EP1453468A2

EP1453468A2 - Method of vaccinating a human patient to prevent metastatic tumors

Info

Publication number: EP1453468A2
Application number: EP02778802A
Authority: EP
Inventors: Ning Y. Chiron Corporation YU; Jian-Min Chiron Corporation LIN; Robert J. Chiron Corporation TRESSLER; Richard E. Chiron Corporation JONES
Original assignee: Chiron Corp
Current assignee: Novartis Vaccines and Diagnostics Inc
Priority date: 2001-11-09
Filing date: 2002-11-08
Publication date: 2004-09-08
Also published as: WO2003041645A3; WO2003041645A2; EP1453468A4; AU2002340439A1

Abstract

Methods of preventing or inhibiting metastatic tumors in an immunocompetent human patient are provided. Specifically, methods of this invention involved identifying one or more solid tumors in a human patient wherein said tumors are accessible by injection techniques. A first does of a pharmaceutical composition is administered to said solid, accessible tumor or tumors in an amount effective to initiate an immune response to the tumor or tumors. The immune response initiated by the methods of this invention may also exhibit immunological memory.

Description

METHOD OF VACCINATING A HUMAN PATIENT TO PREVENT METASTATIC TUMORS

BACKGROUND OF THE INVENTION Field of the Invention

This invention is directed to methods of preventing or inhibiting an immune response to a solid tumor in an immunocompetent human patient. The methods of this invention involve administering a pharmaceutical composition comprising a sufficient amount of a cytotoxic agent, such as a platinate drug, to at least one tumor in a patient to initiate an immune response, particularly an immune response with immunological memory which can prevent or inhibit the growth of other, non-treated tumors and/or tumor metastasis.

State of the Art

Metastatic cancer involves the spread of cancer from an original tumor site to another location within a human patient. For example, cancer cells may be released from the original tumor site and travel through the bloodstream or lymphatic system to lymph nodes near the original tumor, or to regions distant from the original tumor. Alternatively, invasive procedures such as diagnostic biopsies run the risk of physically transporting cancer cells to adjacent and otherwise healthy regions resulting in the risk of metastasis. Metastasis is typically treated with chemotherapy, radiation therapy, biological therapy, hormone therapy, surgery, or a combination of these. In cases where the cancer has metastasized to regions that are inaccessible or inappropriate for surgery and/or radiation, methods that treat the entire body, such as systemic chemotherapy, may be required. Another reason for selecting a systemic treatment regime is the risk that metastatic tumors may be present in other parts of the body, but cannot be detected using conventional techniques, due to their location and/or size.

However, treatment methods such as systemic chemotherapy have several drawbacks. First, it is difficult to obtain a high concentration of the chemotherapy drug, localized within the tumor over time. Using conventional techniques, only approximately 1-2% of an administered chemotherapy dose reaches the tumor. Another disadvantage of systemic chemotherapy is that often chemotherapy drugs are not specific to the cancer cells, and thus may also harm healthy cells throughout the body, resulting in a wide range of serious side effects. While most chemotherapy drugs are intended to disproportionately affect the rapidly proliferating cancer cells, there are also other organs, particularly bone marrow, which involve rapid proliferation of stem cells. Therefore, systemic chemotherapy may also have a particularly pernicious effect on the immune system.

Compositions and methods for localizing chemotherapeutic drugs to a particular tumor site are described in several commonly-assigned U.S. Patents.^1"9 Such treatments include, for example, the injection of cytotoxic agents dispersed in a physiologically acceptable proteinaceous matrix, such as collagen, into the solid cellular growth or area of an existing or removed solid cellular growth. These treatments are useful for directing the activity of the cytotoxic agents toward the tumor cells, while protecting sensitive normal cells from the harmful effects of the drug.

Despite such developments, there remain instances where additional tumors and/or metastasis of the same cell type can go undetectable and/or are inaccessible by injection techniques. Therefore, there is a need for new methods for preventing or inhibiting the growth or development of such tumors and/or metastasis. Preferably, such methods would provide increased efficacy, reduced toxicity, improved specificity and improved quality of life.

SUMMARY OF THE INVENTION This invention is to the novel and surprising discovery that administration of a pharmaceutical composition comprising an effective amount of a cytotoxic agent, such as a platinate drug, to one or more tumors in an immunocompetent subject initiates an immune response to the tumor and prevents or inhibits the growth of other tumors and/or metastasis in the same host. In a preferred embodiment, such immune response has immunological memory.

More particularly, the initiation of an immune response to the tumor permits the prevention and/or inhibition of metastatic tumors in an immunocompetent human patient; permits the treatment of inaccessible tumors in an immunocompetent human patient who has at least one accessible tumor and at least one inaccessible tumor wherein the accessible tumor and the inaccessible tumor are of the same cell type or of similar immunological profile; permits the treatment of a immunocompetent human patient having so many tumors that it is not possible to treat all of the tumors by local techniques such as regional chemotherapy, surgery and/or radiation therapy; and permits the prevention and/or inhibition in the development and/or growth of future tumors in an immunocompetent human patient wherein the future tumors would be of the same cell type or similar immunological profile.

Specifically, the methods of this invention include: (a) identifying one or more solid tumors in an immunocompetent human patient wherein at least one of said tumors is accessible by injection techniques; and (b) administering a first dose of a pharmaceutical composition to said solid, accessible tumor or tumors wherein said pharmaceutical composition comprises an effective amount of a cytotoxic agent to initiate an immune response to said tumor or tumors in said patient. The immune response initiated by the methods of this invention preferably exhibits immunological memory which prevents or inhibits the growth of other, non-treated tumors and metastases of the original tumor in the manner set forth above.

In a preferred embodiment, the methods of the invention may further comprise (c) administering a second dose of said pharmaceutical composition to said accessible tumor or tumors, under conditions wherein said administration initiates or reinforces an immune response to the accessible tumor in said patient, wherein said second dose is administered from about 1 to about 4 weeks after administering the first dose of the pharmaceutical composition. The methods of the invention are preferably used to treat an immunocompetent human patient who has had less than about two prior chemotherapy treatments and/or less than about two prior radiation treatments. The tumor or tumors may be partially or completely surgically excised.

The generation of an immune response against a tumor as described herein can form the basis for treating a second tumor of the same cell type or similar immunological profile as would arise, e.g., from metastasis. This is particularly the case where the second tumor is inaccessible either due to its size or location within the body. Accordingly, another aspect of this invention is directed to a method for treating multiple tumors in an immunocompetent human patient, which method comprises:

(a) identifying two or more solid tumors in an immunocompetant human patient wherein said tumors are at different sites in said patient and are of the same cell type or similar immunological profile and further wherein at least one of said tumors is accessible by injection techniques; and

(b) administering a first dose of a pharmaceutical composition to said accessible tumor or tumors, in an amount effective to initiate an immune response to all tumors, whether treated or not, having the same cell type or similar immunological profile; wherein the pharmaceutical composition comprises a sufficient amount of a cytotoxic agent to initiate said immune response.

In one embodiment, this method is particularly advantageous when at least one of the untreated tumor or tumors is inaccessible.

In another embodiment, this method is particularly advantageous when the immunocompetent human patient has so many tumors of the same cell type or similar immunological profile that treatment of all of said tumors is impractical. The generation of an immune response against a tumor as described herein further forms the basis for treating a patient at risk of having unlocated second tumor or tumors of the same cell type or similar immunological profile as would arise, e.g., from metastasis. This is particularly the case where the second tumor or tumors is/are unlocated due to their small size, e.g., micrometastasis. Accordingly, another aspect of this invention is directed to a method for treating an immunocompetent human patient at risk of unlocated second tumor or tumors of the same cell type or similar immunological profile, which method comprises:

(a) identifying at least one solid tumor in an immunocompetent human patient which tumor is accessible by injection techniques wherein said patient is at risk of unlocated second tumor or tumors of the same cell type or similar immunological profile; and

(b) administering a first dose of a pharmaceutical composition to said accessible tumor or tumors, in an amount effective to initiate an immune response to said tumors in said patient; wherein the pharmaceutical composition comprises a sufficient amount of a cytotoxic agent to initiate said immune response.

Additionally, the generation of an immune response against a tumor as described herein can form the basis for inhibiting the growth or development of future tumors having the same cell type or similar immunological profile. Accordingly, another aspect of this invention is directed to a method for treating an immunocompetent human patient having at least one accessible first tumor and at risk for the growth or development of future tumors of the same cell type or similar immunological profile, which method comprises:

(a) identifying at least one accessible solid tumor in an immunocompetent human patient at risk for the generation of future tumors of the same cell type or similar immunological profile; and

(b) administering a first dose of a pharmaceutical composition to said accessible tumor or tumors, in an amount effective to initiate an immune response to said tumor or tumors in said patient which immune response exhibits immunological memory so as to inhibit the development and/or growth of future tumors of the same cell type or similar immunological profile wherein the pharmaceutical composition comprises a sufficient amount of a cytotoxic agent to initiate said immune response.

DETAILED DESCRIPTION OF THE INVENTION This invention is directed to methods of initiating an immune response to solid tumors in an immunocompetent human patient. The invention advantageously prevents or inhibits the growth of tumors, for example, those that may otherwise be unsuitable or inaccessible by conventional injection techniques. Prior to discussing this invention in further detail, the following terms will be defined:

The term "platinate" or "platinates" refers to cytotoxic agents that contain platinum as a central atom. Examples of platinates include cisplatin, carboplatin, oxaliplatin, ormaplatin, iproplatin, enloplatin, nedaplatin, ZD0473 (cis-amminedichloro(2-methylpyridine)-platinum (II)), BBR3464 and the like.

The term "cytotoxic agent" refers to any agent which can cause cell death and/or apoptosis. Cytotoxic agents include, for example, platinate drugs as well as well known cytotoxic drugs such as alkylating agents, enzyme inhibitors, proliferation inhibitors, lytic agents, DNA synthesis inhibitors, membrane permeability modifiers, DNA intercalators, antimetabolites, or the like. Illustrative drugs include: doxorubicin, epirubicin, bleomycin, fluorouracil, vincristine, vinblastine, etoposide, teniposide, chlorambucil, melphalan, busulfan, carmustine (BCNU), lomustine (CCNU), streptozotocin, thiotepa, dacarbazine (DTIC), methotrexate, cytarabine, azaribine, mercaptopurine, thioguanine, actinomycin D, plicamycin, mitomycin-C, asparaginase, procarbazine, hydroxyurea, topotecan, irinotecan, gemcitabine, temozolamide, capecitabine, tezacitabine, mechlorethamine, cyclophosphamide, mitoxantrone, and tegafur. Other drugs of interest include radiosensitizers, such as SR-2508 and misonidazole; hyperthermia sensitizers, such as lidocaine and marcaine; bioreductive agents, such as mitomycin benzotriazine dioxides and nitroheterocyclic compounds such as benznidazole. See Carter and Livingston, "Drugs Available to Treat Cancer," Principles of Cancer Treatment (Carter et al, eds.) Chapter 10, pp. 111-145, 1982, McGraw-Hill, Inc., New York as well as Cancer Chemotherapy Handbook, Dorr & Van Hoff, Appleton & Lance, Norwalk, Connecticut, USA.

The term "immunocompetent" refers to a human patient whose immune system is sufficiently functional to mount an immune response to an antigen challenge which response possesses immunological memory. Such an immunocompetent human patient does not include a patient who has had radiation, chemotherapy or other immunosuppressant treatment within about

14 days prior to treatment in the methods of this invention. It is well established that repeated treatment of human patients with radiation and/or chemotherapeutic agents as above will compromise the immune systems of such patients. The term "inhibition" includes both stopping and/or reducing growth of a tumor, reducing the size of the tumor and/or inhibiting the growth of new tumors.

The phrase "metastatic tumors" refers to tumors which have arisen from tumor cells transferred from an original tumor site to a different location within a human patient.

The phrase "of common origin" means that the metastasized or secondary tumors have derived from an original tumor site. Typically, cancer cells are released from the primary tumor and travel through the bloodstream or lymphatic system to a different location within a human patient. The cancer cells that form such secondary tumors are similar to those in the original tumor, particularly in that they have similar morphological properties and share common antigenic determinations. In this regard, it is noted that the term "antigenic determinants" is used herein in the same manner and is deemed interchangeable with the term "immunological profile".

The phrase "solid tumor" refers to a tumor mass which is identifiable from surrounding, non-diseased tissue. The phrase "immunological memory" refers to the ability of a human patient to mount an immune response to subsequent antigen challenges which is improved over the patient's response to the initial antigen challenge.

The term "inaccessible" refers to a tumor that either cannot be safely or easily reached via conventional injection techniques and/or a tumor that is so small in size that it can't easily be identified or located.

The term "accessible" refers to a tumor that can be safely and/or easily reached via conventional injection techniques.

The term "injection techniques" relates to any kind of delivery of a pharmaceutical composition to an identified, accessible tumor and includes delivery by a catheter and/or by injection needles.

The phrase "immune response" refers to the recognition, by a human patient's immune system, of an antigen, and the mounting of a response to reduce or eliminate the presence of that antigen. The term "effective amount" or "sufficient amount" refers to an amount of a cytotoxic agent which is sufficient to initiate an immune response.

Methods: The invention provides methods for initiating an immune response to solid tumors in an immunocompetent human patient. More specifically, the methods of this invention include: (a) identifying one or more solid tumors in a human patient wherein said tumors are accessible by injection techniques; and (b) administering a first dose of a pharmaceutical composition to said solid, accessible tumor or tumors wherein said composition comprises an effective amount of a cytotoxic drug to initiate an immune response to said tumor or tumors in said patient. Preferably, the immune response exhibits immunological memory which prevents or inhibits the growth of other (e.g., metastatic) tumors of the same cell type or similar immunological profile.

The methods of this invention may further comprise (c) administering a second dose of a pharmaceutical composition to said accessible tumor or tumors, under conditions wherein said administration initiates or reinforces an already initiated immune response to the accessible tumor in said patient. When a second dose is administered, the pharmaceutical composition may contain the same formulation, or may be a different formulation, as previously administered for the first dose. Thus, the subsequent administrations may be for example, every third day, weekly, or less frequent, such as biweekly or at monthly intervals. Preferably, the second dose is administered from about 1 week to about 4 weeks after administering the first dose of the pharmaceutical composition. Several additional administrations may be provided to the human patient; however, the human patient must be immunocompetent at the time of each administration.

Typically, a human patient will require 14 days after a systemic chemotherapy regime or a radiation treatment prior to regaining the ability to mount an immune response to an antigen challenge. Preferably, the immunocompetent human has had less than about two prior chemotherapy treatments and/or less than about two prior radiation treatments.

As discussed above, in a preferred embodiment, the immune response exhibits immunological memory. Without being limited to any theory, it is believed that the treatment of tumors with intratumoral administration of effective amounts of cytotoxic agents (e.g., with CDDP/epi gel) results in induction of an immune reponse to the tumor, in essence a tumor vaccine. The mechanism of action of this immune induction is presumably mediated by the induction and release of heat shock proteins (HSP) by tumor cells exposed to the cytotoxic drug, coupled with the necrotic (as opposed to apoptotic) response induced by the high drug concentration. In typical therapeutic concentrations achieved by systemic administration of cytotoxic agents, cell death typically occurs through apoptosis, otherwise known as programmed cell death. In the apoptotic cascade, HSP is generated and HSP-tumor protein antigen complexes are formed, but they are typically sequestered within vesicles for non-immune clearance by phagocytic scavenger cells, resulting in immune nonreponsiveness. In contrast, the necrotic response to intratumoral treatment with high concentrations of cytotoxic drugs results in expression of HSP, but with extracellular release of the HSP-antigen complexes. Phagocytic proinflammatory immune cells such as antigen-presenting cells (APCs) and macrophages migrate into the area and take up the free HSP-antigen complexes thereby initiating a tumor- specific immune response in the host.

This process is enhanced further in the presence of cells undergoing apoptosis. With intratumoral administration of the cytotoxic drug, both necrosis and apoptosis occur, depending on the site. The treated tumor is exposed to a concentration gradient of the cytotoxic agent which is highest near the boundary between the dosage form and the surrounding tissue, and decreases as a function of distance due to the diffusion characteristics of the drug. The tumor region closest to this boundary dies by necrosis (non- apoptotic), which is a pro-inflammatory signal for the tumor bearing host. Distal to the boundary, at lower drug concentrations, tumor cells undergo apoptosis, which at low rates is non-inflammatory and non-immunogenic. There is a region within the tumor where a mixture of necrotic and highly apoptotic cells exist. Since this region is already primed for immune recognition and immune response to the necrotic tissue-derived free HSP- antigen complexes, the additional HSP-antigen complexes that are slowly freed as the apoptotic vesicles disintegrate will also add to the immune response.

In one embodiment, the methods of this invention prevent or inhibit the growth of tumors that may otherwise be inaccessible by conventional injection techniques. For example, the methods of the invention may prevent or inhibit the growth of metastatic tumors in one or more distal sites, including for example, the head, neck, brain, liver, lung, and bone marrow. These methods may also inhibit the growth of other established but inaccessible solid tumors of the same cell type or similar immunological profile. In this regard, the treated tumor may be the primary tumor or a tumor mass derived from metastasis of the primary tumor. According to the methods of this invention, the pharmaceutical composition is injected into the lesion, e.g., tumor, or lesion area, e.g., adjacent tissue, or in those situations where the tumor has been removed, tissue adjacent to the previously removed tumor. Administration may be by syringe, catheter or other convenient means allowing for application of a pharmaceutical composition at the tumor site. Injection may be at one or more sites depending on the size of the lesion. Needles from about 18 to about 27 gauge diameter are convenient. For multiple injection, templates with predrilled holes may be employed. The methods of the invention may be used in conjunction with other forms of therapy. For instance, the methods of the invention may further comprise the step of treating the tumor with radiation or heat. Accordingly, the lesions may be irradiated subsequent to administration of the aqueous collagen composition containing the compatible cytotoxic drug. Dose rates may vary from about 20 to 250 cGy/min, usually 50 to 150 cGy/min, depending on the lesion, period of exposure, the condition of the patient, and the like. If radiation is used, radiopotentiators may also be used. Radiopotentiators are anticancer drugs, which when used in conjunction with radiation, lead to greater damage to tumor cells than would be possible using either drug or radiation alone. Hyperthermia (heat) may be used as an adjunctive treatment. Treatment will usually involve heating up to about and including 43° C. for about 5 to 100 minutes.

The methods of the invention may further comprise the step of treating the tumor with radiation or heat. The methods of this invention may also be used in conjunction with surgery. Accordingly, the treated lesions may be excised either in their entirety or in part subsequent to the initial treatment with the cytotoxic agent.

In addition, the patient can optionally receive an immunostimulatory compound administered locally or systemically either prior to, concurrently with or after administration of the pharmaceutical composition comprising the cytotoxic agent. Examples of immunostimulatory compounds suitable for use in this invention include but are not limited to: inter leukins, interferons, colony stimulating factors, lymphokines, tumor necrosis factors and growth factors. More particularly, the immunostimulatory compound may be selected from the group consisting of: alpha interferon, gamma interferon, IL-2, and beta interferon.

Compositions

The pharmaceutical compositions used in the invention comprise a cytotoxic agent, such as a platinate drug, in an amount effective to initiate an immune response. The amount of the cytotoxic agent required to initiate an immune response may be optimized for an individual patient through empirical observations. The factors affecting the optimal dose include the condition of the human patient, the size and type of tumor, the nature of the cytotoxic agent, the co-administration of any other chemotherapy drugs, and the like which is within the skill of the attending clinician. Cytotoxic agents can include any of the aforementioned agents. Platinum-containing drugs, or platinates, are particularly useful.

Platinates suitable for use in the invention include, but are not limited to, cisplatin, carboplatin, oxaliplatin, ormaplatin, iproplatin, enloplatin, nedaplatin, cis-amminedichloro(2-methylpyridine)-platinum (II), BBR3464, and the like and mixtures thereof. Cisplatin is particularly preferred.

The cytotoxic agents of the invention may be administered in any formulation suitable for injection, preferably uniformly dispersed in a physiologically acceptable medium, in the form of a liquid, semi-solid or gel formulation. However, the choice of formulation may be determined by the specific cytotoxic agent, as well as any other optional chemotherapy drugs, agents and/or carriers that may be present. Certain combinations will be more efficacious than others, and can be determined empirically and optimized in accordance with conventional procedures.

In addition to the cytotoxic agent, a number of minor components may also be included to complex and/or protect the stability of the composition, control the pH, alter local cellular permeability, and the like. These agents generally will be present in less than about 2 weight percent of the total composition, usually less than about 1 weight percent, and individually may vary from about 0.001 weight percent to about 1 weight i percent.

Optionally, the cytotoxic agent may be used in combination with one or more other chemotherapy drugs. The drugs may be used individually or in combination, depending upon the nature of the drug, the tumor, and whether cooperative action is pharmacologically indicated. Examples of chemotherapy drugs that may be used in combination with the cytotoxic agent include but are not limited to various alkylating agents, enzyme inhibitors, proliferation inhibitors, lytic agents, DNA synthesis inhibitors, membrane permeability modifiers, DNA intercalators, antimetabolites, or the like. Illustrative drugs include: doxorubicin, epirubicin, bleomycin, fluorouracil, vincristine, vinblastine, etoposide, teniposide, chlorambucil, melphalan, busulfan, carmustine (BCNU), lomustine (CCNU), streptozotocin, thiotepa, dacarbazine (DTIC), mefhotrexate, cytarabine, azaribine, mercaptopurine, thioguanine, actinomycin D, plicamycin, mitomycin-C, asparaginase, procarbazine, hydroxyurea, topotecan, irinotecan, gemcitabine, temozolamide, capecitabine, tezacitabine, mechlorethamine, cyclophosphamide, mitoxantrone, and tegafur. Other drugs of interest include radiosensitizers, such as SR-2508 and misonidazole; hyperthermia sensitizers, such as lidocaine and marcaine; bioreductive agents, such as mitomycin benzotriazine dioxides and nitroheterocyclic compounds such as benznidazole. See Carter and Livingston, "Drugs Available to Treat Cancer," Principles of Cancer Treatment (Carter et al, eds.) Chapter 10, pp. 111-145, 1982, McGraw-Hill, Inc., New York as well as Cancer Chemotherapy Handbook, Dorr & Van Hoff, Appleton & Lance, Norwalk, Connecticut, USA.

In addition, the compositions used in this invention may be encapsulated in liposomes or other controlled rate release compositions so as to provide for separate and distinct rates of release of the drug. In this way, multiphasic compositions can be prepared, so as to provide for sustained release of the drug over long periods of time. Liposomes are prepared from a variety of lamellar-forming lipids including phospholipids, e.g., phosphatidylcholine, phosphatidylethanolamine, etc., gangliosides, sphingomyelins, steroids, e.g., cholesterql, etc. Usually, the weight of the lipids in relation to the weight of drug will range from 1 to 5 liters of entrapped drug per mole of amphipathic lipid. Formation of liposomes with inclusion of various materials is described in Papahadjopoulos, Annals of the N.Y. Academy of Science, 308 (1978); Gregoriadis and Allison, Liposomes in Biological Systems, John Wiley and Sons (1980); Leserman etal, Nature 293:226-228 (1981); Barhet et al, Supramol. Struct. Cell. Bio. Chem. 16:243-258 (1981) and Heath et al, Science 255:8015-8018 (1980).

Alternatively, other methods of encapsulation can be employed where the drug is encapsulated in a biodegradable substance, where the rate of release is related to the thickness of the biodegradable coat.

Optionally, a vasoconstrictive agent, in an amount which constricts capillaries so as to substantially restrict the flow of blood to or through blood vessels near the administration site may be included in the composition. Examples of vasoconstrictive drugs suitable for use in this invention include but are not limited to: catecholamines, epinephrine, norepinephrine, dopamine, epinephryl borate, phenylephrine, amphetamine, metraminol, methoxamine, ergot alkaloids, ergonovine, methylergonavine, methysergide, ergotamines, angiotensins, various prostaglandins and various corticosteroids such as fluocinolone acetonide, betamethasone valerate, and the like. Preferred vasoconstrictive drugs include epinephrine or norepinephrine. Compositions containing cytotoxic agents in combination with a vasoconstrictive drug are described in U.S. Patent Nos. 4,978,332 and

5,597,578, which references are incorporated herein by reference in their entirety.

The use of a vasoconstrictive agent can substantially inhibit migration of the cytotoxic drug from the site of application by alteration of the blood flow serving the rumor/lesion area, so as to maintain the primary effect of the cytotoxic drug at the site of application. When used, the vasoconstrictive drug is generally administered in the range of about 1-100 μg/kg body weight.

Illustrative vasoconstrictive agents suitable for use in this invention include, but are not limited to: (1) sympathomimetics including the catecholamines, norepinephrine, epinephrine, isoproterenol, dopamine, and related compounds such as ephedrine and other phenylisopropylamines, phenylephrine, amphetamine, metraminol, methoxamine; (2) ergot alkaloids including lysergic acid, lysergic acid diethylamine, ergonovine, methylergonavine, methysergide, ergotamine; (3) the angiotensins; (4) various prostaglandins; and (5) various corticosteroids. Vasoconstrictive agents are described in Medical Pharmacology (1984), C. V. Mosby, Company, Chapter 15.

The pharmaceutical compositions used in this invention may further comprise a therapeutically effective amount of an agent affecting tissue architecture. Examples of agents affecting tissue architecture suitable for use in this invention include: papain, chymopapain, trypsin, amylase, collagenase and chymotrypsin.

The pharmaceutical compositions used in this invention may also comprise a therapeutically effective amount of an agent affecting cellular permeability. For example, U.S. Patent No. 5,874,402, which is incorporated herein by reference in its entirety, describes the use of cell membrane permeants with chemotherapeutic agents. The cell membrane permeant may be administered in the same formulation as the cytotoxic drug, simultaneously, or separately. Examples of cell membrane permeants suitable for use in this invention include, but are not limited to: lysoplasmologens, e.g. lysolecithins; surfactants, e.g. polyoxyethylated sorbitans, polysorbates, sorbitan esters etc.; detergents, e.g. sodium dodecylsulfate (SDS); and the like.

Other cell membrane permeants capable of disrupting the integrity of the membrane and suitable for use in this invention include membrane permeants that comprise an extended ring structure which provides for the disruption of the orderly packing of the lipid cell membrane molecules. Cell membrane permeants having extended ring structures include steroidal and triterpenoid glycosides such as saponins, e.g. saponaria officinalis and quillaira saponaria; bile acids, e.g. cholic acid and taurocholic acid; constituents of digitalis, e.g. digitalin, digitonin and digitoxin, as well as derivatives thereof, such as digitogenin, digitoxigenin, digoxigenin, and digoxin; fusidic acid and derivatives thereof, and the like. Cell membrane permeants which disrupt the integrity of the membrane through interaction with membrane constituents, e.g. by binding to membrane components such as steroid, phospholipids and the like, include amphotericin B, melittin, polymyxin B, and the like.

Optionally, the pharmaceutical compositions of this invention may further comprise an adjuvant material. Examples of adjuvants suitable for use in this invention include radioactive pellets, e.g., radionuclides technicium or iridium; radiation sensitizers, e.g., nitroimidazoles and halogenated pyrimidines (BUdR); repair inhibitors, e.g. , methylated xanthines; bioreductive agents, which are activated only in hypoxic cells. Other adjuvants may be immuno stimulators including cytokines, such as the interferons; lymphokines, such as interleukin-2; tumor growth inhibitors, such as tumor necrosis factors and transforming growth factor-β; colony- stimulating factors such as G-CSF and GM-CSF, and the like.

Anaesthetics, such as procaine, may be included in the formulations. Also, other agents which overcome inherent cellular drug resistance, like verapamil, may be included. Other agents affecting tissue architecture include enzymes which can injure the stroma, such as the peptidase papain, chymopapain, trypsin, amylase, collagenase and chymotrypsin.

The pharmaceutical composition of the present invention can be prepared by combining the various components in a sterile environment. Other materials, when present, may be added concomitantly or sequentially. After ensuring the uniform dispersion of the various components in the mixture, the mixture may be sterilized and sealed in an appropriate container.

In the event the various components are unstable or form undesirable complexes when stored in a mixture prior to administration, each component may be dispensed at an appropriate concentration into a separate container for mixing just prior to administration. Those components which are stable together may be dispensed together into a single container for mixture with one or more reagents containing those additional ingredients found to promote instability or to form undesirable complexes.

For liquid formulations, the cytotoxic agent is preferably provided in an aqueous medium, such as saline, phosphate buffered saline, distilled water, etc. Usually, the liquid aqueous medium will be at least 5 weight percent of the entire composition, more usually at least 10 weight percent, and not more than about 75 weight percent, usually not more than about 50 weight percent, so as to provide a flowable mixture. The concentration will vary depending upon the nature of the drug(s), the nature of any carrier material, the presence of other materials, and the like.

Optionally, an additional diluent may be used. The diluent may provide stability, increase solubility, modify viscosity and/or to contribute to cytostatic activity. Diluents suitable for use in this invention are described in U.S. Patent Nos. 5,573,781, for example, which patent is incorporated herein by reference in its entirety. Typical diluents which may be used include alkanol diluents, such as ethanol, isopropanol, butanol, hexanol, octanol and the like, particularly ethanol and isopropanol. When present, the diluent will usually comprise at least about 10 (v/v) %, frequently at least about 20 (v/v) %, more usually at least about 30 (v/v) % and up to about 98 (v/v) % of the carrier compositions.

U.S. Patent Nos. 6,077,545 and 6,224,883 describe an improved diluent for the preparation of cisplatin (cis-diamminedichloroplatinum,

CDDP) suspensions from a lyophilized powder. The diluent is useful where a suspension is preferred over a solution of the drug, particularly for injectable gel formulations. Suitable nonionic surfactants are polysorbates, sorbitan esters, poloxamers, polyethoxylated fatty alcohols, e.g. BRIJ®, polyethoxylated fatty acid esters, e.g. MYRJ®, and the like. Of particular interest are polysorbates, e.g. polysorbate 20 and polysorbate 80. Polysorbate helps to disperse the CDDP crystal clusters which would otherwise remain agglomerated as large, rapidly sinking particles. The surfactant will usually be present in an aqueous diluent at a concentration greater than from about 0.01% weight/volume, usually greater than about 0.05%, preferably at about 0.1%, and usually at a concentration less than 0.75%, more usually less than 0.25%.

In still another embodiment, nonaqueous gels can be used to deliver the cytotoxic drug such as those described in International Patent Application No. WO 98/36776 which is incorporated herein by reference in its entirety. In yet another embodiment of the invention, the pharmaceutical composition may comprise a fatty acid ester matrix, which acts as a depot for the cytotoxic agent, when present. As described in U.S. Patent No. 5,573,781, incorporated by reference in its entirety, such compositions may further comprise an alkanol component, and in some cases, an additional thickening agent. Any thickening agent that does not adversely affect the pharmaceutically acceptable nature or desired properties of the composition may be employed. Thickening agents of interest include: aluminum monostearate, stearic acid, cetyl/stearyl alcohol, guar gum, methyl cellulose, hydroxypropylcellulose, tristearin, cetyl wax esters, polyethylene glycol 4000, and the like. In a preferred embodiment, the pharmaceutical composition of the invention further comprises a proteinaceous composition, further comprising collagen and/or fibrinogen dispersed in an aqueous medium as an amorphous flowable mass. Such proteinaceious compositions are described, for example, in U.S. Patent No. RE 35,748, incorporated by reference in its entirety. Additionally, pharmaceutically elegant proteinaceous collagen compositions, as described in U.S. Patent Nos. 5,750,146 and 5,980,946 may be used, which have a single transition temperature of less than about 45° and possess enhanced retention of the cytotoxic drug at the site of injection. The disclosure of both patents is incorporated herein by reference in their entirety.

In a preferred embodiment, the proteinaceous composition comprises from about 30% to about 100% of collagen and/or fibrinogen dispersed in an aqueous medium as an amorphous flowable mass at a concentration of from about 5 to 100 mg/ml and from about 0.1 to 50 weight percent based on said collagen and/or fibrinogen and the cytotoxic agent, wherein when said proteinaceous composition comprises collagen, said collagen is present as a uniform dispersion of collagen fibrils.

In another preferred embodiment, such proteinaceous composition has a protein component comprising from about 30% to about 100%) of collagen, which protein component is dispersed in an aqueous medium to provide an amorphous flowable mass having a collagen concentration of from about 5 to about 100 mg/mL and wherein the collagen in said proteinaceous composition has a single transition temperature of about 45°C or less.

In yet another preferred embodiment, such proteinaceous composition comprises collagen that is in the form of oxidized collagen. The phrase "oxidized collagen" refers to collagen where a thiomethyl group of one or more of the methionine residues of the collagen has been replaced by a methylsulfoxy and/or methylsulfonyl group. Such oxidized collagen formulations are described in U.S. Patent Application No. 09/858,247, filed on May 15, 2001, entitled "Oxidized Collagen Formulations for Use with Non-Compatible Pharmaceutical Agents," which is incorporated by reference herein in its entirety.

Such proteinaceous compositions substantially inhibit the migration of the drug from the site of injection, so as to maintain the primary effect of the drug in the region of injection. The proteinaceous formulation is generally flowable for injection, but does not migrate significantly once injected into the tissue. Migration can be further inhibited by the use of physiologically acceptable materials which enhance the binding of the drug to the matrix or which modify cellular properties or physiological responses to further regionalize the placement of drug at the injection site. Thus, the proteinaceous composition will be capable of binding the cytotoxic agent covalently or non-covalenfiy, without preventing its therapeutic effect, while retaining the active agents at the site of introduction or retarding transfer of the active agents present from the site of introduction. Preferably, the composition will be comprised of a significant amount of the matrix to provide the desired composition characteristics. The matrix may be comprised of individual or in combination peptides or proteins, e.g., structural proteins such as collagen and fibrinogen, or albumin or other protein which provides for stable placement, or combinations thereof. Preferred materials include collagen, fibrinogen and derivatives thereof.

Collagen is a particularly preferred material. In a preferred embodiment, sufficient amounts of the proteinaceous material are employed in the formulation to provide for a collagen concentration of from about 5 to about 100 mg/mL and preferably from about 5 to about 100 mg/mL. The specific amount of collagen employed is selected relative to the desired viscosity of the collagen composition such that the composition will flow under moderate pressure, but not move significantly after being positioned at a particular site in the patient. Preferably, sufficient collagen is employed such that the composition will have a viscosity of from about 5,000 to about 20,000 centipoise at 20° C and at a shear rate of 15.8 sec^"1. Such formulations have sufficient viscosity that they will be retained at an effective dosage at the site of administration for a reasonable period of time, usually over about 6 hours.

Proteinaceous compositions having at least about 5 weight percent, preferably at least about 10 weight percent, and up to 50 weight percent or more, are of particular interest when used in combination with thrombin or its enzymatic equivalent. In this way fibrinogen is enzymatically modified to fibrin to enhance the non-migratory property of the composition while forming a matrix of fibrils to further stabilize the composition. The thrombin may be mixed with fibrinogen containing proteinaceous composition from a time immediately prior to use or shortly after injection. The amount of thrombin of about 1 to 1000 IU/mg employed will generally range from about 0.1 to 10 weight percent of the fibrinogen present, depending upon the time of use, the rate desired for solid matrix formation, the amount of other components, the effect of the drug on thrombin activity, and the like. In addition, various physiologically acceptable bulking agents or concentrating agents may be optionally employed which serve to provide for drug and protein interactions, with resulting reduction in the rate of drug release. Illustrative materials include inorganic substances, such as hydroxyapatite and organic substances such as carbohydrates, e.g., dextran, agarose, methyl cellulose and cellulose.

The collagen employed in the compositions described herein can be derived from any mammalian host source, such as bovine, porcine or human, or may be prepared, as available, by other techniques, e.g., recombinant DNA techniques. The collagen employed may be natural collagen or may be modified, such as tropocoUagen, atelocoUagen, or the like. The collagen may be non-immunogenic, immunogenic, or only slightly immunogenic.

Various methods of preparing collagen or derivatives thereof in purified foπn for administration to a mammalian host are known in the art. Suitable methods include those recited in, for example, U.S. Pat. No.

3,949,073 and references cited therein. Of interest is bovine collagen which is purified and is obtained from young cows or calves. Purification will normally involve dispersion or precipitation from various media, e.g., dilute acetic acid. In some situations, xenogeneic collagen is employed to enhance an immunogenic response in the area of injection or immunogenic adjuvants may be employed. Additionally, collagen suitable for use herein is also commercially available from a number of vendors. Typically, such commercial sources comprise a proteinaceous material wherein the protein component comprises from about 30% to about 100% of collagen with the remainder of the protein component comprising, for example, fibrogen, albumin or derivatives of such proteins.

The collagen, cytotoxic agent and other optional additives are uniformly dispersed in a physiologically acceptable aqueous medium, such as saline, phosphate buffered saline, distilled water, etc. to form a collagen composition. The aqueous medium will be sufficient to provide for an amorphous dispersion capable of flowing under mild pressure. Usually, the liquid aqueous medium will be at least 90 weight percent of the entire composition, more usually at least 95% weight percent, usually not more than about 99.5 weight percent, so as to provide a flowable mixture. The amount will vary depending upon the nature of the cytotoxic agent, the presence of other materials and the like. Optional additives may also be included in the formulation, although certain additives such as vasoconstrictive or sympathomimetic agents, due to stability problems, may preferably be incorporated into the composition just prior to use. The collagen will be provided in a convenient form, usually admixed with at least a portion of the total aqueous medium to be employed. The composition will be sufficiently workable that upon admixture, a uniform dispersion can be obtained. The cytotoxic agent may be added to the coUagenous dispersion with agitation to ensure the uniform dispersion of the drug. Optional materials, as appropriate, may be added concomitantly or sequentially. Sterilization will usually be achieved using aseptic conditions.

Utility

The methods of the invention are used to prevent or inhibit metastatic cancer in an immunocompetent human patient. The methods of the invention provide increased efficacy, reduced toxicity, improved specificity and improved quality of life over systemic chemotherapy, for example. The methods of the invention are particularly useful to prevent or inhibit tumors that may otherwise be inaccessible by injection techniques, e.g. where the tumors typically either cannot be safely or easily reached via conventional injection techmques and/or are so small in size that they can't easily be identified or located. Thus, tumors located in the head, neck, brain, liver, lung, skin, bone marrow and/or other sites may be inhibited or prevented using the methods of the invention.

Illustrative tumors include carcinomas, sarcomas and melanomas, such as basal cell carcinoma, squamous cell carcinoma, melanoma, soft tissue sarcoma, solar keratoses, Kaposi's sarcoma, cutaneous malignant lymphoma, Bowen's disease, Wilm's tumor, hepatomas, colorectal cancer, brain tumors, mycosis fungoides, Hodgkin's lymphoma, polycythemia vera, chronic granulocytic leukemia, lymphomas, oat cell sarcoma, and the like. The subject methods find particular advantage with tumors or lesions which are clinically relevant such as those solid tumors greater than 100 mm³, more particularly, greater than 150 mm³, in volume. However, the subject methods are also advantageous in the treatment of microtumors arising, for example, from metastasis. Such microtumors can have a size of less than about 100 mm³ and often are as small as 1 mm³.

The methods of this invention are also useful in the treatment of a second inaccessible tumor of the same cell type or similar immunological profile as a first accessible tumor. Such methods entail treatment of the first accessible tumor in the manner described herein so as to produce an immunological response to said tumor which response also is effective against said second tumor.

Still further, the methods of this invention are also useful in the treatment of patients with multiple tumors of the same cell type or similar immunological profile which makes it impractical to treat all of them individually (whether each tumor is accessible or not). In this case, the initiation of an immune response against one tumor will have a therapeutic effect against the other similar tumors. The following examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention. Unless otherwise stated, all temperatures are in degrees Celsius. Also, in these examples, unless otherwise defined below, the abbreviations employed have their generally accepted meaning:

EXAMPLES

The following examples illustrate how the methods of this invention can be used. In these examples, the following abbreviations are employed with the following definitions. If not defined, the abbreviation has its generally accepted meaning. In addition, all temperatures are °C unless indicated otherwise and percentages are weight percents again, unless indicated otherwise.

g — gram mg = milligram mL = milliliter mm³ = cubic millimeter μg = microgram μL — microliter

EXAMPLE 1

A cisplatin/epinephrine injectable gel (cisplatin 4.0 mg/mL, epinephrine 0.1 mg/mL) was prepared from lyophilized Cisplatin for Injection (David Bull Laboratories, Mulgrave, Victoria, Australia), an epinephrine solution (0.152 mg/mL, manufactured for Matrix by Chesapeake Biological Laboratories, Baltimore, MD), and 6.5% bovine collagen gel (Matrix, Fremont, CA).

Female C3H/Sed mice (Charles River Laboratories, Hollister, CA), approximately 3 months old with body weight approximately 24 g, were maintained in isolator cages on a 12-hour light-and-dark cycle. Food and water were available ad libitum. The MBT-2 murine syngeneic bladder tumor cell line was maintained in in vitro culture (RPMI medium supplemented with 10% heat-inactivated bovine serum and containing 100 μg/mL of penicillin and streptomycin) at 37°C in a humidified 5% CO₂ incubator. Log-phase MBT-2 cells were trypsinized and harvested from cell culture flasks, then injected intradermally (2.4 x 10⁵ cells in a volume of 60 μL) into one flank of each of 11 mice. Treatment started when tumors reached approximately 70 mm³ in size. The cisplatin/epinephrine gel was administered intratumorally at 25 μL (cisplatin dose of approximately 4 mg/kg body weight) on Days 9, 11, 13, and 15 post initial tumor cell challenge. This treatment resulted in complete eradication of the tumors by Day 20. A control group of six non-tumor-bearing mice was administered cisplatin/epinephrine gel intradermally on the same schedule as above.

On day 69 the animals were challenged with an intradermal injection of 1 x 10⁵ MBT-2 tumor cells in 50 μL. A second control group of six healthy mice was similarly challenged. Tumors appearing subsequently at the challenge site were measured with Vernier calipers three times per week. Tumor volume (in cubic millimeters, mm ) was calculated according to the formula:

V = π/6 xDι x D₂ x D_3; where Dι_₃ are perpendicular diameters measured in millimeters (mm). Tumor development and growth was monitored until all animals had tumor present or their tumor volume had reached approximately 300 mm³.

TABLE 1 Summary of MBT-2 Tumor Development Following Challenge on Day 69

Total Number Number of Animals with Tumors of Day Tumor Volume (mm³, M ± SE) Treatment Group Animals 77 80 83 85 87 89

1 no initial tumor 6 6 6 6 6 44±3 98±10 176±17 264±32 323±36

2 no initial tumor treated w/ CDDP/epi gel, i.d.

6 6 6 6 6 6

40±5 82±8 153±14 237±21 307±25

3 initial tumor eradicated w/ i.t. CDDP/epi gel treatment

11 5 6 9 9 9 10

8±3 23±7 68±17 124±28 155±33 251±42

In the two control groups, tumors appeared in all animals within 8 days of challenge and grew to an average of more than 300 mm³ by day 18 after challenge. In the group in which the initial tumor burder had been eliminated by intratumoral treatment with the cisplatin/epinephrine gel, the rechallenge with tumor cells resulted in a delay in appearance of new tumors (only 5 of 11 animals exhibited tumors 8 days after challenge) and growth to an average of only 155 mm³ by day 18 after challenge. This suppression of tumor appearance and growth is attributable to a host immune response to the rechallenge that was brought about by treatment of the initial challenge with the intratumoral chemotherapy.

EXAMPLE 2

An experiment similar to Example 1 was carried out, using the RIF-1 syngeneic murine fibrosarcoma as the tumor line. The initial challenge was with 2 x 10⁵ cells in 50 μL, and the later challenge (on day 43 or day 88 after the first) was with 8 x 10⁴ cells in 50 μL.

Table 2a

Summary of RIF-1 Tumor Development Following Challenge on Day 43

Total Number Number of Animals with Tumors of Day Tumor Volume (mm³, M ± SE)

Treatment Group Animals 49 51 53 56 58

1 no initial tumor 5 5 5 5 5

31±5 55±6 86±6 197±12 324±29

2 initial tumor eradicated w/ i.t. CDDP/epi gel 2 3 4 5 5 treatment 5±3 13±6 28±7 73±16 138 ±30

In the control group, tumors appeared in all animals within 6 days of challenge and grew to an average of more than 300 mm³ by day 15 after challenge. In the group in which the initial tumor burder had been eliminated by intratumoral treatment with the cisplatin/epinephrine gel, the rechallenge with tumor cells resulted in a delay in appearance of new tumors (only 2 of 5 animals exhibited tumors 6 days after challenge) and growth to an average of only 138 mm by day 15 after challenge. This suppression of tumor appearance and growth is attributable to a host immune response to the rechallenge that was brought about by treatment of the initial challenge with the intratumoral chemotherapy.

Table 2b Summary of RIF-1 Tumor Development Following Challenge on Day 88

Total Number Number of Animals with Tumors of Day Tumor Volume (mm³, M ± SE)

Treatment Group Animals 94 98 100 102 105 107

1 no initial tumor 5 5 5 5 5 5

24±2 68±5 110±8 202±15 346±23 521±15

2 no initial tumor treated w/ CDDP/ epi gel, i.d. 5 5 . 5 5 5 5

24±4 78±12 122±13 214±17 356±14 568±19

3 initial tumor eradicated w/ i.t. CDDP/epi gel treatment 7 2 7 7 7 7 7

7±3 32±5 65±11 108±13 215±24 334±34

In the two control groups, tumors appeared in all animals within 6 days of challenge and grew to an average of more than 500 mm by day 19 after challenge. In the group in which the initial tumor burder had been eliminated by intratumoral treatment with the cisplatin/epinephrine gel, the rechallenge with tumor cells resulted in a delay in appearance of new tumors (only 2 of 7 animals exhibited tumors 6 days after challenge) and growth to an average of only 334 mm³ by day 19 after challenge. This suppression of tumor appearance and growth is attributable to a host immune response to the rechallenge that was brought about by treatment of the initial challenge with the intratumoral chemotherapy.

Claims

WE CLAIM:

1. A method to initiate an immune response to a solid tumor in an immunocompetent human patient which method comprises: (a) identifying one or more solid tumors in an immunocompetent human patient wherein at least one of said tumors is accessible by injection techniques; and

(b) administering a first dose of a pharmaceutical composition to said solid, accessible tumor or tumors wherein said pharmaceutical composition comprises an effective amount of a cytotoxic agent to initiate an immune response to said tumor or tumors in said patient.

2. The method of Claim 1 , further comprising:

(c) administering a second dose of a pharmaceutical composition to said accessible tumor or tumors, under conditions wherein said administration initiates or reinforces an initiated immune response to the accessible tumor in said patient wherein said second dose is administered from about one to about four weeks after administering the first dose of the pharmaceutical composition.

3. The method of Claim 1, wherein said immune response exhibits immunological memory.

4. The method according to Claim 1, wherein the cytotoxic agent comprises a platinate drug selected from the group consisting of cisplatin, carboplatin, oxaliplatin, ormaplatin, iproplatin, enloplatin, nedaplatin, cis-amminedichloro(2-methylpyridine)-platinum (II), BBR3464, and mixtures thereof.

5. The method according to Claim 4, wherein the pharmaceutical composition comprises a collagen gel and a platinate.

6. The method according to Claim 5 wherein said platinate is cisplatin.

7. The method according to Claim 1, wherein the pharmaceutical composition further comprises an immunostimulatory compound.

8. The method according to Claim 7, wherein the immunostimulatory compound is selected from the group consisting of: interleukins, interferons, colony stimulating factors, lymphokines and tumor necrosis factors.

9. The method according to Claim 8, wherein the immunostimulatory compound is selected from the group consisting of: alpha interferon, gamma interferon, IL-2, beta interferon and GM-CSF.

10. The method according to Claim 1, wherein the pharmaceutical composition further comprises a vasoconstrictive amount of a vasoconstrictive drug.

11. The method according to Claim 10, wherein said vasoconstrictive drug is selected from the group consisting of: epinephrine, norepinephrine, epinephryl borate, catecholamines, dopamine, ephedrine, phenylephrine, amphetamine, metraminol, methoxamine, ergot alkaloids, ergonovine, methylergonavine, methysergide, ergotamine, angiotensins, prostaglandins and vasoconstrictive corticosteroids.

12. The method according to Claim 11, wherein said vasoconstrictive drug is selected from the group consisting of epinephrine or norepinephrine.

13. The method according to Claim 1, wherein the pharmaceutical composition further comprises a therapeutically effective amount of an agent affecting tissue architecture.

14. The method according to Claim 13, wherein said agent affecting tissue architecture is selected from the group consisting of papain, chymopapain, trypsin, amylase, collagenase and chymotrypsin.

15. The method according to Claim 1 , wherein the pharmaceutical composition further comprises a therapeutically effective amount of an agent affecting cellular permeability.

16. The method according to Claim 1, wherein the pharmaceutical composition further comprises an adjuvant material.

17. A method according to Claim 16, wherein the adjuvant material is selected from the group consisting of: radioactive pellets, radiation sensitizers, repair inhibitors, cytokines, and combinations thereof.

18. The method according to Claim 1, further comprising the step of treating the tumor with radiation or heat.

19. The method according to Claim 1, wherein said immunocompetent human has had less than about two prior chemotherapy treatments.

20. The method according to Claim 1, wherein said immunocompetent human has had less than about two prior radiation treatments.

21. The method according to Claim 1 , wherein all or part of the solid tumor or tumors are surgically excised.

22. A method for treating multiple tumors in an immunocompetent human patient, which method comprises: (a) identifying two or more solid tumors in an immunocompetant human patient wherein said tumors are at different sites in said patient and are of the same cell type or similar immunological profile and further wherein at least one of said tumors is accessible by injection techniques; and (b) administering a first dose of a pharmaceutical composition to said accessible tumor or tumors, in an amount effective to initiate an immune response to all tumors, whether treated or not, having the same cell type or similar immunological profile; wherein the pharmaceutical composition comprises a sufficient amount of a cytotoxic agent to initiate said immune response.

23. The method of Claim 22 wherein at least one of the untreated tumor or tumors is inaccessible.

24. The method of Claim 22 wherein the immunocompetent human patient has so many tumors of the same cell type or similar immunological profile that treatment of all of said tumors is impractical.

25. A method for treating an immunocompetent human patient at risk of unlocated second tumor or tumors of the same cell type or similar immunological profile, which method comprises: (a) identifying at least one solid tumor in an immunocompetent human patient which tumor is accessible by injection techniques wherein said patient is at risk of unlocated second tumor or tumors of the same cell type or similar immunological profile; and

26. A method for treating an immunocompetent human patient having at least one accessible first tumor and at risk for the growth or development of future tumors of the same cell type or similar immunological profile, which method comprises: