U.S. patent application number 09/741503 was filed with the patent office on 2002-05-02 for method of cancer treatment.
Invention is credited to Terman, David S..
Application Number | 20020051765 09/741503 |
Document ID | / |
Family ID | 27534096 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020051765 |
Kind Code |
A1 |
Terman, David S. |
May 2, 2002 |
Method of cancer treatment
Abstract
Treatment of solid tumors, including their metastases, without
radiation, surgery or standard chemotherapeutic agents is
described. Ex vivo stimulation of cells, selection of specific
V.beta. subsets of stimulated cells and reinfusion of the V.beta.
subsets of stimulated cells is employed for cancer therapy.
Inventors: |
Terman, David S.; (Pebble
Beach, CA) |
Correspondence
Address: |
MARK S. ELLINGER, PH.D.
Fish & Richardson P.C., P.A.
Suite 3300
60 South Sixth Street
Minneapolis
MN
55402
US
|
Family ID: |
27534096 |
Appl. No.: |
09/741503 |
Filed: |
December 19, 2000 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09741503 |
Dec 19, 2000 |
|
|
|
08491746 |
Jun 19, 1995 |
|
|
|
08491746 |
Jun 19, 1995 |
|
|
|
08189424 |
Jan 31, 1994 |
|
|
|
5728388 |
|
|
|
|
08189424 |
Jan 31, 1994 |
|
|
|
08025144 |
Mar 2, 1993 |
|
|
|
08025144 |
Mar 2, 1993 |
|
|
|
07891718 |
Jun 1, 1992 |
|
|
|
07891718 |
Jun 1, 1992 |
|
|
|
PCT/US91/00342 |
Jan 17, 1991 |
|
|
|
PCT/US91/00342 |
Jan 17, 1991 |
|
|
|
07466577 |
Jan 17, 1990 |
|
|
|
07466577 |
Jan 17, 1990 |
|
|
|
07416530 |
Oct 3, 1989 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/372 |
Current CPC
Class: |
C12N 5/0636 20130101;
C12N 5/0693 20130101; A61P 37/04 20180101; A61P 35/00 20180101;
A61K 38/00 20130101; A61K 2039/5158 20130101; A61K 2039/55544
20130101; C07K 14/315 20130101; C12N 2510/00 20130101; A61K
2039/5152 20130101; A61K 39/001176 20180801; C07K 14/31 20130101;
C12N 2501/23 20130101 |
Class at
Publication: |
424/93.7 ;
435/372 |
International
Class: |
A61K 045/00; A61K
039/00; C12N 005/08 |
Claims
1. A method for inducing a tumoricidal reaction in vivo comprising
contacting cells with superantigens ex vivo, selecting a specific
V.beta. subset of stimulated cells and infusing them into a
tumor-bearing host.
2. A method of human cancer treatment comprising: a) providing a
human cancer patient; b) obtaining hematopoietic cells from said
patient; c) contacting said cells ex vivo with one or more
superantigens to generate stimulated cells; d) selecting a specific
V.beta. subset of said cells; and e) re-introducing said V.beta.
subset of cells into said patient so as to induce an in vivo
therapeutic, tumoricidal reaction.
3. A reagent for treating cancer, comprising a specific V.beta.
subset of T cells sensitized to a growing tumor and stimulated with
superantigens.
4. A method of human cancer treatment comprising: a) providing a
human cancer patient; b) obtaining hematopoietic cells from said
patient; c) contacting said cells ex vivo with one or more
superantigens to generate stimulated cells; d) depleting a specific
V.beta. subset of said cells said V.beta. subset having suppressor
effects on tumoricidal activity; and e) re-introducing said
stimulated cells depleted of said V.beta. subset into said patient
so as to induce an in vivo therapeutic, tumoricidal reaction.
Description
RELATED APPLICATION DATA
[0001] This application is a continuation-in-part application of
copending application Ser. No. 071891,718, filed Jun. 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 Jan. 17, 1990, which is
a continuation-in-part application of Application Serial No.
07/416,530, filed Oct. 3, 1989.
FIELD OF THE INVENTION
[0002] 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.
BACKGROUND
[0003] 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.
[0004] 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 Pharmacological
Basis of Therapeutics (Pergamon Press, 8th Edition) (pp.
1202-1204). This concept of "total cell kill" implies that total
excision of a tumor is necessary for a surgical approach, and
complete 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.
[0005] 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.
[0006] 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 Pharmacological Basis of Therapeutics (Pergamon Press,
8th Edition) (pp. 1209-1216).
[0007] Success with chemotherapeutics as anticancer agents has also
been hampered by the phenomenon 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
Andrew J. Coldman, Cancer Research, 44:3643-3653 (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
predominant drug-sensitive cells. With the drug-sensitive cells
gone, the remaining drug-resistant cells continue to multiply and
eventually dominate the cell population of the tumor.
[0008] 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.
[0009] 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 minimal host toxicity.
SUMMARY OF THE INVENTION
[0010] 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 cells are introduced
into a different host. In still a third embodiment, the cells are
established as a cell line for continuous anticancer use.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] The present invention demonstrates that superantigens can
reliably produce tumoricidal reactions to a wide variety of tumor
types. Moreover, success is achieved with minimal host toxicity
using the in vitro sensitization technique.
[0015] 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 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.
[0016] The present invention offers a method of human cancer
treatment comprising: a) providing a human cancer patient; b)
obtaining hematopoietic cells from said patient; c) contacting said
cells ex 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.
[0017] 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.
[0018] 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 human cancer treatment comprising: a) providing a human cancer
patient, having one or more growing tumors; b) obtaining
V.beta.-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.beta.-expressing T cells; d)
culturing said T cells in a 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 in 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).
[0019] For culturing, the superantigen may comprise the enterotoxin
SEB at concentrations above approximately 0.010 .mu.g/ml.
Preferably, the first culture media contains SEB at a concentration
of approximately 2 .mu.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
[0020] FIG. 1 schematically shows the therapeutic approach of the
present invention.
[0021] FIG. 2 shows a comparison of the primary sequences of the
staphylococcal enterotoxins and their relatives.
DESCRIPTION OF THE INVENTION
[0022] 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 FIG. 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.
[0023] 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.
[0024] 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-.beta. family of
cytokines. In conjunction with positive growth factors, the
negative growth factors could determine metastatic cell growth at
particular sites.
[0025] 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 "self" MHC molecule; allogeneic
antigen-presenting cells are equally effective. It is thought that
the essential requirement is that cells presenting the toxins
express MHC class II molecules, as these molecules specifically
bind the toxins.
[0026] The staphylococcal toxins are believed not to be "processed"
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 T cell receptors for antigen. Most
importantly, there is evidence that the staphylococcal toxins bind
to a site on the V.beta. 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.beta., D.beta., J.beta.,
V.alpha., and J.alpha. 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."
[0027] Antitumor effects may reside in specific subsets of T cells
with V.beta. phenotypes which may or may not have had prior
exposure to tumor. These clones may have been deleted in the course
of life by an antigenic stimulus or they may be genetically absent.
Superantigens have the capacity to activate selective V.beta.
subsets and expand their numbers significantly, alone or together
with IL-2. This stimulation may be carried out ex vivo with T cells
presensitized to the tumor in vitro or in vivo, and simultaneously
or sequentially incubated with various superantigens. These
stimulated T cell subsets can be collected selectively (e.g., with
a fluorescent or magnetic cell sorter), expanded in numbers with
agents such as IL-2 and reinfused into the host, producing a
tumoricidal reaction. It is possible that not one but several
V.beta. clones expanded by superantigens may work additively or
synergistically to enhance the antitumor effect.
[0028] It is possible that certain V.beta. subsets may exert
suppressor effects on the tumoricidal activity. Optimal antitumor
effects might be obtained after expansion of the V.beta. clones
having antitumor activity and depletion of the V.beta. clones
having suppressor activity, followed by reinfusion of the cells
into the host. Selection of the V.beta. clone to be expanded can be
obtained by analysis of V.beta. profiles of tumor infiltrating
lymphocytes as well as from lymph node and peripheral blood V.beta.
T cell profiles. Cytotoxic or tumoricidal activity in vitro of a
given V.beta. subset or enrichment of a V.beta. subset at a
specific tumor location following parenteral administration in vivo
might also assist in identification of the V.beta. subsets with
antitumor activity.
[0029] Tumor specific V.beta. subsets may show different cytokine
secreting profiles depending on the superantigen employed for
stimulation. A preponderance of interferon .gamma. production by
tumor specific T cells stimulated by a given superantigen may
render these cells more potent tumoricidal agents, compared to
another V.beta. subset stimulated by a different superantigen.
[0030] 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-DRAINING LYMPH NODES
[0031] 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 "pre-effector" cells, can differentiate into
functional immune cells upon further in vitro stimulation. Several
culture techniques 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., 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.
SUPERANTIGENS AS STIMULATING AGENTS
[0032] 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.
[0033] 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 1A and 1B).
According to one aspect of the present invention, enterotoxins
isolated from media which are supporting the growth of various
Staphylococcus aureus organisms are used.
[0034] The enterotoxins of Staphylococcus aureus form a group of
serologically distinct extracellular proteins, designated A, B,
C.sub.1, C.sub.2, C.sub.3, D, E and F. These proteins are
recognized as the causative agents of Staphylococcal food
poisoning. Enterotoxin F appears to be important in the
pathogenesis of the Staphylococcal toxic shock syndrome.
[0035] 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.
[0036] The protein sequences and immunological 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 24). 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.
1TABLE 1A Some Properties Of The Enterotoxins Enterotoxin A.sup.a
B.sup.b C.sub.1.sup.c C.sub.2.sup.d Emetic dose (ED.sub.50)
(.mu.g/monkey) 5 5 5 5-10 Nitrogen content (%) 16.5 16.1 16.2 16.0
Sedimentation coefficient (S.sub.20,w) 3.04 2.78 3.00 2.90 (S)
Diffusion coefficient (D.sub.20,w) 7.94 8.22 8.10 8.10 (x 10.sup.-7
cm.sup.2 sec.sup.-1) Reduced viscosity (ml/g) 4.07 3.81 3.4 3.7
Molecular weight 34,700 30,000 34,100 34,000 Partial specific
volume 0.726 0.726 0.728 0.725 Isoelectric point 6.8 8.6 8.6 7.0
Maximum absorption (m.mu.) 277 277 277 277 Extinction (E.sub.1
cm.sup.1%) 14.3 14.4 12.1 12.1 .sup.aF. S. Thadhani et al.,
Biochem., 5:3281 (1966). .sup.bM. S. Bergdoll et al., J.
Bacteriol., 90:1481 (1965). .sup.cC. R. Borja and M. S. Bergdoll,
Biochem., 6:1467 (1967). .sup.dR. M. Avena and M. S. Bergdoll,
Biochem. 6:1474 (1967).
[0037]
2TABLE 1B Physicochemical Properties Of Staphylococcal
Enterotoxins* Enterotoxin Property A.sup.a B.sup.b C.sub.1.sup.c
C.sub.2.sup.d D.sup.c E.sup.f Emetic dose for monkey (.mu.g) 5 5 5
5-10 -- -- Sedimentation coefficient 3.03 2.89 3.0 2.9 -- 2.6
(S.sub.20,w) Molecular weight 27,800 28,366.sup.g 26,000 34,100
27,300 29,600 Isoelectric point 7.26 8.6 8.6 7.0 7.4 7.0 C-terminal
residue Serine Lysine Glycine Glycine Lysine Threonine N-terminal
residue Alanine Glutamic Glutamic Glutamic Serine -- acid acid acid
.sup.aE. J. Schantz et al., Biochem., 11:360 (1972). .sup.bE. J.
Schantz et al., Biochem. 4:1011 (1965). .sup.cC. R. Borja and M. S.
Bergdoll, Biochem., 6:1467 (1967). .sup.dR. M. Avena and M. S.
Bergdoll, Biochem. 6:1474 (1967). .sup.eP. C. Chang and M. S.
Bergdoll, Biochem., 18:1937 (1979). .sup.fC. R. Borja et al., J.
Biol. Chem., 247:2456 (1972). .sup.gData Section in Atlas Protein
Sequence Structure 5:D227, (M. Dayhoff, ed.), National Biomedical
Research Foundation, Washington D.C. (1972) (determined 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
Recent Advances in Staphylococcal Research, (W. W. Yotis, ed.),
Ann. N.Y. Acad. Sci., 236:307-316.
[0038] 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 regions of sequence similarity exist.
[0039] Comparison of the primary sequences of the staphylococcal
enterotoxins and their relatives is shown in FIG. 2. The complete
primary amino acid sequences of 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 remaining toxins. The exfoliating
toxins have properties related to those of the others.
3TABLE 2* Sequence Similarities Among The Pyrogenic Toxins And
Enterotoxins Toxin Sequence 106.sub.--------------------------119
147.sub.---------------------------- -------163 SEA CMYGGVTLHDNNRL
KKNVTVQELDLQARRYL SEB CMYGGVTEHHGNOL KKKVTAQELDYLTRHYL SEC1
CMYGGITKHEGNHF KKSVTAQELDIKARNFL SED CTYGGVTPHEGNKL
KKNVTVQELDAQARRYL SEE CMYGGVTLHDNNRL KKEVTVQELDLQARHYL SPEA
CIYGGVTNHEGNHL KKMVTAQELDYKVRKYL Consensus CMYGGVTLHEGNHL
KKNVTAQELD.sub.Y.sup.LQ- AR.sub.H.sup.RYL TSST-1 IHFQISGVTNTEKL
KKQLAISTLDFEIRHQL *J. J. Iandolo, Ann. Rev. Microbiol., 43:375
(1989).
[0040]
4TABLE 3 Amino Acid Composition Of The Enterotoxins (g/100 g
Protein) Enterotoxin Amino Acid A* B.dagger. C.sub.1.dagger-dbl.
C.sub.2.dagger-dbl. E.sctn. Lysine 11.26 14.85 14.43 13.99 10.83
Histidine 3.16 2.34 2.91 2.87 3.04 Arginine 4.02 2.69 1.71 1.75
4.50 Aspartic acid 15.53 18.13 17.85 18.38 15.10 Threonine 5.96
4.50 5.31 5.80 6.36 Serine 2.99 4.05 4.58 4.81 4.72 Glutamic acid
12.36 9.45 8.95 8.93 12.15 Proline 1.35 2.11 2.16 2.23 1.93 Glycine
2.96 1.78 2.99 2.90 4.10 Alanine 1.94 1.32 1.85 1.61 2.38
Half-cysteine 0.66 0.68 0.79 0.74 0.81 Valine 4.93 5.66 6.50 5.87
4.36 Methionine 0.96 3.52 3.20 3.60 0.45 Isoleucine 4.11 3.53 4.09
4.02 4.30 Leucine 9.78 6.86 6.54 6.13 10.08 Tyrosine 10.63 11.50
9.80 10.27 9.79 Phenylalanine 4.31 6.23 5.35 5.25 4.47 Tryptophan
1.46 0.95 0.99 0.84 1.51 Amide NH.sub.3 1.80 1.66 1.71 1.62 1.66
TOTAL 98.37 100.15 100.00 99.99 100.88 *Schantz et al., 1972.
.dagger.M. S. Bergdoll et al., Arch Biochem Biophys, 112:104
(1965). .dagger-dbl.I. Y. Huang et al., Biochem., 6:1480 (1967).
.sctn.Borja et al., 1972. .paragraph.M. S. Bergdoll et al., Agric.
Food Chem., 22:9 (1974).
[0041]
5TABLE 4.sup..dagger. Amino Acid Compositions Of TSST-1a And
1b.sup.a Amino acid composition TSST-1a residues TSST-1b residues
Amino acid per mole.sup.b per mole.sup.b Clone.sup.b 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 0 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 ND.sup.d ND.sup.d 3 Arginine 4 5 4 TOTAL 197 199 194
.sup..dagger.D. A. Blomster-Hautamaa and P. M. Schlievert, Meth.
Enzym., 165:37 (1988). .sup.aIsolated from strain MN8, as compared
to the inferred amino acid composition of the TSST-1 structural
gene. .sup.bResidues per mole values are based on a molecular
weight of 22,000. .sup.cResidues per mole inferred from the DNA
sequence of the TSST-1 structural gene. Blomster-Hautamaa and
colleagues. .sup.dND. Not determined.
[0042] The toxins shown in FIG. 2 are as follows: SEA to SEE,
Staphylococcus aureus enterotoxins A to E; SPE A and C,
Streptococcus pyogenes toxins A and C; TSST1, 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.
[0043] 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.
[0044] 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).]
[0045] 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); and D. J. Lipman and W. R. Pearson, Science,
227:1435-1441 (1985).
[0046] 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 SUPERANTIGENS
[0047] 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).
[0048] 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 abdominal viscera which stimulate
the emetic and diarrheal response. They are also believed to induce
interferon, tumor necrosis factor, and interleukins 1 and 2.
[0049] 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.
6 TABLE 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 I Leucine L Lysine K Methionine M
Phenylalanine F Proline P Serine S Threonine T Tryptophan W
Tyrosine Y Valine V
[0050]
7TABLE 6 10 20 30 40 50
STR-PKPSQLQRSNLVKTFKIYIFFMRVTL-----VTHENVKSVDQLL- SHDLIYNVS-- : :::
: : : : : : : : :::: :::: :
ESQPDPKPDELHKSS--K-FTGLMENMKV-LYNNDHVSAINVKSINEFF--DLIYLYSIK 10 20
30 40 50 60 70 80 90
----GPNYDKLKTELKNQEMATLFKDKNVDIYGVEYYHLCYLC---------ENAERSAC : :::
: :: : ::: :: : :: :: :: : :
DTKLG-NYDNVRVEFKNKDLADKYKDKYVDVFGANYYQ-CYFSKKTNNIDSHENTKRKTC 60 70
80 90 100 110 100 110 120 130 140 150
LYGGVTNHEGNHLEIPKK----IVVKVSIDGIQSLSFDIEQIKNGNCSRIS-YTVRKYLT :::::
: : : : : : : :: :::: : : : ::
MYGGVTEHGNNQLD---KYYRSITVRVFEDGKNLLSFDVQTNKKKVTAEQLDYLTRHYLV 120
130 140 150 160 160 170 180 190 200 DNKQLYTNGPSKYETGYIKFIPKN-
KESFWFDFFPEPE--FTQSKYLMIYKDNETLDSNTS :: :: : ::::::::: : ::: : : :
: :::::: : :: KNKKLYEFNNSPYETGYIKFIE-NENSFWYDMMPAPGN-
KFDQSKYLMMYNNDKMVDSKDV 170 180 190 200 210 220 220 QIEVYLTTK
:::::::: KIEVYLTTKKK 230
[0051] The present invention contemplates avoiding the undesirable
effects, but nonetheless harnessing the valuable characteristics of
superantigens. Preferably, there is no significant systemic
exposure to superantigens using the ex vivo stimulation approach of
the present invention.
[0052] 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.
[0053] 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.beta. 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
[0054] 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.
[0055] 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 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
37.degree. C. in a 5% CO.sub.2 atmosphere).
[0056] Following the incubation, the stimulated cells are harvested
and washed with synthetic media containing 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.
[0057] 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).
[0058] 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
[0059] 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.beta. 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 various cytokines and mitogens such as anti-CD3
antibody or conconavalin A.
[0060] 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.
[0061] Substances which increase the number of antigen-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.
[0062] Differences of antitumor reactivities between SEA and SEB
stimulated cells probably represent distinct T cell subsets with
V.beta. phenotypes responding to these two superantigens. If a
population of T cells with specific V.beta. phenotype appears to
mediate the antitumor effects, selective depletion of the
ineffective subsets and expansion of the fraction of effective
subset(s) can be carried out with immunomagnetic beads or
monoclonal antibodies. Alternatively, if a major tumor-killing
V.beta. subtype T cell population is found to be deleted, that
population may be reconstituted with T cells transfected with the
specific V.beta. genes by various transfection techniques now in
use in the field. Such a reconstituted T cell clone can be
stimulated with appropriate tumor antigen in vitro or in vivo to
create a presensitized T cell population and then with enterotoxin,
plus antigen presenting cell APC stimulus. After expansion in IL-2,
this reconstituted T cell clone would be expected to restore T cell
function and antitumor activity to the deleted clone.
[0063] 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.beta. repertoire.
[0064] In a canine model using the Protein A collodion charcoal
(PACC) system described in a series of patent applications (for
example, U.S. patent application Ser. No. 331,095, the forerunner
of the present invention), therapeutic success was transferred to
humans in protocols in which objective tumor regressions were
obtained in four of the first five consecutive patients treated.
Thus, the data given herein also is expected to be predictive of
success when the compositions are applied to humans.
EXPERIMENTAL
[0065] 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.
[0066] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); .mu.M
(micromolar); mM (millimolar); N (Normal); mol (moles); mmol
(millimoles); .mu.mol (micromoles); nmol (nanomoles); g (grams); mg
(milligrams); .mu.g (micrograms); L (liters); ml (milliliters);
.mu.l (microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers); .degree. C. (degrees Centigrade);
mAb (monoclonal antibody); MW (molecular weight); U (units); d
(days).
EXAMPLE 1
Production and Isolation of Enterotoxins
[0067] This example describes the preparation of enterotoxins. The
preparation of enterotoxin has been described in detail,
previously; specifically, in patent application, Ser. Number
07/891,718, filed Jun. 1, 1992, the entire contents of which are
hereby incorporated by reference.
[0068] This example describes two purification approaches for
Enterotoxins A and C.sub.2.
[0069] Approach 1:
[0070] A 10 ml culture of Staphylococcus aureus 11N-165 (SEA),
Staphylococcus aureus 361 (Source: Dr. John Iandolo, Kansas State
University, Manhattan, Kans.) (SEC.sub.2) is grown overnight at
37.degree. C. 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-100 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-100. The overall recovery is about 30% for SEC.sub.2 and
40 to 50% for SEA. Both toxins appear homogeneous by sodium
dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).
[0071] Approach 2:
[0072] 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.degree. 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
.mu.g/ml solution is tested in gel diffusion against a known
antisera to SEA and 1 .mu.g/ml is tested in PAGE and endotoxin in
the Sigma-E-Toxate LAL assay.
EXAMPLE 2
Production and Isolation of Enterotoxins
[0073] This example describes a purification approach for
Enterotoxins A and C.sub.1 and D.
[0074] 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 C.sub.1 and D, the supernatants are passed over an
AmberLite-CG-50 column, as described for SED, and the active
fractions pooled. All three toxins are then placed in buffer for
chromatofocusing and then separated 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, SEC.sub.1 and SED is estimated to be 98, 95 and 80%,
respectively. SEA elutes as two peaks at pH 8.8 and 8.6. SEC.sub.1
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 8.0.
[0075] 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 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
[0076] This example describes a purification approach for Alpha
Toxin.
[0077] Staphylococcus aureus Wood 46 strain (Source: Dr. Sidney
Harshman, Vanderbilt University, Nashville, Tenn.) 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 24 h at 37.degree. C.
[0078] The culture supernatant is applied to a glass-pore bead
column and adjusted to pH 6.8. A column of 5.times.20 cm is used
for 3 liter batches and flow rates adjusted to 10-20 ml/min. The
column is washed with 0.01M KHPO.sub.4 pH 6.8 and then the alpha
toxin is eluted with 1.0M KHPO.sub.4 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
[0079] This example describes a purification approach for
Streptococcal Pyrogenic Exotoxin (SPE).
[0080] 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 S.sub.1 is precipitated with 80% saturated ammonium
sulfate. The redissolved pellet is dialyzed and designated Fraction
S.sub.2. This fraction is precipitated with 50-80% ammonium
sulfate, resuspended in phosphate buffered saline (Fraction
S.sub.3), 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.sub.4). 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 (S.sub.5)
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. Both forms show total cross reactivity against
immune sera raised by rabbit immunization with fraction
S.sub.3.
[0081] The Fraction S.sub.5 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
[0082] This example describes a general purification approach for
native enterotoxins.
[0083] 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.
[0084] 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 37.degree. C. The flask is agitated at 300
RPM. The initial pH of the culture is 6.8 and the postincubation pH
8.0. 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
H.sub.2O (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 column and the toxin is
eluted with 0.5 M PB, 0.5 M NaCl pH 6.2. The concentrated, dialyzed
toxin is placed in a column (5 cm.times.75 cm) of CM-sepharose
(pretreated with 0.005 M PB pH 5.6). The column 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 4.degree. C. The final
enterotoxin fraction is a white powder which, when dissolved in
normal saline, is a clear colorless solution. Samples containing 5
and 10 .mu.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 immunocytes, 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.
[0085] PAGE gel analysis of SEB showed a predominant single band at
28,000 m.w. High performance liquid 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 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.
[0086] 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 contamination using Sigma E-toxate LAL assay.
The final product was found to be free of endotoxin with a standard
sensitivity of 0.1 ug endotoxin/mg SEB.
[0087] 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, Calif.), 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 .mu.g/kg enterotoxin B.
[0088] SEA, SEC, SED, SEE, TSST-1 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.
EXAMPLE 6
Isolation of Host Cells: Lymph Nodes
[0089] 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.
[0090] 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, N.Y.), centrifuged and washed. Red cells, if evident, are
lysed by treatment with ammonium chloride-potassium lysing buffer
(8.29 g NH.sub.4Cl, 1.0 g KHCO.sub.3, 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
[0091] 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.
[0092] 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.
[0093] Anterior Method.
[0094] 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.
[0095] 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.
[0096] 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 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.
[0097] Posterior Method
[0098] 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.
[0099] Cell Suspensions
[0100] Spleen cells are mechanically dissociated by using the blunt
end of a 10-ml plastic syringe in buffer. The cell suspension was
passed through a single layer of 100-gauge nylon mesh (Nitex;
Lawshe Industrial Co., Bethesda, Md.) and centrifuged, and the RBC
lysed by resuspension of the cell pellet in ammonium
chloride/potassium lysing buffer, (8.29 g of NH.sub.4Cl, 1.0 g
KHCO.sub.3 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
[0101] In this example, the host cells are obtained from tumor
infiltrating lymphocytes. Lymphocytes infiltrating tumors are
obtained using standard 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, N.C.). The gradient interfaces contain
viable tumor cells and mononuclear cells are washed, adjusted to a
total cell concentration of 2.5 to 5.0.times.10.sup.5 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 .mu.g/ml (Biofluids, Rockville, Md.),
gentamicin 50 .mu.g/ml (GIBCO Laboratories, Chagrin Falls, Ohio),
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 supplied by the Cetus
Corporation, Emeryville, Calif.) can be added at a final
concentration of 1000 .mu./ml.
[0102] Cultures are maintained at 37.degree. C. in a 5%
CO.sub.2-humidified atmosphere. A variety of tissue culture vessels
can be employed, including 24-well plates (Costar, Cambridge,
Mass.). 175 cm.sup.2 flasks (Falcon; Becton Dickinson, Oxnard,
Calif.), 850 cm.sup.2 roller bottles (Corning Glass Works, Corning,
N.Y.), and 750 cm.sup.2 gas-permeable culture bags (Fenwal
Laboratories, Division of Travenol Laboratories, Deerfield, Ill.).
Cultures should be fed weekly by harvesting, pelletting and
resuspending cells at 2.5.times.10.sup.6 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.
[0103] 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.times.10.sup.6/ml in RPMI 1640 medium with 2%
human serum, antibiotics, glutamine, and HEPES buffer. Recombinant
IL-2 is added at 1000 .mu./ml. Cultures are maintained for 3 to 7
days in a humidified 5% CO.sub.2 atmosphere at 37.degree. C.
EXAMPLE 9
Ex Vivo Stimulation
[0104] 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.
[0105] Step One.
[0106] For stimulation, 4.times.10.sup.6 LN cells, in 2 ml of
culture medium containing SEA or SEB, are incubated in a well of
24-well plates at 37.degree. C. in a 5% CO.sub.2 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 .mu.M sodium pyruvate, 2 mM freshly prepared
L-glutamine, 100 .mu.g/ml streptomycin, 100 U/ml penicillin, 50
.mu.g/ml gentamicin, 0.5 .mu.g/ml fungizone (all from GIBCO, Grand
Island, N.Y.) and 5.times.10.sup.-5 M 2-ME (Sigma). The cells were
harvested and washed.
[0107] Step Two.
[0108] The initially stimulated cells are further cultured at
3.times.10.sup.5/well in 2 ml of culture media with Human
recombinant IL-2 (available from Chiron Corp., Emeryville, Calif.;
specific activity of 6 to 8.times.10.sup.6 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
[0109] As noted previously, the present invention involves
stimulating 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.
[0110] 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.times.10.sup.5 MCA 205 tumor cells (i.e.,
methylcholanthrene-induced tumors of B6 origin provided by Dr.
James Yang, Surgery Branch, National Cancer Institute, 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.
[0111] 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.
[0112] 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
enumerated.
[0113] To identify V.beta. 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.beta. mAb. A
preferential stimulation of particular V.beta. T cell subsets by
different microbial superantigenic toxins would suggest the
possibility of antigenic specificity of the responding T cells.
EXAMPLE 11
Immobilized Superantigens for Sustained Delivery After Plasma
Perfusion
[0114] Previous studies have shown that enterotoxins are present in
commercial preparations of protein A produced by either enzymatic
digestion of whole bacteria or by secretion into culture media.
Indeed, the IgG used in affinity chromatography to isolate protein
A has now been shown to contain the complete library of antibodies
to virtually all enterotoxins. Following perfusion with plasma,
plasma products or whole blood over enterotoxins immobilized on
biocompatible support matrices, enterotoxins are released whether
they were immobilized via covalent or non-covalent binding.
[0115] Enterotoxins or superantigens may be immobilized by
non-covalent or covalent methods such as adsorption or carbodiimide
on inert supports such as collodion charcoal or silica, as
previously described (U.S. Pat. No. 5,091,091, issued Feb. 25, 1992
to Terman). After plasma or blood product perfusion, the bound
enterotoxins are released in a graded fashion over a 15 minute to 3
hour period. Toxicity associated with this procedure has been
described in detail previously (Terman, 1984) and is manageable
with corticosteroids and occasionally with low dose dopamine
infusions. Hence, the immobilized enterotoxins may represent
another safe and effective mode of administration of enterotoxins
to patients.
[0116] In this example, enterotoxins are provided for intravenous
adminstration by displacement chromatography from immobilized
surfaces after plasma or plasma component perfusion. Enterotoxins
are immobilized on solid surfaces by carbodiimide chemistry or
adsorbed by adsorptic chemistry on solid supports. Surfaces include
silica, glass, cellulose, agarose, polystyrene and methacrylate.
Perfused fluids can be selected from a group containing albumin,
immunoglobulins or other plasma proteins. For covalent attachments,
the carbodiimide may be incubated with enterotoxin before addition
to the derivatized surface in order to prepolymerize the molecule.
The solid support may be derivatized with a silanizing agent prior
to addition of the polymerized enterotoxins. Other bifunctional
agents may be used such as glutaraldehyde, etc. It is important
that the binding of the enterotoxin to the solid support not be
irreversible so as to interdict displacement of the bound protein
by the perfused fluid.
[0117] Inert matrices such as glass, silica, agarose, polystyrene,
polyacrylamide may be used. Examples of peptide binding using
silica as the inert support and carbodiimide as the coupling agent
are given below.
[0118] The silica is derivatized with the amino group as the
reactive functional sites as follows:
[0119] (a) The silica is acid washed, followed by extensive rinsing
with water and drying. The silica is then reacted with a 5-10%
solution of aminosilane such as .gamma.-aminopropyltriethoxysilane
with pH adjusted to approximately 3.0 for 2 hours at 75.degree. C.
after which the matrix is again washed extensively with water and
dried overnight at 100.degree..
[0120] (b) Carboxyl groups are introduced to the amino-derivatized
material by mixing the silica matrix with succinic anhydride in a
suitable buffer, such as 0.5M phospate buffer with pH adjusted to
6.0 and held for 12-16 hours at room temperature after which the
matrix is extensively washed and dried.
[0121] (c) Hydroxyl groups may be added by addition of a silane
such as .gamma.-glycidoxylpropyltrimethoxysilane for 2 hours at
75.degree. C. The silica matrix is then washed and dried at
100.degree. C.
[0122] (d) The derivatized silica matrix may be reacted with
enterotoxins in the presence of carbodiimide to form a covalent
linkage. The binding reaction for the amino-derivatized matrix is
as follows:
[0123] Enterotoxin is mixed in water in the presence of
carbodiimide. The pH of the solution is adjusted to the range from
3.5 to 4.5, usually about 3.5, and the silica matrix is introduced
and mixed for 5 to 30 hours at room temperature. The matrix is
washed, dried and acid washed at pH 2.0 to 2.5 to remove labile
protein and other substances noncovalently bound, followed by
washing and drying.
[0124] (e) The binding process for carboxyl-derivatized silica is
as follows: A carbodiimide is dissolved in water and the solution
is adjusted to pH 3.5 to 4.5. The silica matrix is introduced and
the solution is stirred for 10 to 25 hours at room temperature. The
silica matrix is then removed and washed with water. The
enterotoxins are then dissolved in water, pH adjusted to 3.5 to 4.5
and the silica matrix added and mixed for 15-30 hours. The silica
matrix is then washed with water and dried, washed once in acid pH
2.0 to 2.5, then washed and dried.
[0125] Enterotoxins may be immobilized on inert solid surfaces by
passive adsorption. Noncovalent coating may involve hydrophobic
interactions, hydrogen bonds, ionic bonds, or protein-protein
interactions. Inert surfaces for adsorption may include polystyrene
balls or beads, silica, collagen or celluloric membranes.
Noncovalent adsorption of proteins proceeds with little difficulty
in a wide range of buffer conditions. The inert common procedures
recommend 50 mM sodium carbonate at a pH between 9.2 and 9.6.
However, a PBS buffer (10 mM sodium phosphate, 0.15 M NaCl), pH
7.2, or a TBS buffer (10 mM Tris, 0.15 M NaCl), pH 8.5 often works
just as well. Components of buffers that compete for hydrophobic
adsorption sites on plastic surfaces should be avoided. Detergents
such as TRITON X-100, TWEEN 20 or NP-40 should be avoided since
they will bind to the surface better than antibody molecules will.
Coating should be done at 4.degree. C. for at least 18 hours. At
37.degree. C., the coating procedure may be complete within 90
minutes.
[0126] The method of adsorption is as follows:
[0127] 1. An enterotoxin solution is prepared from purified
enterotoxins at a concentration of 1-10 .mu.g/ml in 50 mM sodium
carbonate, pH 9.5. For a 96-well plate, a 20-ml solution will be
sufficient to dispense 150 .mu.l per well with enough extra
solution to properly pipette with a multichannel pipetter. For
coating polystyrene beads or balls, enough coating solution is made
to fully immerse the balls in the antibody.
[0128] 2. Antibody solution is added (150 .mu.l) to each well of a
microtiter plate. Alternatively, the polystyrene balls are
submerged in the antibody solution.
[0129] 3. The reaction is incubated at 4.degree. C. for at least 18
hrs, or at 37.degree. C. for at least 90 minutes.
[0130] 4. The plates or balls are washed at least five times with
PBS, pH 7.2, containing 0.05% TWEEN 20. Aliquots of 250 .mu.l of
each wash solution are added to the wells of the microplate. Beads
or balls, are immersed in wash solution and incubated for several
minutes. Then they are removed from the solution.
EXAMPLE 12
Preparation of Polymerized Enterotoxins and Antigen-antibody
Oligomers
[0131] Enterotoxins may be polymerized by incubation with several
well known bifunctional agents such as glutaraldehyde,
carbodiimide, and heterobifunctional agents such as sulfo-SMCC and
the like. The resultant oligomers may then be separated on a size
chromatography column, concentrated and stored for subsequent
use.
[0132] Enterotoxin-antibody complexes can be prepared by incubation
with various dilutions of antibodies. The resulting conjugates may
be further separated by size gel chromatography and sucrose density
gradients. Biological activity of the conjugates can be tested in
murine and human T cell mitogenesis assays.
[0133] 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 minimal host toxicity.
* * * * *