Specific Targeting and Delivery of Therapeutics to Cancer Cells Based on the Tumor Microenvironment Damien Thévenin Department of Chemistry Bioscience in the 21st century (Sep. 14, 218)
Anticancer Drugs and Side Effects Most anticancer drugs have off-target side effects. Severely limit the efficacy of chemotherapy. Clear needs for targeted therapies
Drug Carrier Systems Can improve the therapeutic index by reducing : Side effects in healthy tissues. The overall dose by concentrating the drug in the targeted tissue. Carrier systems include:
Drug Carrier Systems Can improve the therapeutic index by reducing : Side effects in healthy tissues. The overall dose by concentrating the drug in the targeted tissue. healthy tissue Passively target tumors due to the increased permeation of many solid tumors. owever, this effect is small for certain tumors. cancer tissue Specific targeting strategies have been developed
Current Targeting Strategies Most take aim at specific cancer cell surface biomarkers. Example: over-expressed cell surface receptors. Involve the addition of ligands to the carrier system. healthy cell cancer cell Allows specific interaction with cancer cells ow do they get into cells?
Current Targeting Strategies: Relying on Endocytosis Surface receptors are recycled through endocytosis: Cancer Cell Membrane
Current Targeting Strategies: Monoclonal Antibodies Antibodies can be raised against any cell membrane receptors. Cancer Cell Membrane
Current Targeting Strategies: Monoclonal Antibodies Mechanism of action of two FDA-approved antibody-drug conjugates: Approved for: ER2-positive metastatic breast cancer Approved for: odgkin lymphoma and systemic anaplastic large cell lymphoma ess et al. Med. Chem. Commun. (214)
Current Targeting Strategies: Drawbacks 1. ealthy cells also have the same biomarkers. 2. Different cancers have different biomarkers. healthy cell 3. Even in the same tumor, cancer cells can have different biomarkers. 4. Fast evolution of cancer cells --> loss of receptor. Breast cancer cell uptake into normal tissues unacceptable toxicity profiles therapy resistance and disease progression eeds for a more general biomarker
Acidosis: A General Feature of Tumors Tumors: characterized by a lower extracellular p when compared to healthy tissues. Cancer cell p e = 5.5-6.5 ormal cell p e = 7.5 p i = 7.5 p i = 7.2 Acidosis = General biomarker of tumors. ow can we target acidosis?
plip: p(low) Insertion Peptide plip: AAEQPIYWARYADWLFTTPLLLLDLALLVDADEGTG state I in solution state II with lipids p5 ~ 6 state III inserted Bacteriorhodopsin (from alobacterium salinarum) unt et al. Biochemistry (1997) Reshetnyak et al. Biophys. J (27) Reshetnyak et al. PAS (28)
plip: Imaging Tumors in vivo ude mouse with cancer cells expressing the Green Fluorescent Protein (GFP) Cancer Cell low pe GFP Andreev et al. Chim ggi. (29) Andreev et al. PAS (27) Segala et al. Int J Mol Sci (29) plip + GFP
plip: A Targeting and Delivery Agent -S-S- -S-S- Cancer Cell low pe -S
plip plip: Therapeutic Strategies plip plip plip -S-S- DRUG Cancer cell low pe Cancer cell low pe Cancer cell low pe plip plip plip PAR1 -S S- DRUG plip EGFR EGFR Gα ɣ β Cancer Cell Death P P P X Burns, Thévenin (215) Mol. Pharm. Burns et al. (217) Mol. Pharm. Burns, Thévenin (215) Biochem. J. Gerhart J. (218) ACS Chem. Biol.
Strategy #1 Specific Delivery of Auristatin Derivatives
Monomethyl Auristatins: Potent Cytotoxics Monomethyl Auristatins - Family of antimitotic agents. - Derived from Dolastatin 1. - Inhibits tubulin polymerization. - Extremely toxic. - Must be delivered specifically. ) S S S S MMAE + plip-s R' R plip S S Reduction of the disulfide (S-S) breaks inside cells Compound R R Log Po/w IC5 (nm) Dolastatin 1 C33S 3.4.1 MMAE C3 2.2.1-2 S MMAF C.7 15-25 MMAF-Me CMe 2.8.1 Burns, Robinson, Thévenin (215) Mol. Pharm.
plip-mmae: Inhibition of Cancer Cell Proliferation 2 hour treatments Measure cell viability after 72h ela = Cervical cancer cells MDA-MB-231 = Triple negative breast cancer cells plip(wt)-mmae p 7.4 ela p 5. plip(wt)-mmae p 7.4 MDA-MB-231 p 5. % cell viability 1 8 6 4 % cell viability 1 8 6 4 2 2. 1.25 2.5 5. 1.. 1.25 2.5 5. 1. [Conjugate] (µm) [Conjugate] (µm) p- and concentration-dependent cytotoxicity Burns, Robinson, Thévenin (215) Mol. Pharm.
plip-mmae: Inhibition of Cancer Cell Proliferation plip variant (D25E) with a p5 = 6.5 AAEQPIYWARYADWLFTTPLLLLELALLVDADEGTG 352 plip(wt) plip(d25e) plip(d25e)-mmae p 7.4 ela p 5. λ max (nm) 347 342 % cell viability 1 8 6 4 2 337 4 5 6 7 8. 1.25 2.5 5. 1. [Conjugate] (µm) p plip(d25e)-mmae p 7.4 MDA-MB-231 p 5. % cell viability 1 8 6 4 2. 1.25 2.5 5. 1. [Conjugate] (µm) Burns, Robinson, Thévenin (215) Mol. Pharm.
plip-mmae: In vivo Targeting 1 4 6 28 48 48 rs Alexa75-pLIP-MMAE Cr nu/nu mice MDA-MB-231 xenograft Intravenous injection Burns, Robinson, Thévenin (215) Mol. Pharm.
ext Generation Conjugate: MMAF and plip Variants vs. MMAE (Log P o/w = 2.2) crosses cell membrane readily MMAF (Log P o/w =.7) more polar than MMAE > more difficult to cross membranes plip Variant Sequence p5 WT AAEQPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 6.1 D25E AAEQPIYWARYADWLFTTPLLLLELALLVDADEGTCG 6.5 P2G AAEQPIYWARYADWLFTTGLLLLDLALLVDADEGTCG 6.8 R11Q AAEQPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 5.8 R11Q + D14Up AAEQPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 5.6 Aad (α-aminoadipic acid) Gla (Ɣ-carboxyglutamic acid) D14Gla + D25Aad AAEQPIYWARYAGlaWLFTTPLLLLAadLALLVDADEGTCG 6.8 nyango et al. (215) Angewandte Chemie Fendos et al. (213) Biochemistry Barrera et al. (213) PAS
ext Generation Conjugates: Cytotoxicity in ela Cells plip(wt)-mmaf p 7.4 p 5. plip(d25e)-mmaf p 7.4 p 5. plip(p2g)-mmaf p 7.4 p 5. % cell viability 1 5 633 nm 28 nm 23-fold 1 5 944 nm 342 nm 3-fold 1 5.A. 435 nm.a. µm Control.1.1 1 1 [conjugate] (µm) µm Control.1.1 1 1 [conjugate] (µm) µm Control.1.1 1 1 [conjugate] (µm) plip(r11q)-mmaf p 7.4 p 5. plip(r11q);d14(up)-mmaf p 7.4 p 5. plip(d14gla);d(25aad)-mmaf p 7.4 p 5. % cell viability 1 5 2465 nm 469 nm 5-fold 1 5 2441 nm 153 nm 16-fold 1 5 1929 nm 81 nm 24-fold µm Control.1.1 1 1 [conjugate] (µm) µm Control.1.1 1 1 [conjugate] (µm) µm Control.1.1 1 1 [conjugate] (µm) plip(wt)-mmaf over 1-fold more potent than MMAE conjugate! Lead agent for further in vivo studies Burns et al. (217) Mol. Pharm.
plip-mmaf: In vivo Therapy Study Cr nu/nu mice bearing ela tumors (injection of with 5x1 6 cells) Injection of 1 mg/kg i.v. (Days 1, 3, 5 and 8) 1 mice per cohort Faction of initial tumor volume 6 4 2 vechicle plip-mmaf 4 8 12 16 Day Percent survival 1 9 8 7 6 non-treated plip-mmaf 5 5 1 15 2 25 Day 2 treated mice were excluded from calculations because initial tumors were too small: - did not end up growing tumors - 1 is certainly cured!! istopathology of tumors for Ki-67 (a marker of cellular proliferation) - number of cells undergoing cell division is significantly lower in the treated vs nontreated tumors. Burns et al. (217) Mol. Pharm.
plip-mmaf: Studies in immuno-competent mice Toxicology in BALB/c mice 1 mg/kg i.v. (Days 1, 3, 5 and 8) 37 markers Therapeutic Efficacy 4T1 murine breast cancer cells in BALB/c mice 2 mg/kg i.v. (Days 1, 3, 5 and 8) before 3 days 14 days Alanine aminotransferase ALT (U/L) 6 4 2 8 mock plip(wt)-mmaf MMAF-linker Urea itrogen (mg/dl) 3 2 1 25 mock plip(wt)-mmaf MMAF-linker % cell viability 1 75 5 25 p 7.4 p 5. 4T1 cells White blood cell count (cells/µl) 6 4 2 mock plip(wt)-mmaf MMAF-linker monocytes absolute (per µl) 2 15 1 5 mock plip(wt)-mmaf MMAF-linker 8 plip(wt)-mmaf 1 1 [plip(wt)-mmaf] (µm) 4T1 tumors Red Blood Cells (1 3 /µl) 12 8 4 mock plip(wt)-mmaf MMAF-linker White blood cell count (cells/µl) 8 6 4 2 mock plip(wt)-mmaf MMAF-linker Tumor volume (mm 3 ) 6 4 2 plip-mmaf MMAF-linker vehicle 4 8 12 Day