WO1994020124A1 - Method of cancer treatment - Google Patents
Method of cancer treatment Download PDFInfo
- Publication number
- WO1994020124A1 WO1994020124A1 PCT/US1994/002339 US9402339W WO9420124A1 WO 1994020124 A1 WO1994020124 A1 WO 1994020124A1 US 9402339 W US9402339 W US 9402339W WO 9420124 A1 WO9420124 A1 WO 9420124A1
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- WIPO (PCT)
- Prior art keywords
- cells
- superantigens
- tumor
- enterotoxins
- vivo
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- C12N5/0636—T lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55544—Bacterial toxins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
Definitions
- the invention generally relates to the treatment of cancer, and, more specifically, to the treatment of solid tumors, including their metastases, without radiation, 15 surgery or standard chemotherapeutic agents.
- 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.
- epithelial tissues e.g., intestinal mucosa
- reproductive tissues e.g., impairment of spermatogenesis
- 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.
- 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.
- the stimulated cells are introduced into a different host.
- the cells are established as a cell line for continuous anticancer use.
- 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.
- conventional techniques e.g., DMSO, culture media, fetal calf serum, etc.
- an established cell line may be made from cancer-free hosts.
- the cell line can be stored as above.
- 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 minimal host toxicity using the in vi tro sensitization technique.
- 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.
- 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.
- 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.
- 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.
- 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 ⁇ -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 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.
- the method further comprises the step of administering human interleukin 2 to said patient in vivo after re-introducing said cells in step (e) .
- the superantigen may comprise the enterotoxin SEB at concentrations above approximately 0.010 ⁇ g/ml.
- 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.
- 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.
- 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.
- 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.
- tumoricidal reaction means that the tumor cells are killed, and is not meant to be limited to any particular method by which tumor cells are killed.
- 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.
- the stimulated cells while not secreting cytokines themselves, may cause changes in paracrine growth signals.
- metastatic cells receive and process negative paracrine growth signals, e.g., from molecules in the transforming growth factor- ⁇ family of cytokines.
- negative growth factors could determine metastatic cell growth at particular sites.
- 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.
- T cell stimulation by these toxins is believed to be dependent upon presentation by Major Histocompatability Complex (MHC) molecules.
- MHC Major Histocompatability Complex
- 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.
- the staphylococcal toxins are believed not to be
- the invention be limited by the origin or nature of the host cells.
- 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 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 vi tro stimulation.
- pre-effector cells can differentiate into functional immune cells upon further in vi tro 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).
- 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) .
- 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
- 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.
- enterotoxins produced by Staphylococcus aureus 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 IA and IB) .
- enterotoxins isolated from media which are supporting the growth of various Staphylococcus aureus organisms are used.
- enterotoxins of Staphylococcus aureus form a group of serologically distinct extracellular proteins, designated A, B, C lf C 2 , C 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.
- 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.
- 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 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 2-4) .
- SED is moderately related to both groups although it is more similar to the SEA group.
- Emetic dose (ED 50 ) ( ⁇ g/monkey) 5 5 5 5-10
- Emetic dose for monkey ( ⁇ g) 5 5 5 5-10 - -
- strain MN8 Isolated from strain MN8, as compared to the inferred amino acid composition of the TSST-1 structural gene.
- Residues per mole values are based on a molecular weight of 22,000.
- TSST-1 structural gene Blomster-Hautamaa and colleagues.
- ND Not determined.
- 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; TSST1, Staphylococcus aureus toxic shock - associated toxin; ETA and ETB, Staphylococcus aureus exfoliating toxins A and B.
- 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.
- 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.
- 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 TABLE 5 Amino Acid One-letter Symbol
- abdominal viscera which stimulate the emetic and diarrheal response. They are also believed to induce interferon, tumor necrosis factor, and interleukins 1 and 2.
- the increased effectiveness of higher doses of systemically introduced superantigens is correlated with higher toxicity.
- 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.
- 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.
- the criteria for superantigens 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.
- lymph nodes can be used.
- all types of lymph nodes are contemplated (inguinal, mesenteric, superficial distal auxiliary, etc.).
- 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 vi tro 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°C in a 5% C0 2 atmosphere) . 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.
- superantigens e.g
- the stimulated cells may be reintroduced to the host by a number of approaches. Preferably, they are injected intravenously.
- the host may be treated with agents to promote the in vivo function and survival of the stimulated cells (e.g., IL-2) .
- 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.
- 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 various cytokines and mitogens such as anti-CD3 antibody or conconavalin A.
- the present invention contemplates transfecting with superantigen genes into tumor cells to provide powerful augmenting signals to T cell stimulation.
- dual transfeetion with. superantigens and molecules such as B7 is contemplated.
- 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 vi tro 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.
- various superantigens may be employed sequentially to up-regulate the activity of one another.
- SEA which is known to be a powerful cytokine inducer
- 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.
- This example describes two purification approaches for Enterotoxins A and C 2 .
- the eluted toxin is concentrated and rerun on Sephadex-G-100. The overall recovery is about 30% for SEC 2 and 40 to 50% for SEA. Both toxins appear homogeneous by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) .
- Ap roach 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 ⁇ g/ml solution is tested in gel diffusion against a known antisera to SEA and 1 ⁇ g/ml is tested in PAGE and endotoxin in the Sigma-E-Toxate LAL assay
- This example describes a purification approach for Enterotoxins A and Cj 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 C x 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.
- FPLC fast protein liquid chromatography
- SEA elutes as two peaks at pH 8.8 and 8.6.
- SECj 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.
- 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.
- mutagenic agents such as N-Nitroso compounds are capable of augmenting significantly the production of enterotoxins by some strains of Staphylococcus.
- This example describes a purification approach for
- Staphylococcus aureus Wood 46 strain (Source: Dr. Sidney Harshman, Vanderbilt University, Nashville, TN) is used and cultured in yeast extract dialysate medium.
- undialyzed yeast may be used together with casein, glucose, thiamine and nicotinic acid.
- the organism is incubated in medium for 24h at 37°C.
- 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 ml/ in.
- the column is washed with 0.01M KHP0 4 pH 6.8 and then the alpha toxin is eluted with 1.0M KHP0 4 pH 7.5. Fractions are tested for the presence of alpha hemolysin by a rapid hemolytic assay using rabbit erythrocytes as substrate.
- 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 S x is precipitated with 80% saturated ammonium sulfate. The redissolved pellet is dialyzed and designated Fraction S 2 .
- Fraction S 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.
- Fraction (S 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 3 .
- Fraction S 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.
- ELISA enzyme-linked immunoabsorbant assay
- This example describes a general purification approach for native enterotoxins.
- Staphylococcus aureus strain 110-275 is cultured in NZ- Amine A media supplemented with 10 g/liter of yeast extract for 18-20 hours in room air at 37°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.
- 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 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°C. The final enterotoxin fraction is a white powder which, when dissolved in normal saline, is a clear colorless solution.
- SEB 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.
- 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.
- 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-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.
- 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 NH 4 C1, 1.0 g KHC0 3 , and 0.0372 g EDTA/liter, pH 7.4).
- the cells were washed twice with buffer and resuspended for stimulation.
- 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.
- 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.
- 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 si tu, and for this reason it is best divided with the later stage after mobilization of the splenic hilum.
- 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.
- 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.
- the surgeon 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.
- 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
- ammonium chloride/potassium lysing buffer (8.29 g of NH 4 C1, 1.0 g KHC0 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) .
- the host cells are obtained from tumor infiltrating lymphocytes.
- Lymphocytes infiltrating tumors are obtained using standard techniques.
- Solid tumors freshly resected or cryopreserved
- Solid tumors 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 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 ⁇ g/ml (Biofluids, Rockville, MD) , gentamicin 50 ⁇ g/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 inM (MA Bioproducts, Walkersville, MD) .
- Conditioned medium from 3- to 4-day autologous or allogeneic lymphokine-activated killer (LAK) cell cultures can be added at a final concentration of 20% (v/v) .
- Recombinany IL-2 (kindly supplied by the Cetus Corporation, Emeryville, CA) can be added at a final concentration of 1000 ⁇ /ml.
- Cultures are maintained at 37°C in a 5% C0 2 -humidified atmosphere.
- a variety of tissue culture vessels can be employed, including 24-well plates (Costar, Cambridge, MA) . 175 cm 2 flasks (Falcon; Becton Dickinson, Oxnard, CA) , 850 cm 2 roller bottles (Corning Glass Works, Corning, NY) , and 750 cm 2 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 10 6 cells/ml in fresh medium.
- peripheral blood lymphocytes PBL
- RPMI 1640 medium 2% human serum, antibiotics, glutamine, and HEPES buffer.
- Recombinant IL-2 is added at 1000 ⁇ /ml. Cultures are maintained for 3 to 7 days in a humidified 5% C0 2 atmosphere at 37°C.
- Tumor- draining lymph node (LN) cells are obtained as described in Example 7 and stimulated in vi tro in a procedure with an optional second step.
- Step One 4 * 10 6 LN cells, in 2 ml of culture medium containing SEA or SEB, are incubated in a well of 24-well plates at 37°C in a 5% C0 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 ⁇ M sodium pyruvate, 2 M 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 * 10 "5 M 2-ME (Sigma) . The cells were harvested and washed.
- Step Two The initially stimulated cells are further cultured at 3 * 10 5 /well in 2 ml of culture media with Human recombinant IL-2 (available from Chiron Corp., Emeryville, CA; specific activity of 6 to 8 * 10 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) .
- i.v. intravenous
- 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.
- mice 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 * 10 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.
- tumor cells can be routinely passed in vivo in syngeneic mice and used within the third to seventh transplantation generation.
- cells obtained from the mice as in Example 6 are stimulated ex vivo as in Example 9.
- 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) .
- 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.
- 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.
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EP94910831A EP0705104A4 (en) | 1993-03-02 | 1994-03-02 | Method of cancer treatment |
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WO1998045325A1 (en) * | 1997-04-07 | 1998-10-15 | The Rockefeller University | Peptides useful for reducing symptoms of toxic shock syndrome |
EP2887882A4 (en) * | 2012-08-24 | 2016-04-27 | Children S Hospital Of Orange County | Isolation of lymphocytes and delivery to splenectomy patients |
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Non-Patent Citations (4)
Title |
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CAMBRIDGE SCIENCE ABSTRACTS, issued 1993, SCHEGLOVITA et al., "Effect Staphylococcal Enterotoxin A-sensitized spleen Cells on Metastasizing of Mouse Lewis Carcinoma", abstract no. 1477534, Eksp. Onkol., 11(2), pages 54-56, see entire abstract. * |
INFECTION AND IMMUNITY, Volume 57, No. 7, issued July 1989, GARCIA-PENARRUBIA et al., "Selective prolifertion of Natural Killer Cells among Monocyte-Depleted peripheral Blood Mononuclear Cells as a Result of Stimulation with Staphylococcal Enterotoxin B", pages 2057-2065, see at least the Abstract. * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Volume 88, No. 3, issued February 1991, NEWELL et al., "In Vivo T-cell Activation by Staphylococcal Enetrotoxin B prevents Outgrowth of a Malignant Tumor", pages 1074-1078, see at least the Abstract. * |
THE NEW ENGLAND JOURNAL OF MEDICINE, Volume 313, No. 23, issued 05 December 1985, ROSENBERG et al., "Observations on the Systemic Administration of Autologous Lymphokine-Activated Killer Cells and Recombinant Interleukin-2 to Patients with Metastatic Cancer", pages 1485-1492, see at least the Abstract. * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998045325A1 (en) * | 1997-04-07 | 1998-10-15 | The Rockefeller University | Peptides useful for reducing symptoms of toxic shock syndrome |
US6075119A (en) * | 1997-04-07 | 2000-06-13 | The Rockefeller University | Peptides useful for reducing symptoms of toxic shock syndrome |
US7115268B1 (en) | 1997-04-07 | 2006-10-03 | The Rockefeller University | Peptides useful for reducing symptoms of toxic shock syndrome and septic shock |
EP2887882A4 (en) * | 2012-08-24 | 2016-04-27 | Children S Hospital Of Orange County | Isolation of lymphocytes and delivery to splenectomy patients |
US9452206B2 (en) | 2012-08-24 | 2016-09-27 | Children's Hospital Of Orange County | Isolation of lymphocytes and delivery to splenectomy patients |
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