The detection of some, but not all, of the proteins in the pattern does not result in cell killing. Drugs with pattern recognition targeting capabilities can carry out operations that are logically equivalent to the hypothetical cancer-curing machine discussed in a previous section. To consistently cure or control cancer, and do so without significant patient side effects, there is a requirement for some type of PRTT.

There a variety of methods by which PRTT can be achieved. The simplest method involves absolutely no new technology. For decades scientists have made targeted anticancer drugs comprised of a targeting ligand, a linker and a toxic effector agent. The basic structure is illustrated below:

The targeting ligand binds to the target protein on the tumor cell. The complex gets taken up by the cancer cell and the attached poison separates and kills the cell. This is standard stuff. So called “smart bombs” have been around for decades. Commonly the targeting ligand is a monoclonal antibody. A number of such agents have been approved by the FDA for the treatment of leukemia.

Targeted Delivery of a Targeted Toxin, TDTT

TDTT is exactly the same, with one exception. The effector agent is selected so that it is toxic only for cells that have a second target inside the cell. The target pattern is the protein receptor and this second target. For example, consider an effector agent that binds to MCM proteins and then kills the cell. If the cell lacks MCM proteins then it will not be killed. If the cell lacks the cell surface protein receptor then the cell will not be killed. For toxicity to result the entire pattern must be present. The concept of TDTT is illustrated in the diagram below:

The targeting pattern can also be a receptor in the tumor cell environment and an intracellular receptor in the tumor cells as illustrated below:

PRTT based on this approach involves no new science. Conventional chemical approaches to drug targeting can be employed.

PRTT Based on Independently Targeted Synergistic Agents

A second approach that also requires no new technology is based on the targeted delivery of two drugs. The drugs are selected so that each drug alone is essentially non-toxic, but in combination the drugs are very toxic. The approach is illustrated below:

In this approach Agent 1 and Agent 2 are individually nontoxic or of low toxicity but in combination highly toxic. Only cells that express the target pattern of both receptors will be killed. Systems can also be devised in which one or both of the targeting receptors are located in the tumor cell environment rather than only on the tumor cells. This version has a broader applicability and is better suited to comprehensive targeting given the environmental nature of invasiveness. PRTT based on independently-targeted, synergistic agents involves no new chemistry. The key to this method is the use of agents that exhibit pronounced synergistic toxicity. Many pairs of synergistic agents are potentially suitable.

One general class of synergistic agents consists of a targeted prodrug and an enzyme that can specifically toxify the prodrug by activating a trigger. The same type of chemistries that have been developed for antibody-directed–enzyme-prodrug therapy (ADEPT) can be employed. [i] Binary systems that generate low molecular weight, diffusible products that bind irreversibly to cell in the local environment and result in cytotoxicity or permanent arrest of the cell proliferation are especially attractive. To a first approximation, under non-saturating conditions, the reaction rates should be second order. In other words, a 100 fold enhancement in tumor concentration of each targeted agent will therefore give an initial reaction rate that is approximately 10,000 times greater than that in the blood. This means that at sufficiently low drug doses, prodrug activation in the peripheral blood can be insignificant compared to that within the tumor. PRTT based on this approach involves no new science. Conventional chemical approaches to drug targeting can be employed.

Another approach is designed to target patterns comprised on a target receptor and an enzyme.

In this approach the target pattern is comprised of both a triggering enzyme and a target receptor. If only the triggering enzyme is preset than the drug is not concentrated and retained at the site, and toxicity will be minimal. If only the targeting receptor is present than the drug will not be activated into a toxin. PRTT based on this approach involves no new science. Technologies for making the components of the drugs: targeting ligands; linkers; triggers; and toxins are well known. A urokinase-activated, recombinant, diphtheria toxin targeted to a cell surface receptor was recently described. [ii]

A wide variety of enzymes that are involved in the mechanisms of invasiveness can be employed as triggering enzymes including:

• Urokinase
• Plasmin
• Tissue plasminogen activator (tPA)
• Matrix metalloproteinases
• Activated MMP  -2, MMP-9 (Gelatinases)
• MMP-7, MMP-26 (Matrilysins)
• MMP-1, 8, 13 (Collagenases)
• MMP-3, 10,11 ( Stromelysins)
• MMP-14, 15, 16, 25 (membrane types)
• Cathepsins B, D, S, L, K
• Seprase/ Fibroblast Activation Protein
• Heparanases
• Legumain
• C-Proteinase or bone morphogenetic protein-1
• ADAMTS-2
• Myeloperoxidase

Efficient substrates that can serve as the basis of triggers are known for most of these enzymes. For example, prodrugs activated by plasmin, urokinase, and matrix metalloproteinases have been developed. [iii] There are a number of different options and mechanisms by which trigger activation can toxify the drug. A simple mechanism involves liberation of an active toxic agent that can diffuse into cells only after cleavage from the targeting complex. This mechanism has the advantage of being applicable to target patterns located in the tumor cell environment  as well as on the tumor cell. A wide range of suitable cytotoxic agents are available that can be functionally connected to a given trigger and released upon trigger activation.

Making the tumor look like a bacterial infection
A variation on the above approach involves the unmasking by the triggering enzyme of an agent that marks the local tissue for destruction by the immune system. In effect, the targeted, activated agents makes the tumor look like a bacterial infection. This approach is suitable for target patterns that are in the tumor cell environment as well as on tumor cells and also can provide an efficient method to increase the level of triggering enzymes within the tumor environment.

Evolution has endowed the body with the ability to mount effective and almost immediate nonspecific defenses against infectious agents. The body is highly tuned to detect and react to certain molecules derived from, or generated in response to, bacterial infections. [iv] Receptor binding can result in a rapid and massive influx of inflammatory cells to the site. The secondary release of inflammatory factors amplifies the response. Activated white blood cells generate massive amounts of hydrogen peroxide and release large quantities of myeloperoxidase into the extracellular environment. The net result is the production in the extracellular environment of reactive chemical species that kill invading microorganisms. [v] The same intense immune response that is elicited by bacteria can be directed against tumors by the selective targeting of certain known chemicals to tumors. The ability of activated white blood cells to kill tumors is well documented in numerous models. [vi] Truly impressive antitumor activity has been observed in tumors infected with bacteria. [vii] [viii] The same type of inflammatory reaction and tumor destruction could be produced by the tumor selective delivery of effector agents that attract and activate white blood cells, without the risks inherently caused by active bacterial infection.

Malignant cells can evolve that are resistant to the hostile effects of activated white blood cells and white blood cells may enhance the metastatic spread of tumor cells. [ix] However, this should not be an issue within the context of a set of drugs that kill cells expressing target patterns related to invasiveness. Massive signal amplification is possible. To attract and activate one white blood cell will require only a small number of drug molecules. However, one white blood cell can release billions of molecules of enzymes and recruit additional white blood cells to the area. The accumulation and movement of inflammatory cells within the tumor site is a rapid, energy dependent process that contrasts to the relatively slow passive diffusion of drug molecules. In addition, migrating white blood cells may increase the rate of drug permeation in tumors by opening up channels and generating micro-fluid flow.

The targeted delivery of chemotactic agents that attract and locally stimulate white blood cells can provide positive feedback mechanisms to powerfully amplify the delivery and biological activity of drugs targeted to patterns of proteins. In addition to the potential for signal amplification, there is a change of scale. The interactions of targeted drugs and their receptors are at the molecular scale. However, the biological result is determined by interactions that span distances on the scale of hundreds of micron to millimeters. A lone activated white blood cell is of little consequence. The reactive oxygen species and enzymes released can be destroyed or inactivated by a variety of mechanisms. However, as the density and tissue volume of activated white blood cells increases, a critical mass is reached at which there is a virtual inflammatory explosion resulting in a localized tissue destruction or abscess formation. [x]

The targeted delivery of agents that attract white blood cells can provide a highly efficient method for the selective delivery of prodrug-activating enzymes to a tumor.

Some important design specifications and considerations for this approach are given below:

• The targeted chemotactic agent must be devoid of activity until released by the triggering enzyme or triggering agent

• Targeting specificity must be defined by the targeting ligand, not the attached chemotactic agent.

• The chemotactic agent must be sufficiently potent to be biologically active at achievable tumor concentrations.

• The chemotactic agent should activate inflammatory cells.

A number of extremely potent, low molecular weight agents are known that can potentially be used to meet these requirements by binding with high affinity to the FPR, FPRL1, platelet-activating factor receptor, or LTB4 receptors [xi] The key is to connect the chemotactic agent to the targeting group by a trigger that reversibly masks chemotactic activity by precluding binding to the receptors on the inflammatory cells. Trigger activation will release the biologically active chemotactic agent. The development of such agents is well within the scope of existing technology.

PRTT Based on Multi-Site Binding

Nature provides an example of nearly perfect, almost theoretically ideal molecular targeting: anaphylactic shock. Incredibly minute concentrations of antigens can elicit histamine release from mast cells and trigger anaphylaxis. The mechanism is multi-site binding of the antigen to cell surface receptors. This same mechanism can be exploited to develop tumor-specific anticancer drugs that approach the limits of ideal targeting.

The following sections describe some approaches to Pattern Recognition Tumor Targeting that have the potential to enable dose reductions of as a much as one million times relative to non-targeted chemotherapy and thousands of times relative to current targeted approaches.

The enormous difference in functional affinity possible between single site and multi-site binding can provide a basis for pattern recognition targeting.

This approach is illustrated below:

The presence of multiple targeting ligands can enable the drug to engage in multi-site binding and endow the drug with pattern recognition capability. Multi-site binding can increase the functional binding affinity or tightness of binding by thousands of times. However, this enhancement of affinity can occur only if the cell expresses the targeting receptor pattern required for multi-site binding. This means that two site binding can allow drug to bind to cells with the pattern of receptors at drug concentrations thousands of times lower than required for binding to cells with just one of the receptor types.

The ability of multi-site binding to markedly increase functional binding affinity is extensively utilized by nature. [xii] Examples include: immunoglobulin molecules; [xiii] complement C1q;[xiv] antigen mediated histamine release from basophils; [xv] and shiga like toxins and antagonists. [xvi] An increase in functional binding affinity of 200,000 times (compared to single site binding) has been described for peptabodies, a class of proteins with five binding sites. [xvii] A ten billion times increase in affinity has been reported for the binding of a trimer of vancomycin to a trimer of the dipeptide d-Ala-d-Ala. [xviii] In other words, for a molecule that engaged in three site binding the tightness of binding was ten billion times greater.

The enormous increase in binding affinity that can result from multi-site binding can enable cancer cells to be killed at extraordinarily low drug concentrations. This is what happens in anaphylactic shock or severe peanut allergy. [xix] [xx] Nature has provided a wonderful mechanism in multi-site binding that can be utilized to develop anticancer drugs of both extraordinarily high target affinity and pattern recognition capabilities.

The binding of a drug with two targeting ligands to cell surface receptors will occur in two steps: with one site binding followed by binding at the second site. The targeting receptors need not be in close proximity as most proteins are mobile on the cell surface. [xxi] Byron Goldstein and Dr. Carla Wofsy from Los Alamos National Labs, kindly did some mathematical modeling of the predicted binding of drugs with two different kinds of targeting ligands to cells with the target pattern or with only one receptor type. Computer modeling predicted that high specificity for cells with the pattern is possible. In addition, drug binding to cells with the pattern is expected at concentrations that can be thousands of times lower than to cells with only one type of receptor. Finally, once bound, the rate of drug escape from the cells is predicted to be very slow. Multi-site binding can be a very useful tool in PRTT.

Exponential Pattern Recognition Tumor Targeting

Exponential Pattern Recognition Tumor Targeting is a method of pattern targeting designed to enable a massive exponential amplification of the quantity of anticancer drugs that are specifically delivered to tumor cells. Targeting specificity is for the pattern of a targeting receptor and a triggering enzyme. In effect, from one targeting receptor two are created, from two four, etc. Massive amplification of drug delivery to target patterns should be possible. Exponential PRTT can be used to enhance and amplify the previously discussed methods of PRTT. In this method two compounds are co-administered to the patient.

Compound 1 is a drug molecule that is comprised of one or more targeting ligands that can bind specifically and tightly at low concentrations to targeting receptors on the surface of the tumor cell and a masked female adapter. The masked female adapter is a chemical group which, when unmasked can bind tightly and specifically to the male ligand.

Compound 2 is a drug molecule that is comprised of a male ligand that can bind to the unmasked female adapter; a toxic anticancer drug; and two or more masked female adapters.

Exponential PRTT can also be employed against target patterns that are located within the tumor cell environment, including non-cellular components of the extracellular matrix. In this case, the toxins or effector agents delivered must be able to generate a zone of anticancer activity in the local volume that surrounds the targeting elements. This can be accomplished by employing a trigger that upon activation releases effector agents that are rapidly internalized by neighboring cells.

Triggering Enzymes
Targeting specificity in Exponential Pattern Recognition Tumor Targeting derives from the pattern comprised of the targeting receptors on the cell surface and the triggering enzyme. A broad range of enzymes that are enriched on tumor cells or in the microenvironment of tumor cells, such as plasmin, urokinase, matrix metalloproteinases, fibroblast activation protein can be utilized to specifically trigger unmasking of the masked female adaptors. Triggering enzyme can also be delivered to the tumor as previously described. Technology for the development of such triggers is well known.

Male and Female Adaptors
There are very many chemical groups and structures that can be employed as male and female adaptors. Some important design considerations and specifications are given below:

• Chemical stability
• Low toxicity
• Specific and high binding affinity, with a low off rate. (Covalent binding is ideal.)
• The female adaptors must have a suitable site for a masking trigger that precludes male ligand binding
• Trigger activation must be efficient and rapid in the presence of the triggering enzyme or
• Low non-specific protein binding.

Oligo-Nucleotide Analogs as Female Adaptors
Male ligands and female adaptors are available that bind specifically, rapidly, and essentially irreversibly. Complementary peptide oligo-nucleotide analogs are an example. [xxii] These are essentially short pieces of DNA-like molecules that can bind together very tightly. A wide range of chemical components based on existing technology can be utilized to mask the female adaptor. The high binding affinity between the selected male ligands and female adaptors could allow the drugs to be used at extremely low concentrations, yet massively accumulate in the tumor. There are many other types of suitable male and female adaptors.

Exponential PRTT is an extremely versatile technology. Instead of consuming receptors, the targeted drug will in effect increase the target receptor density. The more drug that is delivered, the more drug that can be delivered. In addition, systems can be developed that require two or more triggering enzymes for exponential signal amplification.

Multiple, different, toxic drugs can be targeted and delivered to a single target pattern site as illustrated below:

The result will be a tree like accumulation of different drugs on the cell surface as illustrated below:

The forest fire effect
Amplification of the size of the targeted area can also be obtained to give a forest fire like spreading effect. This can be achieved by using Compound 2 to release a Compound 1 like structure. In this approach, trigger activation releases a Compound 1 like structure and concurrently unmasks a reactive group. The liberated Compound 1 diffuses a short distance and covalently binds by the reactive group to adjacent cells and macromolecules. The net result is the creation of a new receptor site a short distance away. Unmasking of the female adaptors, and binding of additional Compound 2 results in amplification at the new site. The cycle can repeat multiple times.

A variety of triggers can be employed to liberate the Compound 1 and simultaneously generate a reactive chemical group.

A closely related approach can be used to target patterns comprised of two enzymes. In this approach triggering enzyme 1 unmasks a reactive group on Compound 1 that covalently binds it to nearby cells and proteins. Triggering enzyme 2 than unmasks the female adaptor. Amplification can then proceed as described previously. In effect this approach transforms enzyme activity into high affinity targeting receptors.

Exponential amplification can also be dependent upon the presence of a pattern of triggering enzymes. For example, if Compound 2 has the following structure:

then both triggering enzyme 1 and 2 will be required for exponential amplification. Only linear amplification is possible in the presence of enzyme 1 alone or enzyme 2 alone. The difference between linear and exponential amplification is huge, (~ 28 fold after only 6 cycles, and ~100,000 fold after twenty cycles.)

Self-Amplifying Exponential PRTT

Another potentially very useful variation on the theme involves the use of a Compound 2 like structure that is self-amplifying. Compound 1, is targeted to a pattern, and the masked female adaptor is unmasked as in previous approaches. In this version, when Compound 2 binds to the female adaptor, two female adaptors  are unmasked in the process, without the need for enzymatic unmasking. A description of the chemistry of Self-Amplifying Exponential PRTT is beyond the scope of this document.

In Self-Amplifying Exponential PRTT, the very act of male-female adaptor binding will trigger the unmasking of two additional female adaptors. In the absence of a female adaptor this will not occur. Each unmasked female adaptors will in turn trigger the unmasking of two female adaptors. After 20 cycles, approximately one million-fold amplification will result. Forty cycles, in principle would give a 1012 fold amplification and fifty cycles a 1015 fold amplification. In practice other factors will become limiting.

Self-amplifying Exponential PRTT can make it practical to use masked female adaptors that require several different enzyme for unmasking. In other words, a masked female adaptor can be used to detect a pattern of enzymes. In the absence of amplification, this could be impractical. If multiple triggering steps are required, then only trace quantities of unmasked female adaptor may be generated (for kinetic reasons). However, self-amplifying exponential amplification could enable even trace quantities of unmasked female adaptor to be detected and targeted.

Exponential PRTT can transform the challenge of delivering a set of drugs to multiple, different, target patterns into the much simpler task of targeting a set of drugs to one common target: female adaptors. There are many potential practical advantages to this strategy including: a reduction in the number of drugs compounds required and the flexibility and developmental economies afforded by the modular separation of drug targeting specificity from drug cytotoxic activity. This could have very substantial practical advantages.

Transforming a Set of Different Target Patterns into a Common Target: Female Adaptors

Amplification

The degree of amplification with exponential PRTT will be a function of the number of cycles as illustrated in the graph below:

Ultimately the exponential amplification will be limited by the enzyme activity, substrate availability, or time available for amplification. As discussed below even modest amplification can have profound biological effects.

For many drugs there is an approximately linear relationship between the log of the cell survival fraction and the drug concentration. Additive increases in drug concentration can give exponential decreases in the probability of tumor cell survival. This is exemplified by the graph of the surviving fraction of cells versus drug concentration for tirapazamine. [xxiii]

Accordingly, the increased drug delivery enabled by Exponential PRTT can potentially lead to truly massive increases in antitumor activity.

Summary

A variety of different approaches based on well established chemical principles can be employed to make drugs with PRTT capabilities. The basic modular components exist. It is a matter of doing the work and engineering optimization. We see no reason that a set of drugs that can carry out the logical operations of our hypothetical cancer-curing machine cannot be developed and in clinical trials in 5 to 6 years.

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