two
or more different drug resistances would arise spontaneously
in the same cell. V.T. DeVita, Jr., Cancer, 51:1209-1220
(1983). However, it is now known that drug resistance is
due to a membrane transport protein, "P-glycoprotein," that
can confer general drug resistance. M.M. Gottesman and I.
Pastan, Trends in Pharmacological Science, 9:54-58 (1988).
Phenotypically, the tumor cells show, over time, a reduced
cellular accumulation of all drugs. In short, combination
chemotherapy appears not to be the answer.
What is needed is a specific anticancer approach that
is reliably tumoricidal to a wide variety of tumor types.
Importantly, the treatment must be effective with mi n;m~l
host toxicity.
SUMMARY OF THE INVENTION
The invention generally relates to the treatment of
cancer, and, more specifically, to the treatment of solid
tumors, including their metastases, without radiation,
surgery or standard chemotherapeutic agents. In one
embodiment, the invention involves using superantigens,
including SEA and SEB, to stimulate tumor draining lymph
node cells ex vivo, allowing them to differentiate into
tumor specific immune effector cells. The cells are then
reintroduced into the same host to mediate anticancer
therapeutic effects. In another embodiment, the stimulated
WO94/20L~ PCT~S94/0W9
- ~is~37~ ~
cells are introduced into a different host. In still a
third embodiment, the cells are established as a cell line
for continuous anticancer use.
In one embodiment, lymphocytes are obtained early in
life from cancer-free hosts. The cells are stored in
appropriate containers under liquid nitrogen using
conventional techniques (e.g., DMSO, culture media, fetal
calf serum, etc.) until the onset of disease. At this
point, the cells may be thawed, and cultured and stimulated
in the manner of the present invention for reinfusion.
Alternatively, an established cell line may be made
from cancer-free hosts. The cell line can be stored as
above. On the other hand, they may be passed continuously
in culture until use.
The ex vivo stimulation method has decided advantages
over direct intravenous injection of superantigens, namely:
1) the superantigens are ensured of contacting their
appropriate target cell, namely, T lymphocytes; in other
words, stimulation is specific; 2) stimulation in culture
allows for the removal of the stimulating antigens prior to
reintroduction of the cells in the host, i.e., the host is
exposed to only very small amounts of superantigens in vivo;
and 3) lack of systemic exposure to the stimulating antigens
precludes significant interference with naturally occurring
or induced antibodies to superantigens.
The present invention demonstrates that superantigens
can reliably produce tumoricidal reactions to a wide variety
of tumor types. Moreover, success is achieved with m;n;m~l
host toxicity using the in vitro sensitization techni~ue.
In its simplest form, the present invention offers a
method for inducing a tumoricidal reaction in vivo
comprising contacting cells with superantigens ex vivo and
infusing them into a tumor-bearing host. The cells are
typically hematopoietic cells, such as peripheral blood
~0 94/20L~ 7379 PCT~S94/0~39
lymphocytes, spleen cells, tumor-infiltrating lymphocytes or
lymph node cells. Where they are lymph node cells, it is
preferred that they are from a tumor-bearing host. The
superantigens may comprise enterotoxins of Staphylococcus
aureus, or synthetic polypeptides with substantial
structural homology and statistically significant sequence
homology to natural superantigens.
The present invention offers a method of human cancer
treatment compri-sing: a) providing a human c~nc~r patient;
b) obtaining hematopoietic cells from said patient; c)
contacting said cells QX vivo with one or more superantigens
to generate stimulated cells; and d) re-introducing said
stimulated cells into said patient so as to induce an in
vivo therapeutic, tumoricidal reaction. Preferably the
hematopoietic cells are cultured in culture media containing
enterotoxins and the cultured cells are washed prior to re-
introducing said stimulated cells into said patient so as to
essentially avoid introducing enterotoxins in vivo.
The culture cells can be viewed as a reagent for
treating cancer, comprising T cells sensitized to a growing
tumor and stimulated with superantigens. Preferably, the T
cells are suspended in media suitable for intravenous
administration to a human cancer patient, such as a media
comprising a physiological buffered saline solution.
While not limited to any mechanism, it is believed that
culturing the cells in the manner proposed results in subset
enrichment. In this regard, the present invention provides
a method of hllm~n cancer treatment comprising: a) providing
a human cancer patient, having one or more growing tumors;
b) obtaining V~-expressing T cells from said patient that
are sensitized to said growing tumor; c) culturing said T
cells in a first culture media, said media comprising one or
more superantigens so as to specifically stimulate a subset
of V~-expressing T cells; d) culturing said T cells in a
WO94/20L~ PCT~S94/0~39
5~3~9
second culture media, said media comprising human
interleukin 2 so as to cause cell proliferation, thereby
increasing the number of cells in said culture; and e) re-
introducing at least a portion of said T cells into said
patient so as to induce an ln vivo therapeutic, tumoricidal
reaction. In one embodiment, the method further comprises
the step of administering human interleukin 2 to said
patient in vivo after re-introducing said cells in step (e).
For culturing, the superantigen may comprise the
enterotoxin SEB at concentrations above approximately 0.010
~g/ml. Preferably, the first culture media contains SEB at
a concentration of approximately 2 ~g/ml or greater and the
second culture media contains human interleukin 2 at
concentrations above 2 international units per milliliter.
DESCRIPTION OF THE FIGURES
Figure 1 schematically shows the therapeutic approach
of the present invention.
Figures 2A, 2B, and 2C show a comparison of the primary
sequences of the staphylococcal enterotoxins and their
relatives.
DESCRIPTION OF THE INVENTION
The invention generally relates to the treatment of
cancer, and more specifically, the treatment of solid
tumors, including their metastases, without radiation,
surgery or standard chemotherapeutic agents. In one
embodiment, the invention involves a method wherein host
cells are removed and stimulated outside the body, i.e., ex
vivo, with stimulating antigens (see Figure 1). These
stimulated cells are later reintroduced into the same host
to mediate anticancer effects. When administered to
subjects having tumors, the stimulated cells induce a
tumoricidal reaction resulting in tumor regression.
SUBSTITUTE SHEET (RUL~ 26)
s4eo~ S7~7~ ~cTluss4lo~33s
It should be understood that the term, "tumoricidal
reaction,~ as used herein, means that the tumor cells are
killed, and is not meant to be limited to any particular
method by which tumor cells are killed. For example, it may
be that the tumor cells are killed directly (e.g., cell-cell
interaction) or indirectly (e.g., release of cytokines like
interferon) by the reinfused, stimulated cells. On the
other hand, the stimulated cells, while not secreting
cytokines themselves, may cause changes in paracrine growth
signals.
With respect to the latter, it is known that metastatic
cells receive and process negative paracrine growth signals,
e.g., from molecules in the transforming growth factor-~
family of cytokines. In conjunction with positive growth
factors, the negative growth factors could determine
metastatic cell growth at particular sites.
In one embodiment, the stimulating antigens are
selected from among the staphylococcal enterotoxins.
The staphylococcal enterotoxins and toxic shock syndrome
toxin, have extraordinary properties as T cell antigens.
Like other antigens, T cell stimulation by these toxins is
believed to be dependent upon presentation by Major
Histocompatability Complex (MHC) molecules. In contrast to
conventional antigens, however, they apparently do not
require presentation by a l'self" MHC molecule; allogeneic
antigen-presenting cells are equally effective. It is
thought that the essential re~uirement is that cells
presenting the toxins express MHC class II molecules, as
these molecules specifically bind the toxins.
The staphylococcal toxins are believed not to be
Ilprocessedll within antigen-presenting cells to oligopeptides
that are displayed to T cells within the class II antigen-
binding groove. Instead, it is postulated that the intact
protein binds outside the groove and interacts directly with
W094/20~ 3~9 PCT~S94/0~39
T cell receptors for antigen. Most importantly, there is
evidence that the staphylococcal toxins bind to a site on
the V~ segment of the T cell receptor heterodimer that is
distinct from the complex site for binding of self MHC and
foreign peptide antigen. Because the toxins do not bind to
a site constituted by the full array of V~, D~, J~, Va, and
J~ gene products, the frequency of T cells responding to
these molecules exceeds that of conventional peptide
antigens by several orders of magnitude. Hence their name,
"superantigens".
It is not intended that the invention be limited by the
origin or nature of the host cells. Preferably, they are
hematopoietic cells, such as immune cells (e.g., tumor
infiltrating lymphocytes) or cells capable of developing
into immune cells. While they may be isolated from a
variety of sources, such as bone marrow (e.g., from femurs
by aspiration), spleen or peripheral blood (e.g., collected
with heparin and separated by Ficoll/hypaque gradient), as
well as from the tumor (e.g., tumor-infiltrating
lymphocytes). It is preferred that they are obtained from
the lymph nodes. While they may be obtained from normal,
disease-free donors, it is also preferred that they be
obtained from tumor-bearing hosts.
Tumor-Draininq Lymph Nodes
It has been known that tumor draining lymph nodes
contain T cells specifically sensitized to the growing
tumor, although such cells are insufficient to mediate an
antitumor response. These cells, termed llpre-effectorl'
cells, can differentiate into functional immune cells upon
further in vitro stimulation. Several culture techni~ues
have been developed for successful generation of antitumor
effector cells from tumor draining lymph nodes. S. Shu et
al., J. Immun., 139:295-304 (1987). B. Ward et al.,
~ 094/20~ 1S737~ PCT~S94/0~39
J. Immun., 141:1047-1053 (1988). T. Chou et al., J. Immun.,
141:1775-1781 (1988). Initially, irradiated tumor cells
were used to drive the maturation of draining lymph node
cells, and, more recently, anti-CD3 monoclonal antibody and
IL-2 were used. H. Yoshizawa et al., J. Immun., 147:729-737
(1991). However, the results reveal less than complete
killing. While not limited by an understanding of the
mechanism, this may be due to polyclonal stimulation with
the particular stimulating agents used, i.e., generation of
a significant proportion of immune cells with irrelevant
specificity.
Superantiqens As Stimulatinq Aqents
The approach of the present invention is to use more
effective stimulating agents. Again, while not limited by
an understanding of the mechanism, it is believed that so-
called "superantigens" are capable of selectively activating
subsets of T cells responsible for mediating the desired
immune response.
Among the best studied superantigens are enterotoxins
produced by Staphylococcus aureus. These superantigens are
single chain-proteins with~molecular weights ranging from
22,000 to 38,000, and more particularly between 24,000 and
30,000. They are heat stable and resistant to trypsin
digestion (the general properties of the enterotoxins are
given in Table lA and lB). According to one aspect of the
present invention, enterotoxins isolated from media which
are supporting the growth of various Staphylococcus aureus
organisms are used.
The enterotoxins of Staphylococcus aureus form a group
of serologically distinct extracellular proteins, designated
A, B, Cl, C2, C3, D, E and F. These proteins are recognized
as the causative agents of Staphylococcal food poisoning.
W094/20~ 2 ¦57 37 ~ PCT~S94/0~39
-10 -
Enterotoxin F appears to be important in the pathogenesis of
the Staphylococcal toxic shock syndrome.
It is not intended that the present invention be
limited by the origin or nature of the particular
enterotoxin. Indeed, synthetic polypeptides with
substantial structural homology and with statistically
significant sequence homology and similarity to
Staphylococcal enterotoxins and Streptococcal pyrogenic
exotoxins, including alignment of cysteine residues and
similar hydropathy profiles, may also be effective
stimulants ex vivo to induce a tumoricidal reaction when the
stimulated cells are reinfused. In addition to enterotoxins,
such peptides might be derived from, but are not limited to
sequences in additional superantigens such as minor
lymphocyte stimulating loci, mycoplasma and mycobacterial,
Yersinia and Streptococcal Protein M antigens, heat shock
proteins, stress peptides, and mammary tumor viruses.
The protein sequences and ;mml~nological cross-
reactivity of the enterotoxins reveal that they can be
divided into two related groups. The Staphylococcal
enterotoxins A, E and D (SEA, SEE and SED) constitute one
group, and Staphylococcal enterotoxins B and C (SEB, SEC)
and Streptococcal pyrogenic exotoxin A (SPEA) make up the
second group. Amino acid sequences show that SEA and SEE
are almost identical and that SEB, SEC and SPEA share
regions of similar sequence (amino acid sequence
similarities and congruences are given in Tables 2-4). SED
is moderately related to both groups although it is more
similar to the SEA group. There is a striking amino acid
similarity among enterotoxins A, B, C, D and E in the region
immediately downstream from cysteine located at residue 106
in SEA. A second region at residue 147 also shows a highly
conserved sequence.
W094/201~ PCT~S94/0~39
~5~3 j
TAB~E lA
Some Properties Of The Enterotoxins
Enterotoxin
A' B~ c,c C,~
Emetic do~e (ED50)(~g/monkey) 5 5 5 5-10
Nitrogen content (~)16.5 16.1 16.2 16.0
SC~i ~ation coefficient (S,O,~)(S) 3.04 2.78 3.00 2.90
Diffusion coefficient (D~o~)(x 107cm'sec~') 7.94 8.22 8.10 8.10
Reduced viscosity (ml/g) 4.07 3.81 3.4 3.7
Molecular weight 34,70030,000 34,10034,000
Partial specific volume 0.726 0.726 0.728 0.725
Isoelectric point 6.8 8.6 8.6 7.0
Maximum ab~orption (m~) 277 277 277 277
Extinction (E,~) 14.3 14.4 12.1 12.1
' P.S. ~~ah~n; et al., Biochem., 5:3281 ~1966).
b M.S . Bergdoll et al., J. Bacteriol., 90:1481 ~1965).
C.R. Borja and M.S. Bergdoll, Biochem., 6:1467 ~1967).
R.M. Avena and M.S. Bergdoll, Biochem. 6:1474 ~1967).
TABLE lB
Physicochemical Properties Of Staphylococcal Enterotoxins-
Enterotoxin
Property A' B~ Cl' C~ D- E'
Emetic dose for
monkey (~q) 5 5 5 5-10
coefficient (S~O~)3 03 2.89 3.0 2.9 - 2.6
Molecular weight 27,80028,366~26,000 34,10027,300 29,600
Isoeloctric point 7.26 8.6 8.6 7.0 7.4 7.0
C-~A - min-l residueSerineLysine ClycineGlycineLysine Threonine
N-t~nm; n- 1 residue Alanine Glutamic Glutamic Glutamic Serine
E.J. Schantz ~t al., Biochem., 11:360 (1972).
E.J. Schantz et al., Biochem. 4:1011 (1965).
C.R. Borja and M.S. Bergdoll, Biochem., 6:1467 (1967).
R.M. Avena and M.S. Bergdoll, Biochem. 6:1474 (1967).
P.C. Chang and M.S. Bergdoll, Biochem., 18:1937 (1979).
C.R. Borja et al., J. Biol. Chem., 247-2456 (1972).
Data Section in Atlas Protein S~quence S~ u~tu~u 5:D227, (M. Dayhoff, ed.),
National Ri~ - ic~l Research Poundation, Washinqton D.C. (1972) (det~rmin~d from
the amino acid sequence of I.Y. Huang and M.S. Bergdoll, J. Biol. Chem., 245:3493
(1970)).
Modified from M.S. Bergdoll et al. in RQcent Advances in Staphylococcal Research,
(W.W. Yotis, ed.), Ann. N.Y. Acad. Sci., 236:307-316.
.
W094/20~ 2 l S ~ 3 ~ ~ PCT~S94/0~39
-12-
These regions are contained on the peptide fragment of
SEC, and are known to contain the active sites for emesis
and diarrhea. The mitogenic region resides in the C
terminal tryptic fragment of SEC, implying that other
5 regions of sequence similarity exist.
Comparison of the primary sequences of the
staphylococcal enterotoxins and their relatives is shown in
Figures 2A, 2B, and 2C. The complete primary amino acid
sequences o~ the staphylococcal enterotoxins and related
proteins are shown aligned, with the exception of the
sequences of the exfoliating toxins, which are shown aligned
with each other, but not with the re~; n; ~g toxins. The
exfoliating toxins have properties related to those of the
others.
TABLE 2~
Sequence Similarities Among The
Pyrogenic Toxins And Enterotoxins
ToxinSequence
106 119 147 163
SEA CMYGGVTLHDNNRL KKNVl~QELDLQARRYL
SEB CMYGGVTEHHGNOL KKKVTAQELDYLTRHYL
SEC1CMYGGITKHEGNHF KKSVTAQELDIKARNFL
SED CTYGGVTPHEGNKL KKNVTVQELDAQARRYL
SEE CMYGGVTLHDNNRL KKEVTVQELDLQARHYL
SPEACIYGGVTNHEGNHL KKMVTAQELDYKVRKYL
ConsensusCMYGGVTLHEGNHL KKNVTAQELD~QAR~YL
TSST-lIHFQISG-vl~l~KL KKQLAISTLDFEIRHQL
J.J. Iandolo, Ann. Rev. Microbiol., 43:375 (1989).
SUBSTITUTE SHEET (RULE 26)
094/20L~ ; ~i PCT~S94/0~39
TABLE 3
Amino Acid Composition Of The Enterotoxins (g/lOOg Protein)
Enterotoxin
Amino Acid A* Bt Cl~ C2~ E
Lysine 11. 26 14.85 14.43 13.9910.83
Histidine 3.16 2.34 2.91 2.873.04
Arginine 4.02 2.69 1.71 1.754.50
Aspartic acid15.53 18.13 17.85 18.3815.10
Threonine 5.96 4.50 5.31 5.806.36
Serine 2.99 4.05 4.58 4.814.72
Glutamic acid12.36 9.45 8.95 8.9312.15
Proline 1.35 2.11 2.16 2.231.93
Glycine 2.96 1.78 2.99 2.904.10
Alanine 1.94 1.32 1.85 1.612.38
Half-cysteine 0. 66 0.68 0.79 0.740.81
Valine 4.93 5.66 6.50 5.874.36
Methionine 0.96 3.52 3.20 3.600.45
Isoleucine 4.11 3.53 4.09 4.024.30
Leucine 9.78 6.86 6.54 6.1310.08
Tyrosine 10. 63 11.50 9.80 10.279.79
Phenylalanine 4.31 6.23 5.35 5.254.47
Tryptophan 1.46 0.95 0.99 0.841.51
Amide NH3 1. 80 1.66 1.71 1.621.66
TOTAL 98.37 100.15100.00 99.99100.88
* Schantz et al., 1972.
t M.S. Bergdoll et al ., Arch Biochem Biophys, 112: 104
(1965) .
I.Y. Huang et al., Biochem., 6:1480 (1967) .
Borja et al., 1972.
~ M.S. Bergdoll et al., Agric. Food Chem., 22:9 (1974).
WO94/20L~ 2~ 3~ 9 PCT~S94/0~39
TABL~ 4t
Amino Acid Compositions Of TSST-la And lba
Amino acid composition
TSST-la TSST-lb
Amino acidresiduesresidues Cloneb
per moleb per moleb
Aspartic acid 26 27 25
Threonine 21 20 19
Serine 20 20 21
Glutamic acid 20 20 17
Proline 10 8 10
Glycine 13 14 11
Alanine 4 5 3
Half-cysteine 0 ~
Valine 5 5 5
Methionine 0 0 2
Isoleucine 15 15 17
Leucine 14 16 15
Tyrosine 10 8 9
Phenylalanine 7 7 7
Histidine 5 5 5
Lysine 23 24 21
Tryptophan NDd NDd 3
Arginine 4 5 4
197 199 194
t D.A. Blomster-Hautamaa and P,M. Schlievert, Meth.
Enzym., 165:37 (1988).
a Isolated from strain MN8, as compared to the inferred
amino acid composition of the TSST-1 structural gene.
b Residues per mole values are based on a molecular
weight of 22,000.
c Residues per mole inferred from the DNA sequence of the
TSST-1 structural gene. Blomster-Hautamaa and
colleagues.
d ND. Not determined.
~o s4eo~4 21S737 rcT~ss4lo233s
The toxins shown in Figures 2A, 2B, and 2C are as
follows: SEA to SEE, Staphylococcus aureus enterotoxins A
to E; SPE A and C, Streptococcus pyogenes toxins A and C;
TSSTl, Staphylococcus aureus toxic shock - associated toxin;
ETA and ETB, Staphylococcus aureus exfoliating toxins A and
B. Single letter abbreviations for the amino acid residues
are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H,
His; I Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln;
R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
It should be noted that the two Streptococcal toxins
SPEA and C are about as similar to each of the
Staphylococcal groups as they are to each other.
Exfoliative toxins (ETA, ETB) are of similar size to SEB and
SEA with similar modes of action. They share several points
of sequence similarity to the Staphylococcal enterotoxins.
Overall there are several stretches at which similarities
are apparent throughout the total group comprised of
Staphylococcal enterotoxins, Streptococcal pyrogenic
exotoxins and Staphylococcal exfoliative toxins.
The recognition that the biologically active regions of
the enterotoxins and SPEA were substantially structurally
homologous enables one to predict synthetic polypeptide
compounds which will exhibit similar tumoricidal effects.
Table 6 illustrates the amino acid sequence homology of
mature SPEA and Staphylococcus aureus enterotoxin B. The
top sequence is the SPEA-derived amino acid sequence. The
amino acid sequence of enterotoxin B is on the bottom.
Sequences are numbered from the amino acid terminus, with
amino acids represented by standard one character
designations (see Table 5). Identities are indicated by :
and gaps in the sequences introduced by the alignment
algorithm are represented by dashed lines. [See L.P.
Johnson et al., Mol. Gen. Genet., 203:354-356 (1986).]
SUBSTITUTE SHEET (RULE 26)
wo 94/20L~ ~9 PCT~S94/0~39
-16-
One common methodology for evaluating sequence
homology, and more importantly statistically significant
similarities, is to use a Monte Carlo analysis using an
algorithm written by Lipman and Pearson to obtain a Z value.
According to this analysis, a Z value greater than 6
indicates probable significance, and a Z value greater than
10 is considered to be statistically significant. W.R.
Pearson and D.J. Lipman, Proc. Natl. Acad. Sci. (USA),
85:2444-2448 (1988); D.J. Lipman and W.R. Pearson, Science,
227:1435-1441 (1985).
In the present invention, synthetic polypeptides useful
in tumoricidal therapy and in blocking or destroying
autoreactive T and B lymphocyte populations are
characterized by substantial structural homology to
enterotoxin A, enterotoxin B and streptococcal pyrogenic
exotoxins with statistically significant sequence homology
and similarity (Z value of Lipman and Pearson algorithm in
Monte Carlo analysis exceeding 6) to include alignment of
cysteine residues and similar hydropathy profiles.
Toxicity of Superantiqens
Previous approaches utilizing superantigens in cancer
therapy have involved systemic exposure to these agents.
Such early approaches include both plasma perfusion over a
solid support matrix containing superantigens [D. S . Terman
et al ., New Eng. J. Med., 305: 1195 (1981) ] as well as direct
injection of superantigens into a tumor-bearing host. D. S .
Terman, Patent Application Serial No. PCT/US91/00342 (1990);
K.A. Newell et al ., Proc. Nat. Acad. Sci (USA), 88 : 1074
(1991) .
It is believed that all enterotoxins are capable of
inducing fever and shock when given systemically (e.g.,
intravenously). When administered in this manner, they are
presumed to function by affecting emetic receptors in the
~ 094/20124 S~3~9 PCT~S94/02339
: -17-
TABLB 5
Amino Acid One-letter Symbol
Alanine A
Arginine R
Asparagine N
Aspartic acid D
Cysteine C
Glutamine Q
Glutamic acid E
Glycine G
Histidine H
Isoleucine
Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline P
Serine S
Threonine T
Tryptophan W
Tyrosine Y
Valine V
WO94/201~ ~ 3 19 PCT~S94/0~39
-18-
TABL~ 6
STR-PKPSQLQRSNL~K~ KlYl~RVTL-----VTHENVKSVDQLLSHDLIYNVS--
: : : : : : : : : : : : : : : : : : :
ESQPDPKPDELHKSS--K-FT~T.M~N~KV-LYNNDHVSAINVKSINEFF--DLIYLYSIK
----GPNYDKLKTELKNQEMATLFKDKNVDIY~v~YY~LCYLC---------ENAERSAC
: : : : : : : ~ : : : : : : : : : : : : :
DTKLG-NYDNVRVEFKNKDLADKYKDKYVDVFGANYYQ-CYFSKKTNNIDSHENTKRKTC
100 110
100 110 120 130 140 150
LYGGVTNHEGNHLEIPKK----I W KVSIDGIQSLSFDIEQIKNGNCSRIS-YTVRKYLT
: ::::: :: : ::: :: :::: : : : ::
MYGGVTEHGNNQLD---KYYRSITVRVFEDGKNLLSFDVQTNKKKVTAEQLDYLTRHYLV
120 130 140 150 160
160 170 180 190 200
DNKQLYTNGPSKYETGYIKFIPKNKESFWFDFFPEPE--FTQSKYLMIYKDNETLDSNTS
:: :: : ::::::::: : ::: : : : : :::::: : ::
KNKKLYEFNNSPYETGYIKFIE-NENSFWYDMMPAPGNKFDQSKYLMMYNNDKMVDSKDV
170 180 190 200 210 220
220
QIEVYLTTK
::::::::
KIEVYLTTKKK
230
ab~mi n~l viscera which stimulate the emetic and diarrheal
response. They are also believed to induce interferon,
tumor necrosis factor, and interleukins 1 and 2.
Unfortunately, the increased effectiveness of higher doses
of systemically introduced superantigens is correlated with
higher toxicity. In this regard, direct administration of
increasingly effective, anti-cancer doses in animals has
been followed by shock and death within 12-24 hours.
The present invention contemplates avoiding the
undesirable effects, but nonetheless harnessing the valuable
characteristics of superantigens. Preferably, there is no
~0 94/20L~ 2 - r PCT~S94/0~39
3,~
-19 -
significant systemic exposure to superantigens using the ex
vivo stimulation approach of the present invention.
It should be noted that the ex vivo approach also
allows for the presence of minor impurities in the
preparation that would be unacceptable in preparations for
direct administration. While these impurities might be
toxic (or even lethal) in vivo, they can simply be washed
away along with the superantigen itself following ex vivo
culture.
In sum, the criteria for superantigens, and in
particular, superantigen purity are: 1) mitogenic activity
in a tritiated thymidine proliferation assay, 2) stimulation
of interferon release, 3) V~ cell reactivity, 4) amino acid
profile (see above), 5) HPLC and PAGE (21-28,000 MW); 6)
negative in the limulus amebocyte lysate (LAL) test for
endotoxin; 7) negative in a hemolytic assay for the presence
of alpha-hemolysin.
Ex Vivo Stimulation
As noted above, a number of cell types can be used.
When cells from lymph nodes are used, all types of lymph
nodes are contemplated (inguinal, mesenteric, superficial
distal auxiliary, etc.). For ex vivo stimulation, they are
removed aseptically and single cell suspensions are prepared
by teasing under sterile conditions. Cell preparations then
may be filtered (e.g., through a layer of nylon mesh),
centrifuged and subjected to a gentle lysing procedure, if
necessary.
Tumor-draining lymph node cells may be stimulated in
vitro using a number of protocols. For example, a
sufficiently large number of lymph node cells (i.e., a
number adequate to show a tumoricidal reaction upon
reinfusion) are exposed to superantigens (e.g., SEA, SEB,
etc.) and diluted in synthetic culture media (e.g., RPMI
WO94/201~ ~ 9 PCT~S94/0~39
-20-
1640 with typical supplements) for the appropriate period of
time (e.g., two days). Any number of standard culture
techniques can be employed (e.g., 24-well plates in an
incubator at 37C in a 5% CO2 atmosphere).
Following the incubation, the stimulated cells are
harvested and washed with synthetic media cont~;ning no
superantigens. At this point, the cells may be cultured
further with other agents if desired (e.g., IL-2). In any
event, the.cells are counted to determine the degree of
proliferation and resuspended in appropriate media for
therapy.
The stimulated cells may be reintroduced to the host by
a number of approaches. Preferably, they are injected
intravenously. Optionally, the host may be treated with
agents to promote the in vivo function and survival of the
stimulated cells (e.g., IL-2).
Of course, the stimulated cells may be reintroduced in
a variety of pharmaceutical formulations. These may contain
such normally employed additives as binders, fillers,
carriers, preservatives, stabilizing agents, emulsifiers,
and buffers. Suitable diluents and excipients are, for
example, water, saline, and dextrose.
Alternate Embodiments
Tumor resensitized lymphocytes may become anergized in
the course of tumor growth in vivo and become refractory to
activation or expansion by the superantigens with T cell V~
specificity. various cytokines may partially reverse T
memory cell anergy, namely, IL-2, IL-4, or IL-1 plus IL-6.
These cytokines may promote T cell proliferation and may
represent an essential "second signal" typically provided by
antigen presenting cells. Hence, responsiveness of tumor
sensitized lymphocytes may be restored by co-culturing with
094/20L~ S ~ ~ PCT~S94/0~39
-21-
various cytokines and mitogens such as anti-CD3 antibody or
conconavalin A.
While the preferred embodiment involves culturing ex
vivo, other approaches are also contemplated. In one
embodiment, the present invention contemplates transfecting
with superantigen genes into tumor cells to provide powerful
augmenting signals to T cell stimulation. In another
embodiment, dual transfection with superantigens and
molecules such as B7 is contemplated. Moreover, various
cytokines and antibodies which are known to enhance T cell
proliferation and secretion such as interleukin 1,
interleukin 2, interleukin 4, interleukin 6, anti-CD3 or
anti-CD2 may be employed simultaneously or sequentially with
enterotoxins in vivo or in vitro to augment antitumor
effects of the enterotoxins.
Substances which increase the number of anitgen-
presenting cells, as well as substances which induce up-
regulation of class II molecules on antigen-presenting cells
or T cells, such as Y interferon, ICAM molecules and the
like, used in vitro or in vivo could create additional
binding sites for superantigen presentation to the T
lymphocyte population and augment T lymphocyte proliferative
and secretory function as well as anti-tumor effects.
Finally, various superantigens may be employed
sequentially to up-regulate the activity of one another.
For example, SEA, which is known to be a powerful cytokine
inducer, may be used in vitro or in vivo to up-regulate
class II molecules before the use of SEB or SEC, which are
potent T cell stimulants. The up-regulated class II binding
sites created by SEA would be occupied by SEB, providing
significantly increased antigenic presentation to the T cell
v~ repertoire.
WO941201~ PCT~S94/0~39
~ 22-
EXPERIMENTAL
The following examples serve to illustrate certain
preferred embodiments and aspects of the present invention
and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the
following abbreviations apply: eq (equivalents); M (Molar);
uM (micromolar); mM (millimolar); N (Normal); mol (moles);
mmol (millimoles); ~mol (micromoles); nmol (n~nom~les); g
(grams); mg (milligrams); ~g (micrograms); L (liters); ml
(milliliters); ~l (microliters); cm (centimeters); mm
(millimeters); ~m (micrometers); nm (n~nometers); C
(degrees Centigrade); mAb (monoclonal antibody); MW
(molecular weight); U (units); d(days).
EXA~PLE 1
Production And Isolation Of Enterotoxins
This example describes two purification approaches for
Enterotoxins A and C2.
Approach l: A lO ml culture of Staphylococcus aureus
llN-165 (SEA), Staphylococcus aureus 361 (Source: Dr. John
Iandolo, Kansas State University, Manhattan, KS) (SEC2) is
grown overnight at 37C. The removal of enterotoxin from
the supernatant is carried out using QAE-Sephadex. The
toxin is then eluted batchwise from the ion exchanger and
recovered by filtration on a sintered glass funnel. The
eluates are concentrated by ultrafiltration. The toxin is
then passed through a Sephadex-G-lO0 column. Two peaks
absorbing at 280 mm are eluted, with the latter containing
the enterotoxin. The eluted toxin is concentrated and rerun
on Sephadex-G-lO0. The overall recovery is about 30% for
SEC2 and 40 to 50% for SEA. Both toxins appear homogeneous
by sodium dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE).
~ 094/20L~ ~ $~ PCT~S94/0~39
3,~
Approach 2: Staphlococcus aureus Strain FRI-722 is
grown in a 3% enzyme-hydrolyzed casein and 7% yeast extract
at pH 6.6 at a temperature of 35-37 C. The mixture is
gently agitated for 16-20 hours. The culture is filtered
through a 0.2 micron filter and the filtrate pH is adjusted
to 5.6. The filtrate is diluted 1:5 to 1:10 with deionized
water, incubated with a cation exchange resin and stirred
for 1 h. The resin is collected and the bound protein is
eluted with high ionic strength buffer. The eluate is
concentrated and dialyzed then reincubated with a second
cation exchange resin. The SEA is eluted with a low ionic
strength to high ionic strength buffer gradient. The
fraction containing SEA is concentrated, dialyzed and loaded
onto a gel filtration system. The fraction containing SEA
is concentrated and dialyzed against PBS pH 7.2. The final
solution is filter-sterilized and frozen. Total protein is
determined spectrophotometrically at 280/260 nm. A 5 ug/ml
solution is tested in gel diffusion against a known antisera
to SEA and 1 ug/ml is tested in PAGE and endotoxin in the
Sigma-E-Toxate LAL assay.
EXAMPLE 2
Production And Isolation Of Enterotoxins
This example describes a purification approach for
Enterotoxins A and C1 and D.
This approach utilizes fast protein liquid
chromatography (FPLC) and high resolution chromatofocusing
Mono P column. Enterotoxins in media are concentrated and
passed over a Sephadex-G-75 column. The toxin containing
fractions are pooled. For C1 and D, the supernatants are
passed over an AmberLite-CG-S0 column, as described for SED,
- and the active fractions pooled. All three toxins are then
placed in buffer for chromatofocusing and then separated
W094/20~ PCT~S94/0~39
2~3 ~
-24-
using the MONO P column FPLC system. Since all of the toxins
have isoelectric points in the range of 7 to 9, the
polybuffer PBE-96 is used for elution. The purity of SEA,
SEC1 and SED is estimated to be 98, 95 and 80%,
respectively. SEA elutes as two peaks at pH 8.8 and 8.6.
SECl also elutes as two peaks at pH 8.3 and 7.9, and SED
elutes as three peaks at pH 8.6, 8.3 and 8Ø
Enterotoxins may also be produced in mutant strains of
Staphylococcus aureus by expression of an enterotoxin
producing gene in another bacteria or cell. Genetic
material which appears to be in the chromosomal plasmid, or
phage portion of the bacteria may be used for gene insertion
procedures. Complete molecules or fragments with amino acid
sequence homology to the parent enterotoxin may be produced
lS with this technology. (Reviewed in Iandolo, J.J., Annu.
Rev. Microbiol., 43:375 (1989). Moreover, mutagenic agents
such as N-Nitroso compounds are capable of augmenting
significantly the production of enterotoxins by some strains
of Staphylococcus.
EXAMPLE 3
Production And Isolation Of Enterotoxins
This example describes a purification approach for
Alpha Toxin.
Staphylococcus aureus Wood 46 strain (Source:
Dr. Sidney Harshman, Vanderbilt University, Nashville, TN)
is used and cultured in yeast extract dialysate medium.
With the glass-pore bead method undialyzed yeast may be used
together with casein, glucose, thiamine and nicotinic acid.
The organism is incubated in medium for 24h at 37C.
The culture supernatant is applied to a glass- pore
bead column and adjusted to pH 6.8. A column of 5 x 20 cm
is used for 3 liter batches and flow rates adjusted to 10-20
094/20L~ ~ PCT~S94/0~39
-25-
ml/min. The column is washed with O.OlM KHPO4 pH 6.8 and
then the alpha toxin is eluted with l.OM KHPO4 pH 7.5.
Fractions are tested for the presence of alpha hemolysin by
a rapid hemolytic assay using rabbit erythrocytes as
substrate.
EXAMPLE 4
Production And Isolation Of Enterotoxins
This example describes a purification approach for
Streptococcal Pyrogenic Exotoxin (SPE).
Streptococcus NY-5 strain (Source: ATCC 12351) has
been the most widely used for toxin production and studies.
A list of various strains to produce toxins A, B, and C has
been published. The Kalbach S84 type 3 strain (Source:
Dr. Joseph E. Alouf, Institute Pasteur-Unite Associee,
Paris, France) is cultured and the supernatant is
concentrated and stirred in calcium phosphate gel. Fraction
S1 is precipitated with 80% saturated ammonium sulfate. The
redissolved pellet is dialyzed and desi~nated Fraction S2.
This fraction is precipitated with 50-80% ammonium sulfate,
resuspended in phosphate buffered saline (Fraction S3), and
gel filtered on a Bio-Gel P-100 column. The fraction
corresponding to the volume eluted between 160 and 240 ml is
collected and concentrated by ultrafiltration to about 20 ml
in an Amicon PM10 Membrane (Fraction S~). Fraction S4 is
then submitted to preparative isoelectric focusing (IEF)
performed with a 100 ml column. The material which focuses
at around pH 4.8 in a narrow peak is collected and dialyzed
in an Amicon cell using PBS to eliminate ampholines and
sucrose. The Fraction (Ss) constitutes purified pyrogenic
exotoxin. Another electrophoretic form of SPE with a pI of
4.2 is often separated simultaneously with that of pI 4.8.
2~ 79
WO94/20L~ PCT~S94/0~39
-26-
Both forms show total cross reactivity against immune sera
raised by rabbit ;mml~nization with fraction S3.
The Fraction Ss shows a single band by SDS-PAGE
corresponding to a molecular weight of 28K. Bioassays for
determination of activity include erythematosus skin test in
rabbits or guinea pigs lymphocyte blast transformation. The
toxin may also be detected by enzyme-linked immunoabsorbant
assay (ELISA) or hemagglutination inhibition.
EXAMPLE 5
Production And Isolation Of Enterotoxins
This example describes a general purification approach
for native enterotoxins.
Current methods for purification of all of the
enterotoxins utilize ion exchange materials such as CG-50,
carboxymethyl-cellulose and the Sephadexes (gel filtration).
The preparation of the SEB used for these studies is as
follows.
Staphylococcus aureus strain I10-275 is cultured in NZ-
Amine A media supplemented with 10 g/liter of yeast extract
for 18-20 hours in room air at 37C. The flask is agitated
at 300 RPM. The initial pH of the culture is 6.8 and the
postincubation pH 8Ø The culture is filtered through a
DC-10 Amicon filter (pore size 0.1 micron). The final
filtrate is adjusted to pH 5.6. The filtrate is tested for
the presence of SEB in radial immunodiffusion using known
antisera to SEB. Eighteen to 20 liters of culture
supernatant fluid are diluted with deionized, distilled H2O
(1:5 to 1:10) and the pH adjusted to 5.6, CG-50 resin
(Malinkrodt) (800 ml), preequilibrated to pH 5.6 in 0.03 M
phosphate buffer, pH 6.2 (PB) is added and the mixture
stirred for one hour. The resin is allowed to settle and
the supernatant fluid is decanted. The resin is placed in a
~WO94/20L~ ~ ~ PCT~S94/0~39
-27-
column and the toxin is eluted with 0.5 M PB, O.5 M NaCl pH
6.2. The concentrated, dialyzed toxin is placed in a column
(5 cm x 75 cm) of CM-sepharose (pretreated with 0.005 M PB
pH 5.6). The colu-m-n is washed with the same buffer and the
enterotoxin eluted by treating the column stepwise with PB
0.03 M pH 6.0, 0.045 M pH 6.25, 0.06 M pH 6.5 and 0.12 M pH
7.2. The fractions containing the enterotoxin are combined,
concentrated with polyethylene glycol (200 ml wet volume of
packed resin), and dialyzed against 0.5 M NaCl 0.05 M PB pH
7.2. The concentrated enterotoxin solution (5 ml) is placed
in a column of Sephacryl S-200 (pretreated with 0.5 M NaCl,
0.05 M PB, pH 7.2). The column is eluted with the same
buffer. The fractions containing the enterotoxin are
combined and dialyzed against 0.01 M PB, 0.15 M NaCl pH 7.2.
The enterotoxin B concentration is approximately 1 mg/ml.
The solution is filter sterilized, frozen and lyophilized.
Samples are stored in lyophilized form at 4C. The final
enterotoxin fraction is a white powder which, when dissolved
in normal saline, is a clear colorless solution. Samples
containing 5 and 10 ~g/ml are tested in a double diffusion
immunoprecipitation assay using known standards of SEB and
mono-specific antisera. A single precipitation line is
noted which showed a line of identity with known SEB. Using
a tritiated thymidine mitogenic assay with human and murine
;mml]nocytes~ SEB showed significant mitogenic activity
comparable to that of SEA. SEB was found to be devoid of
contaminating alpha hemolysin assessed in a rabbit
erythrocyte hemolytic assay.
PAGE gel analysis of SEB showed a pre~om;n~nt single
band at 28,000 m.w. High performance li~uid chromatography
(HPLC) profiles were obtained on a MAC PLUS controlling a
Rainin Rabbit HPLC with a Hewlett Packard 1040 A Diode array
detector and a Vyadac Protein and Peptide C18 column. The
profile for purified enterotoxin B was a sharp peak without
WO94/20L~ PCT~S94/0~39 ~
~ ~7 3~ 9
-28-
significant shoulder. There was minimal trace
contamination. Amino acid analysis was carried out with a
Bechman 6300 amino acid analyzer and displayed residues
consistent with known SEB standards. The sterility of the
preparations was demonstrated by negative cultures in
thioglycolate medium and soybean-casein digest. Protein
determinations were carried out by a spectrophotometric
method.
The:sterility of the preparation was demonstrated by
negative cultures using (a) fluid thioglycollate medium and
(b) soybean-casein digest. A sample containing 1 mg/ml of
SEB was tested for endotoxin cont~m;n~tion using Sigma E-
toxate LAL assay. The final product was found to be free of
endotoxin with a st~n~rd sensitivity of 0.1 ug endotoxin/mg
SEB.
Toxicity testing was carried out in two Hartley strain
guinea pigs weighing less than 450 grams, and two female C57
black mice (Simonson Laboratories, Watsonville, CA),
weighing less than 22 grams. Each animal was observed for 7
days with no significant change in condition or weight after
intraperitoneal injection of 0.5 ml of 26 ~g/kg enterotoxin
B.
SEA, SEC, SED, SEE, TSST-l and Streptococcal pyrogenic
exotoxin in the studies were prepared by the previously
described methods. The identity, purity and sterility of
these preparations were tested in a fashion similar to that
for SEB.
WO94/201~ ~ PCT~S94/0~39
i'S73,~
-29-
EXAMPLE 6
Isolation Of Host Cells: Lymph Nodes
As noted previously, the invention involves, in one
embodiment, a method wherein host cells are removed and
stimulated outside the body, i.e., ex vivo, with stimulating
antigens. These cells may be isolated from a variety of
sources. In this example, they are obtained from the lymph
nodes.
Inguinal, mesenteric, or superficial distal axillary
lymph nodes are removed aseptically. Single cell
suspensions are prepared by teasing (e.g., with 20-gauge
needles) followed by pressing mechanically with the blunt
end of a 10-ml plastic syringe plunger in buffer under
sterile conditions. The cell preparations were filtered
through a layer of No. 100 nylon mesh (Nytex; TETKO Inc.,
Elmsford, NY), centrifuged and washed. Red cells, if
evident, are lysed by treatment with ammonium chloride-
potassium lysing buffer (8.29 g NH4Cl, 1.0 g KHCO3, and
0 . 0372 g EDTA/liter, pH 7 .4) . The cells were washed twice
with buffer and resuspended for stimulation.
EXAMPLE 7
Isolation Of Host Cells: Spleen Cells
In this example, the host cells are obtained from the
human spleen. Either a left subcostal incision or midline
incision may be used for resection. The spleen is mobilized
initially by dividing the ligamentous attachments, which are
usually avascular. The short gastric vessels then are
doubly ligated and transected. This permits ultimate
dissection of the splenic hilus with individual ligation and
division of the splenic artery and vein.
~ ~ ~ 7 3 7 ~ PCT~S94/0~39
-30-
The sequence of technical maneuvers necessary to remove
the spleen varies somewhat, depending on the surgeon~s
election to approach the splenic hilum either anteriorly or
posteriorly. The anterior approach is somewhat slower.
Anterior Method. On entering the abdomen, the stomach
should be thoroughly emptied by suction through a
nasogastric tube already in place, if this maneuver has not
been accomplished preoperatively. An opening is made in the
gastrosplenic omentum in an avascular area, and by
retracting the stomach upward and anteriorly through this
opening the upper part of the pancreas can be visualized.
The tortuous splenic artery can be seen along its upper
margin; it is, at the option of the surgeon, ligated.
The next step in the procedure is division of the lower
two-thirds of the gastrosplenic omentum. This is
accomplished by dividing the vascular omentum between clamps
and ligating the cut ends subsequently. The gastrosplenic
omentum is frequently infiltrated with a considerable amount
of adipose tissue and tends to slip away from clamps,
especially if traction is applied to the instruments. The
upper portion of this omentum also contains the vasa brevia
and large venous tributaries joining the left gastroepiploic
vein. To avoid hemorrhage from these sources, suture
ligation rather than simple ligatures should be utilized in
this area. Access to the upper portion of the gastrosplenic
omentum is difficult with the spleen in situ, and for this
reason it is best divided with the later stage after
mobilization of the splenic hilum.
Following division of the splenic vasculature, the
splenorenal, the splenocolic, and the splenophrenic
ligaments are divided. All except the last mentioned are
generally avascular and pose no particular technical
problems in division. The remnants of the splenophrenic
094/201~ S737~ PCT~S94/0~39
ligament left behind may have to be underrun with running
chromic catgut suture for hemostasis. The spleen is
displaced from the abdomen and delivered through the
incision. The only remaining attachments still in place is
the upper third of the gastrosplenic ligament which is now
carefully divided between ligatures, completing the
splenectomy procedure.
Posterior Method. The posterior approach of removing
the spleen is much more expeditious than the anterior
approach, but blood loss is usually more substantial than in
the anterior approach. After entering the abdomen the
surgeon makes an incision in the avascular splenorenal
ligament and then inserts three fingers behind the hilum of
the spleen which is easily mobilized by blind dissection.
Hemorrhage from the splenic hilum during this process can be
avoided by placing the incision on the splenorenal ligament
closer to the kidney and away from the spleen. By rapidly
dividing the splenophrenic and the splenocolic ligaments, it
is now possible to deliver the spleen through the incision.
Any hemorrhage from the splenic hilum or from the ruptured
spleen itself is very easily controlled at this point by
manual compression of the splenic hilum or placement of a
noncrushing clamp, taking care not to injure the tail of the
pancreas. The gastrosplenic ligament and the presplenic
fold when present can now be divided and suture ligated in a
deliberate manner.
Cell Suspensions. Spleen cells are mechanically
dissociated by using the blunt end of a lO-ml plastic
syringe in buffer. The cell suspension was passed through a
single layer of lO0-gauge nylon mesh (Nitex; Lawshe
Industrial Co., Bethesda, MD) and centrifuged, and the RBC
lysed by resuspension of the cell pellet in ammonium
WO94/201~ ~ ~ ~ 7 3 7 ~ PCT~S94/0~39
-32-
chloride/potassium lysing buffer, (8.29 g of NHqCl~ 1.O g
KHCO3 and 0.0372 g of EDTA/L pH 7.4; Media Production
Section, National Institutes of Health, Bethesda, MD). The
cells were again filtered through nylon mesh, washed two
times, and resuspended in culture medium (see below).
EXAMPLE 8
Isolation Of Host Cells: Infiltrating Cells
In this example, the host cells are obtained from tumor
infiltrating lymphocytes. Lymphocytes infiltrating tumors
are obtained using st~n~rd techniques. Solid tumors
(freshly resected or cryopreserved) are dispersed into
single cell suspensions by overnight enzymatic digestion
[e.g., stirring overnight at room temperature in RPMI 1640
medium containing 0.01% hyaluronidase type V, 0.002% DNAse
type I, 0.1% collagenase type IV (Sigman, St. Louis), and
antibiotics]. Tumor suspensions are then passed over Ficoll-
Hypaque gradients (Lymphocyte Separation Medium, Organon
Teknika Corp., Durham, NC). The gradient interfaces contain
viable tumor cells and mononuclear cells are washed,
adjusted to a total cell concentration of 2.5 to 5.0 x 10~
cells/ml and cultured in complete medium. Complete medium
comprises RPMI 1640 with 10% heat-inactivated type-
compatible human serum, penicillin 50 IU/ml and streptomycin
50 ~g/ml (Biofluids, Rockville, MD), gentamicin 50 ~ug/ml
(GIBCO Laboratories, Chagrin Falls, OH), amphotericin 250
ng/ml (Funglzone, Squibb, Flow Laboratories, McLean, VA),
HEPES buffer 10 mM (Biofluids), and L-glutamine 2 mM (MA
Bioproducts, Walkersville, MD). Conditioned medium from 3-
to 4-day autologous or allogeneic lymphokine-activated
killer (LAK) cell cultures (see below) can be added at a
final concentration of 20% (v/v). Recombinany IL-2 (kindly
~094/20~ ;~ PCT~S94/0~39
~ ~S~
-33-
supplied by the Cetus Corporation, Emeryville, CA) can be
added at a final concentration of 1000 ~/ml.
Cultures are maintained at 37C in a 5% CO2-humidified
atmosphere. A variety of tissue culture vessels can be
employed, including 24-well plates (Costar, Cambridge, MA).
175 cm2 flasks (Falcon; Becton Dickinson, Oxnard, CA), 850
cm2 roller bottles (Corning Glass Works, Corning, NY), and
750 cm2 gas-permeable culture bags (Fenwal Laboratories,
Division of Travenol Laboratories, Deerfield, IL). Cultures
should be fed weekly by harvesting, pelletting and
resuspending cells at 2.5 x 106 cells/ml in fresh medium.
Over an initial period (e.g., 2 to 3 weeks) of culture, the
lymphocytes will selectively proliferate, while the
remaining tumor cells will typically disappear completely.
To make LAK cell cultures, peripheral blood lymphocytes
(PBL) are obtained from patients or normal donors. After
passage over Ficoll-Hypaque gradients, cells are cultured at
a concentration of 1 x 106/ml in RPMI 1640 medium with 2%
human serum, antibiotics, glutamine, and HEPES buffer.
Recombinant IL-2 is added at 1000 u/ml. Cultures are
maintained for 3 to 7 days in a humidified 5% CO2 atmosphere
at 37C.
EXAMPLL 9
Ex Vivo Stimulation
This example describes an approach to stimulate host
cells in vitro with superantigens for reinfusion. Tumor-
draining lymph node (LN) cells are obtained as described in
Example 7 and stimulated in vitro in a procedure with an
optional second step.
WO94t20L~ ~S~ ~ PCT~S94/0~39
Step One. For stimulation, 4 x 106 LN cells, in 2 ml
of culture medium containing SEA or SEB, are incubated in a
well of 24-well plates at 37C in a 5% CO2 atmosphere for 2
days. The culture media comprises RPMI 1640 medium
supplemented with 10% heat inactivated fetal calf serum, 0.1
mM nonessential amino acids, 1 ~M sodium pyruvate, 2 mM
freshly prepared L-glutamine, 100 ~g/ml streptomycin, 100
U/ml penicillin, 50 ~g/ml gentamicin, 0.5 ~g/ml fungizone
(all from GIBCO, Grand Island, NY) and 5 x 10-5 M 2-ME
(Sigma). The cells were harvested and washed.
Step Two. The initially stimulated cells are further
cultured at 3 x 105/well in 2 ml of culture media with Human
recombinant IL-2 (available from Chiron ~orp., Emeryville,
CA; specific activity of 6 to 8 x 106 U/mg protein; units
equivalent to 2-3 International U). After 3 days incubation
in IL-2, the cells can be collected, washed, counted to
determine the degree of proliferation, and resuspended in
media suitable for intravenous (i.v.) administration (e.g.,
physiological buffered saline solutions).
EXAMPLE 10
Immunotherapy
As noted previously, the present invention involves
stimulating cells ex vi~o, allowing them to differentiate
into tumor specific immune effector cells. The cells are
then reintroduced into the same host to mediate anticancer
therapeutic effects.
In this example, 8 to 12 week old female C57BL/6J (B6)
mice (Jackson Laboratory, Bar Harbor, ME) are injected i.v.
with approximately 3 x 105 MCA 205 tumor cells (i.e.,
methylcholanthrene-induced tumors of B6 origin provided by
Dr. James Yang, Surgery Branch, National Cancer Institute,
~ 094/201~ 7~7? PCT~S94/0~39
: -35-
Bethesda, MD) suspended in 1 ml of media to initiate
pulmonary metastases. These tumors can be routinely passed
in vivo in syngeneic mice and used within the third to
seventh transplantation generation.
On day 3, cells obtained from the mice as in Example 6
are stimulated ex vivo as in Example 9. Specifically, LN
cells draining progressively growing MCA 205 fibrosarcoma
for 12 d are stimulated with graded concentrations of SEA or
SEB for 2 d followed by culture in 4 U/ml of IL-2 for 3 d.
The antitumor efficacy of superantigen stimulated cells
is assessed by reinfusion. Mice may also be treated with
exogenous IL-2 to promote the growth of transferred cells
(i.p, with 15,000 U IL-2 in 0.5 ml buffered saline twice
daily for 4 consecutive days to promote the in vivo function
and survival of the stimulated cells). On day 20 or 21, all
mice can be randomized, sacrificed, and metastatic tumor
nodules on the surface of the lungs ~nllm~rated~
To identify V~ phenotypes of cells in the tumor-
draining LN before and after SEA and SEB stimulation, cells
can be stained with a collection of anti-v~ mAb. A
preferential stimulation of particular v~ T cell subsets by
different microbial superantigenic toxins would suggest the
possibility of antigenic specificity of the responding T
cells.
From the above, it should be clear that the present
invention provides a method for the treatment of cancer,
and, more specifically, for the treatment of solid tumors,
including their metastases, without radiation, surgery or
standard chemotherapeutic agents. The ex vivo stimulation
method has decided advantages over direct intravenous
injection of superantigens. Most importantly, success is
achieved with m; n;m~l host toxicity.
