Requirements for the Cure of Cancer: Formulating a

Plan of ActionWorkshop sponsored by the

Van Andel Institute Jan. 10-11, 2007

FROM PRINCIPLES TO PRACTICE

SESSION VI(B) The development of technologies for

targeting cells that express target patterns

Arnold Glazier MD

General Design Considerations

The challenges of multi-drug therapy

Ideal Drug Targeting

• The tumor would act like a black hole for drug

• All drug in the blood flow to the tumor would be irreversibly retained

• No drug accumulation in non-target sites

Ideal Drug Targeting

• Based on a typical blood flow of 0.15 –0.6 ml/min/gm and 24 hours, maximum average tumor levels would be about 200-800 times the average blood level

• The biological effects can be even orders of magnitude higher

Multiplicative Increases in Concentration can give Exponential Increases in Effect

Surviving Cell Fraction versus Drug Concentration

TirapazamineBrown JM, Wouters BG.; . Cancer Res. 1999 Apr 1;59(7):1391

Examples of Almost Perfect Targeting Exist

• Hormone/ receptor binding• Peanut allergy / anaphylactic shock• Nerve gas

Approaches Towards Ideal Drug Targeting

• Specific, high affinity or irreversible binding• Slow “off rates” of drug from receptors• Administering the drugs at the lowest

concentration needed to saturate “drug accessible” receptors

• Decreasing nonspecific binding • Increasing the quantity of drug receptors

(exponential PRTT)• Prolonging treatment time

Principles that can be applied towards achieving these goals are well known. (Multi-site binding, slow binding, covalent binding, etc..)

Major Issues

• Chaotic and uneven blood flow• Limited drug penetration into tumors• Slow rates of drug diffusion• Episodic target pattern expression• On a given day only parts of a tumor

will be drug accessible

The drugs need to be given continuously for prolonged periods of time. (6 months?)

The Aim Should be to Deliver Drug to “Drug Accessible” Target Patterns • The important pathology that sustains cancer

occurs within a limited zone around blood vessels.• Areas close to blood vessels will be drug

accessible. • Drug accessible cells will be killed, new layers of

cancer cells will be exposed and killed over time in an “onion peeling effect”

• Therapy needs to be sufficiently intense so that the rate of cell loss exceeds the rate of cell production

A Minor, Sustained Decrease in the Probability of Cancer Cell Survival can have Profound Effects

Data: Berman JJ, Moore GW; Anal Cell Pathol. 1992 Sep;4(5):359-68

Drugs Targeted to a Comprehensive Set of Target Patterns will Inhibit

• Angiogenesis• Vasculogenic mimicry• Vascular co-option

This will achieve Dr. Folkman’s vision by effectively depriving tumor cells of new blood supply, constraining growth and allowing time for the “onion peeling” killing effect to work.

Non-synchronous Expression of Target Pattern Elements

Targeting specificity should be for• Invasiveness alone, or• Invasiveness and the potential for

proliferation

Elements of these classes of target patterns are expressed concurrently.

Effector Agents Should be Cell Cycle Independent

G2/mitotic-specific cyclin-B1 in colon cancer http://www.proteinatlas.org/

The Microenvironmental Nature of Invasiveness

There is a requirement for approaches that generate a zone of anticancer activity in the local volume that surrounds target patterns

Major RequirementsThe need for:• Pattern specificity• Signal amplification• Multiple, redundant mechanisms of cell killing or

inactivation• Prolonged therapy• The ability to simultaneously give multiple drugs• Chemical stability• Lack of antigenicity• Modularity in design

The Logic Function of PRTT Drugs

Are all the elements of the pattern present ?

Yes No

Kill Cell Spare Cell

BA C

Specificity is for the pattern, not the individual elements.

4. Catalysis of a reaction

5. Dissolution or precipitation

1. Binding

3. Breakage of chemical bonds

2. Chemical bond formation

Medicinal Chemistry Boils Down To:

Modular Building Blocks• Targeting ligands• Triggers• Triggering agents• Effector agents• Linkers and scaffolds• Male and female adaptors• Masking groups• Molecular clocks• Intracellular transport ligand• Solubility modifiers

These components exist and are within the scope of current technology.

Targeting Ligands

Ligand Receptor Complex

Ligands are chemical groups that bind together like a lock and key to target receptors.

A Urokinase Selective Ligand

HNHN N

HN NH2

NHOH

O

O

HO

Tamura S Y., et al., Bioorganic Med Chem Lett, 10:983-987 (2000)

Kd is in the low nanomolar range.

Triggers and Triggering Agents

Triggers are chemical groups then when acted upon by a triggering agent undergo a chemical change.

Enzymes and non-enzymes can serve as triggering agents.

Trigger

Drug molecule

Triggering Agent Chemically altered drug

Applications of Triggers

• To turn on or off a chemical process• To activate a toxin• To inactivate a toxin• To unmask a ligand• To release a toxin

Effector Agents

• Toxic agents that kill cells• Agents that irreversibly block the

potential for cell proliferation• Agents that trigger an immune

response• Agents that amplify a response

Linkers and ScaffoldsToxin

Trigger

Targeting Ligands

Linkers

Scaffold

Structural elements that provide the backbone of the drug

Rigidity, multiple sites for linker attachment,solubility, spatial separation of components, low toxicity

Cyclodextrins as Scaffold

Male and Female Adaptors

The male and female parts bind specifically and tightly.

In the ideal case the binding is irreversible.

Masking Groups

A masking group blocks a receptor.A triggering agent can unmask the receptor.

MaskedReceptor

UnmaskedReceptor

Triggering Agent

Molecular Clocks

Molecular clocks provide an adjustable time delay between a triggering event and a chemical change.

Chemical change

Trigger

Triggering Agent

Intracellular Transport Ligands

Tumor CellTumor Cell

Cell Receptor

Transport into Cell

Drug

IntracellularTransport Ligand

Intracellular transporter groups can also work by physical, non-receptor mediated mechanisms.

DrugDrug

Drug

A wide range of pattern targeting technologies can be developed by combining these modular building blocks in logical ways.

PRTT ApproachesPRTT Approaches• Targeted delivery of a targeted agentTargeted delivery of a targeted agent• Targeted delivery of a trigger activated drug• Independently targeted synergistically toxic drugsIndependently targeted synergistically toxic drugs• Multi-site bindingMulti-site binding• Exponential Pattern Recognition TargetingExponential Pattern Recognition Targeting• Combinations of the aboveCombinations of the above• OtherOther

Targeted Delivery of a Targeted Cytotoxic Agent

This method is the simplest and requires no new drug technology

Targeted Delivery of a Targeted Targeted Delivery of a Targeted Cytotoxic AgentCytotoxic Agent

Drug

The Pattern is a Surface Receptor and Intracellular Target

For cell killing both must be present

Targeted Delivery of a Targeted Targeted Delivery of a Targeted Cytotoxic AgentCytotoxic Agent

Internalizationlinker cleavage

No Toxicity

Toxicity

Without pattern

With patternInternalizationlinker cleavage

Cell

Cell

Cell

Cell

The cytotoxic agent is toxic only if its target is present

The Targeting Receptor Can Also be in the Tumor Cell Microenvironment

  

The Target Pattern of a Receptor in the Microenvironment and an Intracellular Target

CellCell

Intracellular target y

Receptor x inmicroenvironment

Linker cleavage

ToxicityWith pattern

Cell Cell

Drug internalization

in

Targeted Delivery of a Trigger Activated Drug

Targeted Delivery of a Trigger Activated Drug

No Toxicity Toxicity No Toxicity

Cell

The Pattern is a Triggering Enzyme and a Receptor

Trigger

Toxin

TriggeringEnzyme

TriggeringEnzyme

Toxin

Trigger

Cell Tumor Cell Cell

Receptor

Only cells that have both the target receptor and the triggering enzyme will be killed.

A Urokinase-Activated GMCSF Receptor Targeted Diphtheria Toxin

Diphtheria toxin

TriggerUrokinase activates

Binds to GMCSFReceptor on cells

Ralph J. Abi-Habib, Shihui Liu, Thomas H. Bugge, Stephen H. Leppla, and Arthur E. Frankel; Blood, 1 October 2004, Vol. 104, No. 7, pp. 2143

The drug targets the pattern of urokinase and GMCSF receptor.

Targeting the Microenvironment

The drug is targeted to the microenvironment, released by the triggering enzyme, diffuses to the tumor cell and kills it.

The Pattern can be a Receptor and Triggering Enzyme in the Tumor Cell Microenvironment

Toxin

Trigger TriggeringEnzyme

Tumor Cell

Receptor

Trigger

Toxin

Tumor Cell Toxin

Cell Death

Advantages of Releasing a Toxin into the Tumor Microenvironment

• Invasiveness is a property of both the cancer cell and its microenvironment

• A zone of toxicity is created making it easier to kill all the cancer cells

Approaches that produce a zone of toxicity are strongly preferred.

Paired, Independently Targeted, Synergistically

Toxic Drugs

Paired, Independently TargetedSynergistically Toxic Drugs

Agent 2Agent 1

No Toxicity Toxicity No Toxicity No Toxicity Toxicity No Toxicity

Agent 1 Agent 2Agent 1

Tumor cellNormal cellType A

Normal cellType B

Agent 2

Individually, Agent 1 and Agent 2 are Individually, Agent 1 and Agent 2 are Nontoxic, But Toxic in Combination:Nontoxic, But Toxic in Combination:

Multi-Site Binding

Multi-Site Binding and Pattern Multi-Site Binding and Pattern RecognitionRecognition

Multi-site binding can give an enormous Multi-site binding can give an enormous increase in the tightness of binding increase in the tightness of binding compared to single site bindingcompared to single site binding

Vancomycin

Ala-Ala

Tri-Vancomycin

Tri- Ala-Ala

A Ten Billion Times Increase in Affinity due to Three Site Binding

Rao J, Lahiri J, Isaacs L, Weis RM, Whitesides GM; Science 280:708-11 (1998)

Kd = 10 –17Kd = 10 – 6

Multi-Site Binding

At low concentrations the drug can bind tightly to cells with the target At low concentrations the drug can bind tightly to cells with the target pattern without binding to cells that express only one element of the patternpattern without binding to cells that express only one element of the pattern

Toxin

Tumor cell

Toxin

Normal cell

Tight BindingTight Binding No Binding

Advantages of Multi-Site Binding

• Specificity for the pattern• Potency• Slow off rate• Immense reductions in the dose

of drug required• Reductions in side effects

Exponential Pattern Recognition Targeting

From one receptor create two, from two create four ….

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.

Exponential Pattern Recognition Targeting

In this method specificity is for the pattern of a receptor and a triggering enzyme.

Components of Exponential Pattern Recognition Targeting

Toxin

Targeting Ligand

Masked Female Adapter

Male Ligand

The male and female parts bind with very high affinity.

1 2

Tumor cell

Toxin

3

Tumor cell

Toxin

Toxin +

Tumor cell

Triggering-Enzyme

Tumor cell21

Toxin

Tumor cell

Triggering-Enzyme

4

Unmasked Female Adaptor

Compound 2

Compound 2

Compound 1

Two Unmasked Female Adaptors

5

Toxin

Toxi

n

Toxin

Tumor cell

After Multiple Cycles:

The Mechanism of Exponential PRTT

Triggering Enzymes Unmask the Female Adaptor

• Many enzymes that are over-expressed by tumors can be utilized

• The triggering enzyme can also be independently targeted to tumor cells

Exponential Pattern Recognition Targeting

1.) Component 1 binds to cell receptors. 2.) Triggering enzyme(s) unmask female adapter.

Tumor cell

Triggering enzyme

Tumor cell

Toxin

3) Component 2 binds to the unmasked female adaptor.4) The triggering enzyme unmasks twice as many new female adaptors.

Triggering enzyme

Tumor cell

Toxin

Tumor cell

Toxin

Toxin

Toxin

Toxi

nToxin

Tumor cellRepetition of the cycle can deposit a large quantity of drug in a tree like structure

Massive Amounts of Drug can be Delivered to a Tumor Cell

5 10 15 201

10

100

1 103

1 104

1 105

1 106

Amplification

Number of Cycles

The quantity can increase exponentially

Self-Amplifying Exponential Pattern

Recognition Targeting

Self-Amplifying Exponential PRTT

The very binding of a male ligand and female adaptor creates two new female adaptors without the need for a triggering enzyme.

ToxinToxin

Tumor cellTumor cell

Masked Female Adaptors Unmasked Female Adaptors

Female Adaptor

Male Ligand

Spontaneous

UnmaskedFemale Adaptor

Unmasked Female Adaptor

UnmaskedFemale Adaptor

Male and FemaleCovalently Bound

MaskedFemale Adaptor

MaleAdaptor

MaskedFemale Adaptor

Masked Female Adaptor

Masked Male Adaptor

Masked Female Adaptor

Bulky Group

Unmasked Female Adaptor

Female Adaptor from a second molecule

UnmaskedFemale Adaptor

Bulky Group

Female Adaptors can Transform Different Patterns into a Common Target

Pattern 2

Pattern 3

Pattern 4

Pattern 5

Pattern 6

Pattern 1

Pattern 7

Different Target Patterns

A Common Target

Female Adaptors

This can enable the efficient delivery of multiple drugs to each target pattern and prevent the development of drug resistance.

 Multiple Toxins Can be Delivered to a

Single Target Pattern

ToxinToxin Toxin

A wide range of possibilities and emergent properties can arise with drugs that interact with each other.

Amplification and positive feedback can be achieved by delivering enzymes to adaptors which in turn unmask additional adaptors.

An other approach is to deliver a marker to the target patterns that make it look to the immune system like a bacterial infection.

Massive signal amplification is possible along with a change in scale.

To attract and activate one neutrophil requires only a small number of

chemotactic molecules.

Each neutrophil can deliver billions of molecules of:

• Hydrogen peroxide• Myeloperoxidase• MMP-9• Urokinase• Elastase• Catepsins

The system exhibits positive feedback:

• Myeloperoxidase activates neutrophils• ROS inactivate protease inhibitors• Ros activate MMP’s• Ros stimulate MMP production• Cathepsins

The protease released can also activate MMP-2, MMP-9, and plasminogen.

The net result could be a massive signal amplification in and around the target pattern and…

a change in scale.