Patent Application
20050113290
The invention relates to a method in the general field of immunotherapy, wherein a particular therapeutic effect is sought to be achieved with respect to particular cells or organisms in humans or animals through the use, either directly or indirectly, of soluble radioactive toxic agents which are introduced into the living host. The soluble radioactive toxic agents being adapted to be converted into insoluble radioactive toxic precipitates by the action of previously introduced non-mammalian enzymes bound to the non-endocytosing receptors of targeted cells. These radioactive toxic precipitates generate supra-lethal regions of radiation, called Hot-Spots, which are capable of non-selectively killing cells in the targeted region. In particular, the present invention relates to a method of the treatment of cancer.
1. Field of the Invention
A considerable portion of world-wide research efforts in the treatment of cancer is currently devoted to killing cancer cells by means of various cell killing agents. Despite the fact that numerous drugs, radioactive compounds, and the like have been shown to be capable of killing cancer cells, these agents fail to treat cancer successfully because of their inability to circumvent three universally present obstacles: (1) the agents do not kill all the cancer cells because they do not exhibit cytotoxic specificity for all the cancer cells, (2) the agents also kill normal cells because they do not exhibit cytotoxic specificity exclusively for cancer cells, and (3) the agents are not potent enough to kill resistant cancer cells or to overcome the ability of cancer cells to adapt and become resistant to the cell killing agents. An appreciation of these three obstacles is necessary to understand why current treatments fail and to understand the rational and methodology of the proposed invention.
Fifty years of intense research has shown that there is a wide heterogeneity in every characteristic that has been measured in cancer cells. These characteristics include cell size, buoyancy, anaerobic metabolism, enzyme composition, growth rate, gene errors, differential gene expression, chromosome number, and chromosome errors. The heterogeneity is also expressed by the presence of some cancer cells that are super-sensitive and others that are super-resistant to being killed or treated by any therapeutic agent. Within the same tumor population a fraction of cells will be sensitive to a given therapeutic agent and will be killed when that agent is administered, a fraction of cells will be resistant to the agent and will not be killed, and a fraction will adapt and become increasingly more resistant to subsequent therapeutic regimens. The resistant cells will continue to divide and spread to distant locations in the body to form metastatic tumors.
The wide heterogeneity in sensitivity to any particular therapeutic agent leads to the high probability that the systemic application of any therapeutic agent will cause partial remission of the tumor by killing the super-sensitive cancer cells, but will not be able to achieve a complete cure because it cannot kill the super-resistant cancer cells. Previous attempts at cancer therapy have generally ignored the negative therapeutic consequences of these divergent cells. There has been an intuitive and optimistic belief than an approach achieving partial remission in its early phase will give a complete cure after it has been fine-tuned. This optimism contradicts the biological principle, supported by a large amount of data, that every large population of cells or organisms is heterogeneous, and that cancer cells, which have a genetic instability, exhibit a particularly high degree of heterogeneity. Therefore, it is not surprising that the past history of cancer therapy approaches has been a monotonous sequence of short periods of hope, because killing same cancer cells leads to a remission, followed by prolonged periods of disappointment, because some cancer cells survive, seed, and continue to grow in the living host and subsequent treatments are less effective at killing the cancer cells of these metastatic tumors. It is likely that the latest field of oncogenes and other gene manipulations, as applied to cancer therapy, will also follow the same pattern. This prediction is based on the fact that there is a heterogeneity of gene errors and gene expression in the cancer cell population, and with time, more and more cells, with more and varied genetic and chromosome errors accumulate in the cancer cell population. No simple genetic correction, even if it could be applied successfully to all the cancer cells growing in the body, is likely to repair every cell.
2. Prior Art
The first serious deficiency of current cancer therapeutic approaches is that they do not take into account, and are unable to deal with, the heterogeneity of the cancer cell population. The inability of current approaches to circumvent this heterogeneity is illustrated by the failure of immuno-therapeutic approaches that rely on antigenic receptors on the surface of cancer cells to deliver therapeutic agents.
All current attempts at cancer therapy (apart from the treatment of thyroid cancer with radio-iodide) depend on killing each and every individual cancer cell by their direct individual interaction with the candidate therapeutic agents or applied environmental condition. In order to describe the need for this direct interaction, these strategies can be loosely called “sniper killing,” i.e. each cell to be killed must be targeted directly. Sniper killing agents include cytotoxic drugs, binary reagents made by attaching cancer targeting agents to cytotoxic drugs, augmented immune response, hormonal therapy, genetically engineered products (like interferon), manipulations of oncogenes, or products coded by these genes.
In order for these sniper killing strategies to be successful in treating cancer, it would be necessary for the cancer cells to have an exploitable characteristic which is present on all cancer cells, for that characteristic to be absent from all (or at least most) normal cells, and for that characteristic to not adaptively change and become non-exploitable.
It is known that cancer cells exhibit on their surface numerous receptors, including antigenic receptors, to which selected molecules such as specific antibodies, hormones, and peptides can bind. Antibodies, hormones, and peptides can be used as targeting agents for the cancer cells that express those particular antigenic receptors. Ideally, all cancer cells would express the receptor, and the number of non-cancerous cells which express the receptor would be very small. In the ideal model, binary reagents (an example of a sniper strategy) which are composed of targeting agents and cytotoxic agents would be preferentially directed to the cancer cells. However, in practice, binary reagents do not result in the delivery of the cytotoxic agent to all cancer cells in the tumor population because some cancer cells do not exhibit the particular antigenic receptor. The binary reagent will not attach to these antigenic receptor deficient cancer cells, and therefore these cells will be unaffected by the treatment and will be left to proliferate in the host. High-dose sniper killing, even when employed at dose levels which kill many normal cells, fail to kill all cancer cells because some cancer cells are antigenic receptor deficient, some cancer cells are super-resistant even before the treatment begins, and some cancer cells adapt to the therapeutic agent, survive, and become resistant to future treatments. All these sniper strategies have failed, and are doomed to fail in the future, because they cannot deal with the fact that some normal cells also express the characteristics which is the target for the sniper killing, and because they cannot deal with the universally present heterogeneity and adaptive ability of cancer cells.
The recent development of highly pure and highly immuno-specific monoclonal antibodies, hormones, and peptides which can act as specific targeting agents for particular antigenic receptors has greatly increased the ability to direct cell killing agents specifically to cancer cells and thereby minimize any adverse effects on non-cancerous cells. Paradoxically, this current direction of isolating and producing such highly specific targeting agents (for the purpose of minimizing the possibility that such antibodies, and the cytotoxic agents carried thereby, might attach to non-cancerous cells) is, in one sense, counter-productive, since the number of cancer cells within the tumor population which will exhibit an affinity for such highly specific targeting agents will be reduced.
Notwithstanding the above-mentioned advances in the development of highly specific targeting agents to deliver the cell killing agents specifically to targeted cells, and the demonstrated cell killing ability of the particular delivered agents, therapeutic success through the use of binary reagents composed of targeting agents and toxic agents has not been achieved, and should not have been expected. Unfortunately, in practice these therapies have been far less successful than they were hoped to be.
The second serious deficiency of binary reagents to carry cytotoxic agents to target cancer cells is that the so called “cancer targeting agents” of which the binary reagents are made, also target a significant number of normal cells. These targeted normal cells are also killed by the administration of binary reagents, cause unacceptable destruction of normal tissues, serious illness of the patient, and limit the aggressiveness of the attack which can be launched against the cancer.
The third serious deficiency of binary reagents to carry cytotoxic agents to target cancer cells, particularly cytotoxic radioactive isotopes, is that they cause significant systemic toxicity because the targeting agent carrying the cytotoxic agent is a large molecule which causes them to have a long residence time in the blood circulation, and causes them to be taken up non-specifically by normal cells.
The fourth serious deficiency of binary reagents to carry cytotoxic agents to cancer cells is that even those cancer cells which the targeting agents attaches to, outright killing of the cancer cell is often not accomplished. In large part this is due to the inherent limitations of the treatment method, i.e., the absolute quantity of cytotoxic agent which can be coupled to the targeting agent is smaller than that required to actually kill the cancer cell (the small quantity of cytotoxic agent which can be attached is limited to avoid destroying the targeting ability of the targeting agent and to avoid adversely altering the distribution of the binary reagent in the host). While the amount of cytotoxic agent which can be brought to bear on cancer cells through the use of binary reagents may be sufficient to damage some of the cells, the damage often is temporary or, indeed, simply results in the emergence of mutant cells which are still cancerous and have become resistant to the effects of the cytotoxic agent.
The fifth serious deficiency of the binary reagents to carry cytotoxic agents is that it is impossible to make a valid choice of the most appropriate targeting agent to make the binary reagent for each cancer in each patient. Furthermore, it is not possible to predict the outcome of the therapy prior to administering the binary reagent at the necessary cytotoxic dose level.
Despite the three obstacles and the deficiencies described above, the treatment of thyroid cancer with radio-iodide is successful in a high proportion of cases. This high rate of success is not due to a fundamental difference between cancer cells of the thyroid and cancer cells which have arisen from other tissues. The successful treatment of thyroid cancer is due to the fact that normal and malignant thyroid cells have a unique biological function which allows them to store iodine. Thus, when patients with thyroid cancer are treated with radio-iodide, a fraction of the cancer cells take up sufficient quantities of isotope and store the isotope long enough to generate micro-regions of intense radiation in which all the cells in each micro-region are killed. These intense radiation fields, call Hot-Spots, are generated exclusively in the normal and malignant thyroid tissue. The radiation field in the Hot-Spots extends beyond the cells taking up the isotope and kills hundreds of neighboring cells thereby creating overlapping micro-regions of supra-lethal radiation (overlapping Hot-Spots) exclusively in the thyroid tissue. Inside these Hot-Spots, the radiation is so intense that all the cancer cells in the tumor are killed, including the cells that do not take up the radio-isotope.
Two types of strategies have been employed to amplify and localize the effect of cytotoxic agents on targeted cells in order to circumvent the five deficiencies described and in order to simulate the operating conditions that make the treatment of thyroid cancer so successful. The first strategy is to form the cytotoxic agents on the surface of the targeted cells, and the send strategy attempts to accumulate the cytotoxic agents outside cells in the extra-cellular fluid. The first strategy to amplify and localize the effect of cytotoxic agents on targeted cells is to accumulate agents on the surface of targeted cells. Accumulation of radioactive toxic materials on the surface of targeted cells has been achieved by a single step direct delivery of radio-labeled targeting agent which can bind directly to the non-endocytosing receptors of targeted cells. The single step method consists of the administration of a radio-labeled protein or peptide targeting agent that can directly bind to the targeted non-endocytosing cell receptors. In order to overcome the problem of systemic toxicity caused by the long residence time of the targeting agent in the circulating blood (the long residence time being caused by the slow diffusion out of the blood capillaries of the targeting agent which is a large molecule) a three step indirect delivery of radio-isotopes to the non-endocytosing receptors of targeted cells has been used. In the three step method, the isotope carrying molecule is small enough to circulate rapidly throughout the body fluids and thus have a short residence time in the blood. The three-step, indirect delivery method consists of: (i) administering a bispecific reagent which will bind to the non-endocytosing target cell receptors, the bispecific reagent comprising an antibody having a substantial affinity for the non-endocytosing receptors on cancer cells and further having an antibody or peptide which has a substantial affinity for, and can bind to, the small radioisotope carrying molecules; (ii) waiting for a period of time to allow for the elimination from the body of bispecific reagent not specifically bound to the non-endocytosing receptors on targeted cancer cell receptors; and (iii) after the elimination of all bispecific reagent, not specifically bound to the targeted cell receptor is completed, administering a small isotope carrying molecule. The small isotope carrying molecule circulates in the blood, diffuses rapidly into the extra-cellular fluid, and binds to the non-endocytosing receptors via the bound bispecific reagent (Corvalan et al., 1987 Cancer immunology and immunotherapy, 24, 133-137; Corvalan et al., 1988, Int J. of Cancer, 2 Supple. 2-25; Glennie et al. 1988, J. of Immunology, 141, 3662-3670; Stickney et al., 1989, In Fourth Int. Conf. on Monoclonal Antibody Immunoconjugate For Cancer, p 29, San Diego,-UCSD).
The second strategy to amplify and localize the effect of cytotoxic agents on targeted cells is to form the cytotoxic agents outside targeted cells in the extra-cellular fluid. The formation of cytotoxic agents outside targeted cells in the extra-cellular fluid of the targeted regions has been achieved by the enzymatic conversion of a pro-drug into an active drug by a method called Antibody Dependent Enzyme Pro-Drug Therapy (ADEPT). The enzyme which makes the conversion is one moiety of a bispecific reagent, the other moiety being an antibody with a binding affinity to the non-endocytosing receptors on surface of targeted cancer cells. Since the enzyme moiety is bound to the surface of the targeted cells, the conversion from pro-drug to the active drug takes place in the extra-cellular fluid.
The active drug diffuses into the immediate micro-region to have its pharmacological cytotoxic effect on the non-target cancer cells in the micro-region. For example, alkaline phosphatase converts the pro-drugs mitomycin phosphate into an active mitomycin C derivative and etoposide phosphate into an active etoposide (Senter et al, 1989, Cancer Research, 49, 5789-5792), beta-lactamase converts a cephalosporin derivative of 4-de-succetylvinblastine-3-carboxhydrazide into an active cytotoxic drug (Meyer et al, 1993, Cancer research, 53, 3956-3963), and activates cephalo-doxorubcin (Rodrigues et al, 1995, Cancer Research, 55, 63-70), DT diaphorase followed by a non-enzymatic reaction with a thioester activates the mono-functional alkylating agent CB1954 into an active agent which can cause ctotoxicity by cross-linking DNA (Knox et al, 1993, Cancer and Metastasis Reviews, 12, 195-212); carboxypeptidase G2 can convert a nitrogen mustard prodrug into an active drug (Springer and Niculescu-Duvaz, 1995, Anticancer Drug Des. 10, 361-372); nitroreductase can activate CB 1954 (Knox et al, 1995, Biochem. Pharmacol., 49, 1641-1647); and dinitrobenzamide (Anlezark et al, 1995, Biochem. Pharmacol., 50, 609-618); to form cytotoxic derivatives, and alpha-galactosidase can activate prodrugs of anthracycline (Azoulay et al, 1995, Anticancer Drug Des., 10, 441-450).
The three step ADEPT approach fails to successfully treat cancer for the following reasons: (a) the bispecific reagent is bound to the non-endocytosing target cancer cells and also to some normal cells because the targeting agent moiety does not exhibit exclusive cytotoxic specificity for cancer cells which reduces the tumor specificity of the non-mammalian enzyme location and pro-drug conversion, (b) the antigenic receptors of the target cells are in a constant state of flux which prevents the bispecific reagent from remaining bound for a sufficient period of time to allow all bispecific reagent not bound specifically to the target cell receptors to be eliminated from the body prior to administering the pro-drug; (c) the soluble active drug which is made by the enzyme diffuses away from its site of production to have a cytotoxic action on healthy normal cells; (d) the cells on which the bispecific reagent is bound, and where the active drug is formed, are the first cells to be killed because they receive the highest concentration of the active drug. When these cells are killed, the enzyme will no longer be in a position to convert the pro-drug into an active drug and, therefore, the production of active drug is self limiting; and (e) the shape and volume of the micro-region in which there is a sufficiently high concentration of the active drug to kill cells is variable and ill-defined because the diffusion parameters of the soluble active drug are dependent on the particular status of the blood capillaries and extra-cellular structures in the cancer, the parameters of the diffusion varying from one location of the tumor to another.
The two strategies described above fail to generate Hot-Spots because the number of cytotoxic chemical or radioisotope agents which are delivered is small, the number being directly proportional to the relatively small number of antigenic receptors on the surface of the target cells. In addition, the agents or isotopes do not remain in the correct location for long enough to achieve an aggressive attack on the cancer, and furthermore, they cause systemic toxicity because the agents circulate in the blood for a long period of time. Finally these strategies also fail to locate the attack specifically to the tumor, because the location where the agent or isotopes are delivered or where the active drug is made is dependent on only a single cancer associated characteristic on the cancer cell surface, and every single characteristic found on cancer cells is also found on some normal cells.
The present invention mimics for non-thyroid cancers, the Hot-Spot killing which makes the treatment of thyroid cancer successful; however, since no other malignant tissue has the same natural iodide involving process as the thyroid, the mimicking requires the construction of a special, multi-step, sequential process to achieve “Hot-Spots” in non-thyroid cancers. The basic process of the present invention consists of sequential steps which act independently and together with naturally occurring characteristics of the cancer and normal cell populations to generate overlapping Hot-Spots virtually exclusively in the tumor without causing significant systemic toxicity. Cancer cells within these Hot-Spots are eradicated, the eradicated cells include cancer cells that are not targeted, cancer cells that are resistant and even super-resistant, and cancer cells that would otherwise adapt and become resistant to therapy.
An object of the present invention is to provide a method for the treatment of cancer that directs supra-lethal doses of radiation in the form of micro-regions of radiation, called Hot-Spots, virtually exclusively to the cancer. All the cells, numbering in the thousands, within each Hot-Spot are killed, therefore, the method of the present invention does not require each individual cancer cell to be targeted in order for every cancer cell in the living host to be killed. As a consequence the method of the present invention is not defeated by the heterogeneity of cancer cell receptors and the imperfect nature of current targeting agents. Because the Hot-Spots are located virtually exclusively in the region of the cancer, the present invention does not kill healthy cells and will not cause significant systemic toxicity.
It is another object of the present invention to provide a method for accumulating at the non-endocytosing receptors of target cells non-mammalian enzymes capable of converting a soluble therapeutic agent into an insoluble precipitate. The non-mammalian enzyme is accumulated via the administration of bispecific reagents comprised of a non-mammalian enzyme moiety and a targeting agent moiety with a substantial affinity for the non-endocytosing receptors of target cancer cells.
It is a further object of the present invention to accumulate non-toxic and toxic therapeutic precipitates in the extra-cellular fluid of the cancer region. Precipitates are accumulated via the administration of a soluble precipitable material which is adapted to be converted into an insoluble precipitate by the non-mammalian enzyme moiety of the bispecific reagent.
It is still a further object of the present invention to provide a method for the immunological treatment of any cell population or organism for therapeutic purposes.
It is an additional object of the present invention to provide a bispecific reagent adapted to have a non-mammalian enzyme moiety and a targeting agent moiety, the bispecific reagent being adapted to bind to the non-endocytosing receptors of targeted cells. The bispecific reagent having a targeting agent moiety adapted to have a substantial affinity for the non-endocytosing receptors on target cancer cells, the enzyme moiety being adapted to convert a soluble therapeutic agent into an insoluble precipitate.
The present invention is a method for treating a heterogeneous population of cancer cells in a living host by at least one of a first therapeutic agent and a second therapeutic agent. The living host being composed of normal cells growing in a normal extra-cellular matrix, the normal extra-cellular matrix having at least collagen and fibronectin, the heterogeneous population of cancer cells growing in a cancer-altered extra-cellular matrix having at least cancer-altered antigenic epitopes 99 (
As shown in
The normal cells of the living host endogenously making and containing products including at least sulphated glycosaminoglycans, natural intra-cellular enzymes in the lysosomes, and natural intra-cellular material including DNA, histone, and complexes of DNA-histone, the DNA, histone, and complexes of DNA-histone having antigenic epitopes. The normal cells including at least two sub-populations of normal cells. As shown in
The method of the invention comprising a plurality of steps including at least introducing into the living host a first bispecific reagent.
As shown in
In accordance with the method of the present invention, after all unbound first bispecific reagent has been eliminated from the living host, the first therapeutic agent, which is a soluble precipitable agent, is administered to the living host. The first therapeutic agent adapted to be converted in the extra-cellular fluid adjacent to the first bispecific reagent by the first enzyme moiety of the first bispecific reagent into an insoluble non-digestible precipitate which is the first extra-cellular precipitate. The first extra-cellular precipitate being capable of remaining in the extra-cellular fluid adjacent to the first bispecific reagent for an extended period of time.
Since the enzymatic conversion of the first therapeutic agent takes place exclusively in the extra-cellular fluid, there is no need for the first therapeutic agent to gain entrance into the intra-cellular compartment of the body. Entrance of the first therapeutic agent into cells throughout the living host would increase the volume of distribution of the first therapeutic agent (the intra-cellular compartment has a volume at least 2-3 times that of the extra-cellular fluid) and would increase the duration of time that the first therapeutic agent would circulate in the living host, both these factors—the volume of distribution and the long circulation time—could increase the systemic toxicity caused by the first therapeutic agent. For these reasons it is of significant advantage for the first therapeutic agent to be cell impermeant by being a molecule larger than 1000 daltons and/or being anionic. Alternatively the first therapeutic agent can be made cell impermeant by attaching one of a number of cell impermeant molecules at least including peptides or polymers having a molecular size greater than 1,000 daltons and anionic chemicals including thiols.
As shown in
The first therapeutic agent can be a soluble precipitable agent made by converting chemical X to a soluble XY. The bond attaching X to Y is cleaved by the first enzyme moiety of the first bispecific reagent to create the highly reactive intermediate molecule Xa. The Xa molecule is readily and extremely rapidly oxidized to form Xb. In the oxidized form, Xb spontaneously and covalently self-condenses or dimerizes to create a new molecule which is insoluble and immediately and spontaneously forms a precipitate. Because a new molecule is formed by the dimerization, the core structure of the precipitate has a neo-antigenic epitope which is not present on the original XY, Xa, Xb or anywhere else in the body.
A specific example of a first therapeutic agent to be used is the application of a indoxyl-lactam as examples of XY, which is soluble and can be administered to the living host as a free molecule. The lactam (Y) of the indoxyl-lactam (XY) is cleaved by beta-lactamase enzyme (being the first enzyme moiety of the first bispecific reagent) to liberate a highly reactive intermediate indoxyl (Xa). As shown in
The oxidation and dimerization of indoxyl proceeds at a slower rate in the acidic pH which is often present in the extra-cellular fluid of the tumor tissue, compared to the rate of oxidation and dimerization in the relatively neutral pH found in the extra-cellular fluid of normal tissues. The slower rate of oxidation and dimerization may allow some of the soluble indoxyl molecules and intermediates to diffuse away from the first enzyme moiety prior to the indoxyl oxidizing, dimerizing, and precipitating. A controlled diffusion away from the first enzyme would have the advantage of distributing the first extra-cellular precipitate more evenly throughout the tumor tissue, thus increasing the size of the Hot-Spots that are later generated and reducing the problem of tumor heterogeneity. On the other hand, if the diffusion away from the first enzyme was extreme, the diffusion could allow the soluble indoxyl molecules to diffuse into the blood or lymphatic capillaries where they could dimerize, precipitate, and deliver precipitates to normal tissues and reduce the subsequent radiation dose to the tumor. In order to obtain the advantaged controlled diffusion, and to circumvent the problem of the indoxyl diffusing into the blood modifications can be made to the indoxyl-lactam so that the rate of diffusion of indoxyl into blood capillaries is greatly reduced. Since charged molecules move much slower through the extra-cellular fluid than neutral molecules (Clauss and Jain, 1990 Cancer Research, 50, 3487-3498) (positively charged molecules tend to interact with negatively charged extra-cellular structures, and negatively charged materials are effectively repelled by the many negatively charged extra-cellular structures), molecules having a charge can be covalently attached to the indoxyl-lactam to reduce the rate of diffusion of the soluble indoxyls and intermediates. This can be achieved by attaching a charged molecule to the benzene ring of the indoxyl by reductive amination, involving an amino group on the benzene ring and the reducing end (aldehyde) of the charged molecule. The result is an alkyl amino group, similar to that formed when polylysine is lactosylated by reductive amination. The resultant bond is incapable of being cleaved by mammalian enzymes, and the charged molecule will control the rate of movement of the released indoxyl to be optimum. The attachment of the charged molecule to the benzene ring of the indoxyl-lactam will not interfere with the ability of the beta-lactamase enzyme to cleave the indoxyl-lactam bond, or the ability of the indoxyl to be oxidized and to dimerize and precipitate.
A further modification can be made by covalently attaching two indoxyl-lactam molecules together at a position on the benzene ring to make a bi-indoxyl-lactam. Cleavage of the two lactam bonds of the bi-indoxyl-lactams by beta lactamase creates a bi-indoxyl molecule which will dimerize with two other bi-indoxyl molecules, and so on, to create a self-assembling linear insoluble polymer. The attachment of the two indoxyl-lactams can be either direct, or indirect via a digestible or non-digestible spacer molecule. The spacer molecule can be one of several kinds such as a poly (ethylene oxide) polymer with hetero-bifunctional reactive groups at its terminals (Yokoyama et al, 1992, Bioconjug. Chem 3, 275-276), a non-degradable copolymer [N-(2-hydroxypropyl) methacrylamide] which is non-immunogenic, non-toxic, and has a versatile chemistry which allows for a range of side chains and pendant chemicals such as lactose, mannose, and radio-labeled tyrosinamide to be introduced (Maeda et al, 1992, Bioconug, Chem. 3, 351-362; Seymour, 1992, Critical Reviews in Therapeutic Drug Carrier Systems 9, 135-187; Primm et al, 1993, J. Drug Target. 1, 125-131), or a hydrophobic hexamethylene spacer group (Ouchi et al, 1992, Drug Des. Discov. 9, 93-105). The formation of an insoluble linear polymer has substantial advantages in reducing the ability of the substance to move by diffusion and convection in the extra-cellular fluid of the tissue. Additional indoxyls can be made (a) by attaching chemicals to position 3 and which will precipitate by the action of the non-mammalian enzyme moiety of the bispecific reagent, (b) all substituents at position 4, 5, 6, and 7, including hydroxyl groups; (c) phenyl at position 5, and all its derivatives, (d) benzloxy at position 5 and all its derivatives and (e) 5,5-bi-indoxyls, with or without spacers.
The first therapeutic agent can be radio-labeled to be trace-labeled or to be radioactive-therapeutic. For example, the indoxyl can be radio-labeled with radioiodine or other radio-isotopes. Radio-iodination of the indoxyl compounds can be achieved in three ways: (i)
An additional method of converting first therapeutic agent into an insoluble material which precipitates in the extra-cellular fluid is where the first therapeutic reagent has a soluble moiety and an insoluble moiety, the soluble moiety having a solubilizing effect on the insoluble moiety and being cleaved by the first enzyme moiety of the first specific reagent, the solubilizing effect of the soluble moiety being thereby dissipated and the remaining material, being insoluble, spontaneously forming a precipitate.
In accordance with the method of the present invention, continuing the introducing of the first therapeutic agent into the living host increases the amount of the accumulation of the first extra-cellular precipitate in the extra-cellular fluid to form a plurality of antigenic epitopes adjacent to the first bispecific reagent which is proportional to the amount of accumulation. As shown in
The method of the present invention includes additionally introducing into the living host a second bispecific reagent.
Following the delivery to the living host of the second bispecific reagent, some of the second bispecific reagent will be in body fluids or bound non-specifically to non-target cells or to extra-cellular structures. It is known that, with time, circulating antibodies and antibodies bound non-specifically, are naturally eliminated from the body of the living host more quickly than antibodies that are bound to specific targets (Henkel et al., 1985, Clinical Immunology and Immunopathology, 35, 146-155: Goldberg et al., 1988, Archives Of Pathology Laboratory Medicine, 112, 580-587). Therefore, the elimination of the second bispecific reagent from the living host can be achieved simply by allowing a lapse of time; the elimination of the second bispecific reagent can be hastened, as has been described previously for the second step of the
EPT process (Sharma et al, 1994, Cancer Supplement, 73, 1114-1120), by the administration a galactosylated antibody specific for the second enzyme moiety of the second specific reagent. The galactosylated antibody can inactivate the enzyme and/or facilitate the clearance of the second enzyme moiety-galactosylated antibody complexes via the galactose specific receptors in the liver.
The elimination, from the living host, of all the second bispecific reagent not specifically bound to the first extra-cellular precipitate prior to the administration of the additional therapeutic agent is necessary to prevent the additional therapeutic agent from being converted into the new form in locations other than in the tumor. After all unbound second bispecific reagent has been eliminated from the living host, the additional therapeutic agent is additionally administered to the living host, where it circulates freely in the body fluids and is converted by the second enzyme moiety of the second bispecific reagent into a new form.
In accordance with the method of the present invention, after all unbound second bispecific reagent has been eliminated from the living host, the additional therapeutic agent, which is a soluble radioactive toxic agent, is additionally administered to the living host, the additional therapeutic agent adapted to be converted, in the extra-cellular fluid adjacent to the first extra-cellular precipitate, by the second enzyme moiety of the second bispecific reagent bound to the first extra-cellular precipitate, into a radioactive toxic new form. The new form of the additional therapeutic agent being capable of remaining in the extra-cellular fluid adjacent to the first extra-cellular precipitate for an extended period of time sufficient to kill non-selectively all cells adjacent to the first extra-cellular precipitate. Since the first extra-cellular precipitate is retained in the extra-cellular fluid for an extended period of time and since the second bispecific reagent is bound thereto, the continued administration of the additional therapeutic agent enables the second enzyme moiety to convert a substantial amount of the additional therapeutic agent into the radioactive toxic new form. The radioactive toxic new form remaining in the extra-cellular fluid adjacent to the first extra-cellular precipitate for an extended period of time thereby creating an intense field of radiation—called a Hot-Spot—which kills non-selectively all cells adjacent to the first extracellular precipitate.
Since the enzymatic conversion of the additional therapeutic agent takes place exclusively in the extra-cellular fluid, there is no need for the additional therapeutic agent to gain entrance into the intra-cellular compartment of the body. Entrance of the additional therapeutic agent into cells throughout the living host would increase the volume of distribution of the additional therapeutic agent (the intra-cellular compartment has a volume at least 2-3 times that of the extra-cellular fluid) and would increase the duration of time that the additional therapeutic agent circulates in the living host. Both these factors—the increased volume of distribution and the increased circulation time—would increase the systemic toxicity caused by the additional therapeutic agent. For these reasons it is of significant advantage for the additional therapeutic agent to be cell impermeant by being a molecule larger than 1000 daltons and/or being anionic. Alternatively the additional therapeutic agent can be made cell impermeant by attaching one of a number of cell impermeant molecules at least including peptides or polymers having a molecular size greater than 1,000 daltons and anionic chemicals including thiols.
In accordance with the present invention, there are at least three different methods of retaining the new form of the additional therapeutic agent in the tumor region. Each method of the invention involves the step of delivering into the living host the second bispecific reagent and additionally administering to the living host the additional therapeutic agent which is converted by the second enzyme moiety of the second bispecific reagent into a new form which is retained for an extended period of time in the extra-cellular fluid adjacent to the first extra-cellular precipitate by at least three different methods.
As shown in
The second therapeutic agent to be used in the first method can be a soluble radioactive toxic precipitable agent made by converting chemical X to a soluble XY. The bond attaching X to Y is cleaved by the second enzyme moiety of the second bispecific reagent to create the highly reactive intermediate molecule Xa. The Xa molecule is readily and extremely rapidly oxidized to form Xb. In the oxidized form, Xb spontaneously and covalently self-condenses or dimerizes to create a new molecule which is insoluble and immediately and spontaneously forms a radioactive toxic second precipitate. Because a new molecule is formed by the dimerization, the core structure of the second precipitate has a neo-antigenic epitope which is not present on the original XY, Xa, Xb or anywhere else in the body.
A specific example of a second therapeutic agent to be used in the first method is the application of a radioactive indoxyl-lactam as examples of XY, which is soluble and can be administered to the living host as a free molecule. The lactam (Y) of the indoxyl-lactam (XY) is cleaved by beta-lactamase enzyme (being the enzyme moiety of the bound bispecific reagent) to liberate a highly reactive intermediate indoxyl (Xa). As shown in
The oxidation and dimerization of indoxyl proceeds at a slower rate in the acidic pH which is often present in the extra-cellular fluid of the tumor tissue, compared to the rate of oxidation and dimerization in the relatively neutral pH found in the extra-cellular fluid of normal tissues. The slower rate of oxidation and dimerization may allow some of the soluble indoxyl molecules and intermediates to diffuse away from the second enzyme moiety of the second bispecific reagent prior to the indoxyl oxidizing, dimerizing and precipitating. A controlled diffusion away from the second enzyme moiety would have the advantage of distributing the radioactive toxic second precipitate more evenly throughout the tumor tissue, thus increasing the size of the Hot-Spots and reducing the problem of tumor heterogeneity. On the other hand, if the diffusion away from the second enzyme moiety was extreme, it could allow the soluble indoxyl molecules to diffuse into the blood or lymphatic capillaries where it could dimerize, precipitate, and deliver radioactive precipitates to normal tissue and reduce the radiation dose to the tumor. In order to obtain the advantage of controlled diffusion, and to circumvent the problem of the indoxyl diffusing into the blood, various modifications can be made to the indoxyl-lactam so that the rate of diffusion of indoxyl into blood capillaries is greatly reduced. Since charged molecules move much slower through the extra-cellular fluid than neutral molecules (Clauss and Jain, 1990, Cancer Research, 50, 3487-3493) (positively charged molecules tend to interact with negatively charged extra-cellular structures, and negatively charged materials are effectively repelled by the many negatively charged extra-cellular structures), molecules having a charge can be covalently attached to the indoxyl-lactam to reduce the rate of diffusion of the soluble indoxyls and intermediates. This can be achieved by attaching a charged molecule to the benzene ring of the indoxyl by reductive amination, involving an amino group on the benzene ring and the reducing end (aldehyde) of the charged molecule. The result is an alkyl amino group, similar to that formed when polylysine is lactosylated by reductive amination. The resultant bond is incapable of being cleaved by mammalian enzymes, and the charged molecule will control the rate of movement of the released indoxyl to be optimum. The attachment of the charged molecule to the benzene ring of the indoxyl-lactam will not interfere with the ability of the beta-lactamase enzyme to cleave the indoxyl-lactam bond, or the ability of the indoxyl to be oxidized and to dimerize and precipitate.
A further modification can be made by covalently attaching two indoxyl-lactam molecules together at a position on the benzene ring to make a bi-indoxyl-lactam. Cleavage of the two lactam bonds of the bi-indoxyl-lactams by beta lactamase creates a bi-indoxyl molecule which will dimerize with two other bi-indoxyl molecules, and so on, to create a self-assembling linear insoluble polymer. The attachment of the two indoxyl-lactams can be either direct, or indirect via a digestible or non-digestible spacer molecule. The spacer molecule can be one of several kinds such as a poly (ethylene oxide) polymer with hetero-bifunctional reactive groups at its terminals (Yokoyama et al, 1992, Bioconjug. Chem. 3, 275-276), a non-degradable copolymer [N-(2-hydroxypropyl) methacrylamide] which is non-immunogenic, non-toxic, and has a versatile chemistry which allows for a range of side chains and pendant chemicals such as lactose, manose and radio-labeled tyrosinamide to be introduced (Maeda et al, 1992, Biocong, Chem. 3, 351-362; Seymour, 1992, Critical Reviews in Therapeutic Drug Carrier Systems, 9, 135-187; Primm et al, 1993, J. Drug Target 1, 125-131), or a hydrophobic hexamethylene spacer group (Ouchi et al, 1992, Drug Des. Discov. 9, 93-105). The formation of an insoluble linear polymer has substantial advantages in reducing the ability of the substance to move by diffusion and convection in the extra-cellular fluid of the tumor tissue. Additional indoxyls can be made (a) by attaching chemicals to position 3 and which will precipitate by the action of the non-mammalian enzyme moiety of the bispecific reagent, (b) all substituents at position 4, 5, 6, and 7, including hydroxyl groups; (c) phenyl at position 5, and all its derivatives, (d) benzloxy at position 5 and all its derivatives and (e) 5,5-bi-indoxyls, with or without spacers.
An additional method of converting a soluble second therapeutic agent into an insoluble material which precipitates in the extra-cellular fluid is where the second therapeutic reagent has a soluble moiety and an insoluble moiety, the soluble moiety having a solubilizing effect on the insoluble moiety. The soluble moiety being cleaved by the non-mammalian enzyme of the bound bispecific reagent, the solubilizing effect of the soluble moiety being thereby, dissipated and the remaining material, being insoluble, spontaneously forming a precipitate.
As shown in
A specific example of a non-mammalian enzyme-substrate system to be used for this method is chondroitinase ABC as the second enzyme moiety, and radio-labeled chondroitin sulphate attached to a short polypeptide as the third therapeutic agent, as shown in
As illustrated in
The administration of large molecules results in higher concentrations of large molecules to be present in tumor tissue compared to normal tissue (Seymour, 1992, Critical Reviews in Therapeutic Drug Carrier Systems, 91, 135-187). Therefore, the concentration of the administered precipitating antibody, being a large molecule, will be higher in the tumor than in normal tissues. The higher concentration of the precipitating antibody in tumor tissues enables a larger amount of the new form of the third therapeutic agent to bind and complex to the precipitating antibody and thereby form a precipitate, being the third extra-cellular precipitate which is retained in the extra-cellular fluid adjacent to the first extra-cellular precipitate for an extended period of time.
A specific example of a non-mammalian enzyme-substrate system to be used in the third method is chondroitinase ABC as the second enzyme moiety, and radio-labeled chondroitin sulphate (CS) as the additional therapeutic agent. As described in method two, the chondroitinase ABC converts the chondroitin sulphate into a new form, the new form being a soluble material and having a neo-antigenic epitope not found on the chondroitin sulphate (Christner et al, 1980, J. Biol. Chem., 255, 7102-7105). Prior to the administration of the chondroitin sulphate, a precipitating antibody capable of binding to the neo-antigenic epitope of the chondroitin sulphate is administered to the living host. The complex, formed by the administered precipitating antibody binding to the neo-antigenic epitope of the chondroitin sulphate forms a precipitate, the precipitate being the third extra-cellular precipitate which remains in the extra-cellular fluid adjacent to the first extra-cellular precipitate for an extended period of time sufficient to kill non-selectively all cells adjacent to the first extra-cellular precipitate.
The intensity of the radiation field that will be generated by each of the three methods that use the second enzyme moiety to convert the additional therapeutic agent into a new form capable of generating Hot-Spots is very high. A large number second enzyme moiety molecules, for example beta-lactamase or chondroitinase ABC, will be bound to the first extra-cellular precipitate via the first bispecific reagent. The number of enzyme molecules is proportional to the amount of precipitate which had accumulated in the extra-cellular fluid adjacent to the first extra-cellular precipitate. Since it is possible to accumulate any required amount of first extra-cellular precipitate, the number of bound second bispecific reagents can be made very large, therefore, the number of second enzyme moiety molecules can also be made very large. The more second enzyme moiety molecules that are present, the higher will be the rate at which the additional therapeutic agent is converted into the new form. The higher the rate of conversion, the more intense the radiation field adjacent to the first extra-cellular precipitate will be and less intense the level of systemic toxicity in the living host will be.
The present invention exploits a number of naturally occurring biological mechanisms of the loving host to increase the specificity of the location of Hot-spots to the tumor. Following the administration of the first therapeutic agent, and prior to the delivery of the bispecific reagent and/or the additional administration of the second therapeutic agent, several mechanisms operate to transfer first extra-cellular precipitate in normal tissue (but not cancer tissue) to a location where first extra-cellular precipitate cannot act as a platform from which a Hot-Spot can later be generated. Macrophages in the normal tissue can transfer the first extra-cellular precipitate to an intra-cellular location by phagocytosis, thereby preventing Hot-Spots from developing. In contrast, macrophages in cancer tissue are inhibited by the cancer cell driven aberrant environment (Boetcher and Leonard, 1974, J. Nat. Cancer Inst. 52, 1091-1096; Snyderman et al, 1978, J. Nat. Cancer Inst. 60, 737-742; Norman, 1985, in Macrophage Biology, p. 285-298, Allan R Liss Inc.; Braun et al, 1993, Cancer Research, 53, 3362-3368) and will not phagocytose the first extra-cellular precipitate as effectively.
In addition, normal epithelial cells that line the boundary between the inner and outer environment of the body exfoliate into the lumen of the organ when they are killed (Ishikawa et al, 1993, 17 suppl. pS 104-110; Montefort et al. 1993, Eur. Respir. J., 6, 1257-1263; Sisson et al, 1994, Am. J. Respir. Crit. Care Med. 149, 205-213). The exfoliation of these cells effectively transfers the first bispecific reagent and any first extra-cellular precipitate to a location where the first extra-cellular precipitate cannot act as a platform from which Hot-Spots can later be generated. In contrast, cancer cells which arise from these boundary cells only grow inside the body of the host and cannot exfoliate to the external environment. Consequently, virtually all cancer epithelial cells (but not normal epithelial cells) that have received the first bispecific reagent and which have generated a first extra-cellular precipitate will be in a correct location to generate Hot-Spots.
Similarly, normal endothelial cells that are damaged or killed become detached and enter the blood stream (Dini et al, 1995, J. Cell. Sci. 108, 967-73) and any bispecific reagent bound to them will be engulfed quickly by the macrophages which line the sinusoids of the liver and spleen, thereby making the first extra-cellular precipitate unavailable to generate Hot-Spots from.
Particles which are injected into the extra-cellular fluid move by convective flow from the extra-cellular fluid of tissues into the lymph drainage channels which drain into to the regional lymph nodes, where the particles are quickly and effectively engulfed by the very active macrophages which line the lymph flow pathway. First extra-cellular precipitates and second and third extra-cellular precipitates behave in a similar way and suffer the same fate. The movement of precipitates can occur in normal tissue thereby reducing the number of Hot-Spots which would otherwise have been generated in normal tissue. The movement of the first extra-cellular precipitate in normal tissue enables the first extra-cellular precipitate to be engulfed by macrophages in the regional lymph glands prevents Hot-Spots from developing in normal tissues, whereas movement of radioactive second and third precipitates in normal tissues which enables the radioactive precipitates to be engulfed by macrophages in the regional lymph glands causes Hot-Spots to be generated in the regional lymph glands, which is a much more desirable and clinically safe location for radiation damage to occur, compared to normal parenchymal tissue. In contrast, cancer tissue lacks an effective lymphatic drainage system (Jain, 1987, Cancer Research, 47, 3039-3051; Jain and Baxter, 1988, Cancer Research, 48, 7022-7032; Clauss and Jain, 1990, 50, 3487-3492) and first, second, and third extra-cellular precipitate movement into the lymphatic system cannot take place from tumor tissue and there will be no reduction in the number of Hot-Spots which will be generated in tumor tissue.
Lymphatic vessels are sometimes present in tumor tissue (reflecting the heterogeneity of non-malignant cells within the tumor tissue) which could lead to movements of the first, second and third extra-cellular precipitate into regional lymph glands. This movement can be prevented by “tethering” the first and second extra-cellular precipitate and the soluble new form of the second therapeutic agent to stable structures which are substantially more present in tumor tissue. Tethering of the first extra-cellular precipitate can be achieved by administering bispecific reagents to the living host prior to the administration of the additional therapeutic agent. The bispecific reagents tether the extra-cellular precipitate to at least one of three different stable structures in the extra-cellular fluid including the third antigenic receptor of the second target cancer cells, the antigenic epitopes of the cancer-altered extra-cellular matrix, and the antigenic epitopes of the relocated natural intra-cellular material. The tethering bispecific reagents are comprised of two moieties, the first moiety having an affinity for one of the first antigenic epitope, second antigenic epitope, and neo-antigenic third epitope of the extra-cellular precipitate. The second moiety having an affinity for at least one of three different stable structures in the extra-cellular fluid including the third antigenic receptor of the second target cancer cells, the antigenic epitopes of the cancer-altered extra-cellular matrix, and the antigenic epitopes of the relocated natural intra-cellular material.
Specificity of Hot-Spot location is also increased because the binding moiety of the second bispecific reagent is adapted to bind exclusively to the antigenic epitopes of the first extra-cellular precipitate and the first extra-cellular precipitate is a material not naturally present in the body, therefore, the binding moiety of the second bispecific reagent with its second enzyme moiety can have a high and specific affinity for the antigenic epitopes of the first extra-cellular precipitate with little or no cross reaction to natural structures of the living host. For example, the cellulose binding domain peptide, from which the second bispecific reagent can be made binds virtually irreversibly to cellulose which is one candidate material for the first extra-cellular precipitate.
Increased specificity of Hot-Spot location can also be achieved by “tethering” the new form of the additional therapeutic agent in a manner similar to that employed to tether the first extra-cellular precipitate.
For example,
For example,
The effectiveness of the fifth, eighth, and eleventh bispecific reagents to tether the first extra-cellular precipitate, the second extra-cellular precipitate, and the new form of the third therapeutic agent respectively depends on the presence in the cancer region of the natural intra-cellular material of cells, in particular the presence of DNA, histone, and complexes of DNA-histone.
In order to increase the effectiveness of the ability of the fifth, eighth, and eleventh bispecific reagents to tether the first extra-cellular precipitate, the second extra-cellular precipitate, and the new form of the third therapeutic agent respectively, the present invention provides the additional step of administering to the living host a cell killing process capable of selectively killing cells in the cancer region and thereby increasing the amount of DNA, histone, and complexes of DNA-histone that will be present in the cancer region.
Theory and data support the view that the low dose selective killing of these super-sensitive cells can be readily achievable. In fact, the frequent presence of dead cells in histological preparations of cancer tissue suggests that some tumor cells are so super-sensitive that they have been killed by various natural host factors which operate at the physiological low levels which prevail in the tumor-bearing subject. It is even possible that in some tumors, the natural host killing is so frequent that no external agent need be administered to achieve the necessary selective killing of the super-sensitive fraction. The presence of super-sensitive cancer cells, reflecting as it does the universal heterogeneity and genetic instability of cancer cell populations, may be the most common, the most specific, and with respect to the method of the invention, the most exploitable characteristic of any cancer. The low dose selective killing of these super-sensitive cancer cells can be considered to be analogous to, or an enhancement of the natural, continuous, and selective killing of some cancer cells by the defense system of the body.
As discussed earlier, the on-going natural killing of cancer cells by the natural immune system of the body may be sufficient to provide the intra-cellular material necessary for tethering via the fifth, eighth, and eleventh bispecific reagents.
As shown in
In accordance with the present invention, selective killing of a fraction of cancer cells is achieved by exploiting the heterogeneity of the tumor cell population. The universal and wide heterogeneity which is found in tumor cell populations is well known. It is expressed in every parameter which has been measured in cells, including a heterogeneity in their sensitivity or resistance to being killed by an untoward environment. This untoward environment includes all current agents which are used in an attempt to kill cancer cells, as well as the environment created by the natural system of the living host. As a result of this heterogeneity, it is likely that there will always be some cancer cells which are super-resistant to being killed prior to the administration of any of the current therapeutic agents; however, heterogeneity of the cancer cells expresses itself, not only by the presence of super-resistant cells, but also by the presence of cells which are super-sensitive to being killed. These cancer cells have so many gene errors that they are killed by low doses of the current therapeutic agents or by other cell killing processes. Normal cells do not have these gene errors so that few normal cells (if any) are super-sensitive. Therefore, few normal cells are killed by the very low doses of agents which kill super-sensitive cancer cells.
Current research and therapy regimens ignores the presence of these super-sensitive cancer cells because these cells are of no scientific interest or practical value in the context of current therapies. In contrast, the approach of the present invention exploits the presence of super-sensitive cancer cells and kills them selectively by administering the cell killing process. The cell killing process may include the administration of low doses of anti-cancer agents which are currently available, and preferably which cause lysis of cell membranes.
Death of cells eliminates the normal permeability restrictions to molecules that are characteristic of intact cells and enables molecules like trypan blue and antibodies to gain access to intra-cellular material. For example, labeled anti-myosin antibody can bind to cardiac myosin when the cardiac cells have been killed (Khaw et al., 1987, J. Nuclear Med., 28, 1671-1678), and anti-histone-DNA antibody can bind to histone-DNA complex when cancer (or other) cells are killed (Epstein et al. 1988, Cancer Research, 48, 5842-5848).
The cell killing process capable of selectively killing cells which have a high sensitivity to being killed by, the natural system of the living host, and having a high sensitivity to being killed by the cell killing process. The cell killing relocates the natural intra-cellular material of cells, including DNA, histone, and complexes of DNA-histone, to the extra-cellular fluid of the cancer, thereby making it available for tethering by the fifth, eighth, and eleventh bispecific reagents.
The cell killing process including at least one of the administration any one of numerous anti-cancer cytotoxic drugs or cellular agents at a low dosage so that only cells with the characteristic of having a high sensitivity to being killed by the cell killing process would be killed. The cell killing process also includes non-toxic agents, such as hormones or anti-hormones, or a procedure, such as orchidectomy, which leads to an alteration in the hormonal status of the living host and causes a cell killing process called apoptosis which is directed against cells of a particular cell lineage which are sensitive to the hormonal status of the living host. For example, orchidectomy and/or the administration of anti-androgens causes the apoptotic killing of a large number of normal prostate cells and a variable number of prostatic cancer cells. Regardless of which cell killing process is employed, the cell killing process is capable of selectively killing at least cells with the characteristic of having a high sensitivity to being killed by the cell killing process.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 08782590 | Jan 1997 | US |
| Child | 10949671 | Sep 2004 | US |
