Antibody Drug Conjugates: Conjugates: Drug Antibody Principle,Applications, and Future Directions Future and Raffaele Colombo Raffaele

Department of Chemistry Antibody Drug Conjugates: Conjugates: Drug Antibody Principle,Applications, and Future Directions Future and Raffaele Colombo Raffaele

Pittsburgh – 11/22/2014 – Pittsburgh 2 Department of Chemistry Timeline Adapted from Adapted Drug Discov Today Discov Drug . 2014 , 19 ,869-81

3 Department of Chemistry • • • • and 150 more are ADCs in the preclinical stage development will products ADCs launched of to become that Industry participants estimate that that estimate participants Industry ADCs 30 than More that predicts firm research market The predict companies pharmaceutical major of Leaders > 60% of all existing drugs will be modifiedbe will drugs existing all of 60% > cell specific ADC factsADC r in are in less than 20than years inless exceed $5billion exceed advanced between by

2018

clinical

sales 100 4 Department of Chemistry Classical types of cancer treatment • • • • ypod n meod upeso hd curd fe epsr. oi S. profound treat Louis to used that be exposure. lymphoma could agent after this suggested that nitrogen occurred reasoned Gilman victims Alfred had to and Goodman suppression the exposed myeloid were of and lymphoid soldiers examination when Medical WW1 mustard. after prevent discovered to Initially try to treatment Systemic cells. invasion, growth, metastasis. and cancer kill to signals receiving from cells the divisionand growth for necessary preventing by growth beams. energy high with tumors. Chemotherapy: Treatments: Hormonal Radiation: Surgery: fe te is ln o tetet o mn solid many for treatment of line first the often

il h cne cls iety y aaig them damaging by directly cells cancer the kill a term used for a wide variety of drugs used drugs of variety wide a for used term a

eind o nii cne cell cancer inhibit to designed 5 Department of Chemistry Vinca Taxanes Alkaloids ANTIMICROTUBULE

Traditional chemotherapy AGENTS 5-Fluoro-uracile Mercaptopurine Azathioprine Capecitabine Fludarabine Cytarabine Cyclofosfamide Nitrosoureas Ifosfamide

ANTIMETABOLITES

ALKYLATING-LIKE CHEMOTHERAPY

AGENTS Tetrahydrofolate Platinum Drugs Methotrexate ANTITUMOUR ANTIBIOTICS

Podofylotoxins Camptothecines

Antracyclines

6 Department of Chemistry • • • •

h mxmm oeae dose tolerated maximum the agents chemotherapy efficacy tumor killing reduces the activity of chemotherapy chemotherapy of activity the reduces duration Systematic administration of these drugs results in bothin results drugs these of administration Systematic h blne ewe tee w atos iis the limits actions two these between balance The h tetet otn consist often treatments The The Traditional chemotherapy ai clearance rapid and tolerability and damage healthyto tissues , each administered administered each , of single-agent chemotherapy single-agent of f ml mlcls further molecules small of f obnto of combination of n fr a for and at or nearor at limited 7 Department of Chemistry • •

eutn i a o teaetc ne ad serious and index side effects cells therapeutic membrane low a mucous in resulting and marrow, bone blood, cells normal ih ae f mutation populations. cell tumor drug-resistant overgrow of rate high genetic their of because of Efficacy is further limited by the occurrence or developmentor occurrence the by limited further is Efficacy their by limited is Efficacy drug resistance Efficacy ofEfficacy chemotherapy . ( especially to rapidly growing cells such as such cells growing rapidly to especially : : instability tmr el cn eet and select can cells tumor , o-pcfc oiiy to toxicity non-specific , heterogeneity

and, ) 8 Department of Chemistry • •

MED: Minimum EfficaciousMED: Minimum Dose dose; MTD:tolerated Maximum tumor eradication complete remission Significantly Significantly >99% DRUG DOSE EFFICACIOUS DOSE (MED) TOXIC DOSE (MTD) Chemotherapy Chemotherapy Therapeutic Window Window Therapeutic of the cells in the tumor have to be killed to achieve a achieve to killed be to have tumor inthe cells the of Traditional Therapeutic window greater degree of cell kill required to achieve achieve to killrequired cell of degree sufficient number of EFFICACIOUS DOSE (MED) cancer cell without cancer cell Inability killto a causing toxicity TOXIC DOSE (MTD) ADCs / ADCs SMDCs Therapeutic Window Window Therapeutic 9 Department of Chemistry tumor cells via tumor-specific and/or over-expressed antigens/receptors antigens/receptors and/or over-expressed via tumor-specific cells tumor can ADCs/SMDCs MED: Minimum EfficaciousMED: Minimum Dose dose; MTD:tolerated Maximum DRUG DOSE • • EFFICACIOUS DOSE (MED)

Reduce normal tissue drug exposure exposure drug normal tissue Reduce tumor to delivery drug Increase TOXIC DOSE (MTD) Chemotherapy Chemotherapy Therapeutic Window Window Therapeutic Traditional Therapeutic window selectively deliver a drug potent cytotoxic EFFICACIOUS DOSE (MED) Increase potency Increase selectivity TOXIC DOSE (MTD) ADCs / ADCs SMDCs Therapeutic Window Window Therapeutic to 10 10 Department of Chemistry Nature Reviews Cancer, Antibody strategies 2001 ,

1 ,118-129 11 Department of Chemistry ADCs consist of a of consist ADCs coupled antigen cell What isWhat an ADC?

toa

monoclonal antibody ADC cytotoxic drugcytotoxic

mAb

viaa linker drug chemically chemically linker

12 12 Department of Chemistry • • are: aspects Important • • • ADCs consist of a of consist ADCs

The delivery vehicle The molecular target Cytotoxic agentCytotoxic or payload The linker Chemically conjugation coupled (including the mechanism of release) release) of mechanism the (including What isWhat an ADC? toa monoclonal antibody cytotoxic drugcytotoxic ( mAb (antigen) (antigen) (method, site, stoichiometric) stoichiometric) site, (method, )

(mechanism, potency) (mechanism,potency) viaa linker chemically chemically 12 13 Department of Chemistry Validated targets are: key role in role key Cancer cellspossess • • • • have: should target cancer ADC Ideal

antibody engagement engagement antibody accessibility rapid internalization and trafficking low abundant / absent Choosing a good target targets that fulfill all oftargets that these criteria tumor growth and progression and There are a limited number of expression expression to intravascular macromolecules intravascular to macromolecules uniform specific molecular markers CD30 and Her2 on healthy tissues expression on the cell surface surface cell the on expression to lysosomesupon that play a play that 14 14 Department of Chemistry clinical stage, for example: example: for stage, clinical in evaluated being currently are targets new of number A Target antigen Nectin-4 Nectin-4 VEGF-A GPMNB HER2 EphA2 EphA2 CD138 CD20 CD20 AGS-5 AGS-5 CD22 CD52 CD74 CD74 CD19 Le Le Choosing a good target Pancreatic cancer, prostate cancer, epithelial tumors tumors cancer, epithelial cancer,Pancreatic prostate B-cell lymphoma,B-cell non-Hodgkin lymphoma Colorectal cancer, kidney,lung, Colorectal Bladder, breast, lung, and pancreatic pancreatic andlung, Bladder,breast, HER2-positive breast cancer Prostate cancer, ovarian Prostate cancer Breast cancer, melanoma melanoma cancer, Breast non-Hodgkin lymphoma non-Hodgkinlymphoma non-Hodgkinlymphoma Multiple myeloma myeloma Multiple myeloma Multiple B-cell lymphoma lymphoma B-cell Cancer type Solid tumors tumors Solid glioblastoma

15 Department of Chemistry is the the is plasma inthe found antibody of class abundant most The Immunoglobulins Antibody structure

γ ( IgG ). (ca.150 chains light two andchains heavy Each - - chain: polypeptide IgG

Light Heavy are composed of two different different two of composed are IgG kDa molecule consists of two two of consists molecule orL chain(ca. 25 to ato different antigen Each variants can bind or H chain(ca.orH 50 in total) intotal) kDa kDa ) )

16 Department of Chemistry Monoclonal antibodies canbe: antibodies Monoclonal the mass represent antibody monoclonal The HAMA Murine Chimeric Humanized Human (human anti-mouse antibody) anti-mouse (human

of most ADCs ADCs most of Rapid clearance Antibody structure improved efficacy and improved safetyefficacy more ofthan 90% 17 17 Department of Chemistry constant ( variable( Fragment vH cL crystallizable ), light-chain variable( ), light-chain ), the complementarity determining regions ( regions determining complementarity ),the Antibody structure region ( region Fc ), fragment antigen-binding ( antigen-binding ),fragment vL ), heavy-chain constant ( ), constant heavy-chain CDRs cH Fab ) ), light-chain ),light-chain ) heavy-chain ) 18 18 Department of Chemistry approved and mainly in combination with other therapies! therapies! other with incombination mainlyand approved first the Insince 32 years Generic Name Panitumumab Alemtuzumab Alemtuzumab Bevacizumab Trastuzumab Ofatunumab Pertuzumab Pertuzumab Ipilimumab Cetuximab Cetuximab Rituximab 2 chimeric,2 humanized, 4 3 human fully Nude Nude antibody approved FDA Brand Name Herceptin Herceptin Campath Vectibix Vectibix Rituxan Erbitux Erbitux Avastin Arzerra Arzerra Perjeta Perjeta Yervoy

Ab CTLA-4 EGFR1 EGFR1 HER-2 HER-2 Target VEGF VEGF CD20 CD20 CD52 was evaluated, only nine have beenhave nine only evaluated, was Chronic lymphocytic leucemia leucemia Chroniclymphocytic leucemia Chroniclymphocytic Colon, head and neckand Colon,head Lymphomas Melanoma Cancer(s) Cancer(s) Multiple Multiple Breast Breast Breast Colon mAbs 2009 2006 2004 2004 2001 2012 1998 1998 Year Year 1997 1997 2011

19 Department of Chemistry Most ADCs have an average of of anhaveaverage ADCs Most (2, additions 4, or8) 6 drug to Conjugation adducts to Conjugation - - to attached been have Drugs

the the

sulfhydryl portion of reduced cysteine residues ε with with -amino terminus of lysine residues Chemical conjugation different stoichiometry and location reduced cysteines reduced lysine Lysine H N eius eut i a in results residues O NH 2 mAb 4 drugs4 per antibody Cysteine H N using native using O SH results in results aminoacids even-numbered itiuin of distribution : :

20 Department of Chemistry - - developed by: year, recent In not yet been not yet evaluated in clinical trials The impact ofsite-specific conjugation methods has - - - to attached been have Drugs

orthogonal chemistry methods methods chemistry orthogonal introducing novel introducing enzymatic conjugation conjugation enzymatic introducing the the

sulfhydryl portion of reduced cysteine residues ε -amino terminus of lysine residues Chemical conjugation site-specific conjugation conjugation site-specific non-natural unpaired cysteine mAb aminoacids using native using residues residues methods have been been have methods

aminoacids ht lo for allow that : :

21 Department of Chemistry the reachADC the tumor after only drug the release should linker The maximize tumor exposure drugsto to key the Stability conjugates the of activity and bioavailability the of nature The Thioether Disulfide Peptide Hydrazone Linker

of the ADCs before they reach the target cells is cells target the reach they before ADCs the of degradation for intracellular designed and Non-reducible intracellular an with exchange disulfide through to cleaved be Designed as such proteases to enzymatically be Designed the cytoplasm within compartments acidic in degradation and for stability serum Designed Release mechanism iiie h oftre killing off-target the minimize the linker plays an important role

ADC LinkerADC thiol , as glutathione such cathepsin B hydrolysed

by proteolytic lysosomal

and in 22 Department of Chemistry than standard chemotherapy agents were needed. needed. were agents chemotherapy standard than agents cytotoxic potent that apparent become has it decades, 2 last the In additional are transport) facilitated ADCs. of design the for considerations or diffusion (via are that lysosomal agents cytotoxic Identifying the as such elsewhere, activity cytoplasm ornucleus. cell-killing its exerts usually is drug The concentration in present be must ADC an of drug released The proteases inside the tumor cells to cause cell death. death. cell cause to cells tumor the inside ADC payloadADC released in the lysosome the in released and are are and 0-00 tms oe potent more times 100-10000 able to exit exit to able resistant the lysosome the sufficient but oftenbut highly

to 23 Department of Chemistry h ACatgn ope cud e quickly, be could internalized orpoorly moderately complex ADC/antigen The the molecule cell-surface of type theon depends internalization of efficiency The endocytosis through internalized be may complex ADC/antigen The 2. Endocitosy Mechanism of action

1. Binding 1. Binding but it’s mainly used as used mainlyit’s but an activities, of anticancer own its possess may component ADC antibody monoclonal The surface. the of ADC component antibody monoclonal The binds to the target antigen target the to binds mAb binds to to binds site-directed delivery on the cell the on

24 Department of Chemistry tumor cells target inside cytotoxic the of release the activate and Internalization the activated, be internalized be must ADC to cytotoxic the for order In drugs cytotoxic Lysosomal 4. Release erdto rsls n ees o the of release in results degradation Mechanism of action 3. Degradation h lssms r bt aii ad ih in rich and acidic both proteolytic are lysosomes The complex. by followed is complex ADC/antigen the of Internalization lysosomal enzymes. degradation lysosomal degradation of the 25 Department of Chemistry Mechanism of action Images from: Images http:// 5. Celldeath neat wt ciia clua poess to processes cellular death cell induce critical with then it interacts released, been has cytotoxic the Once www.biooncology.com /research-education /research-education 26 Department of Chemistry Drugs drugs Aim : improve tumor selectivity : ( : First First generation of ADCs desacetyl )vinblastine, methotrexate, )vinblastine,methotrexate, of clinically used anticancer used clinically of doxorubicine

27 Department of Chemistry Release Linker : esters, amides, hydrazones hydrazones amides, :esters, First First generation of ADCs : esterases, peptidases, hydrolysis hydrolysis peptidases, :esterases, 28 Department of Chemistry Non-small-cell lung unconjugated drugs drugs unconjugated Potency: Antibody: Murine First First generation of ADCs moderately potent and often less active than the the than active less often and potent moderately murine or chimeric (HAMA in >50% of the patients) the of in>50% (HAMA orchimeric murine Murine Lung and colon adenocarcinoma Breast Murine Chimeric 29 Department of Chemistry Non-small-cell lung - - Evidence of tumor localization, BUT:

immune response to the the to response immune and/orresponse clinical benefit therapeutic of evidence little First First generation of ADCs Phase I Lung and colon adenocarcinoma Phase I mAb and to the drugs (mainly drugs the to and Breast

Phase I Phase II Vincas ) 30 30 Department of Chemistry sd uie niois i cud e ovd using solved be could it “humanized” antibodies: murine used immunogenicity of Problem • • The linkers were:

too stable (amides) enough stable not ➙ ➙

➙ drug notreleased ➙ drug the of release premature low potency/efficacy potency/efficacy low index therapeutic lower and toxicity systemic or Lessons learned fully humanfully (esters and hydrazones) hydrazones) and (esters : antibodies oe wt ery Ds that ADCs early with noted

31 Department of Chemistry be efficient. be notmay moiety, drug active the release to processing intracellular or b) 10 (typically canbind antibody the which a) the of Delivery required The - - potency: Lacksufficient of

internalization moderate number of antigen of number moderate conjugated drugs drugs conjugated not modes different ubr f molecules of number enough concentration toeffect

cytotoxic drugscytotoxic Lessons learned cell kill of cell-surface bound antigen–antibody complex, antigen–antibody bound cell-surface of f ellr pae f h ucnuae and unconjugated the of uptake cellular of could be very high ( high very be could f mdrtl ptn cttxc drug cytotoxic potent moderately a of in the tumor cells cells tumor inthe by an ADC is limited by 2 factors: by limited is anADC by molecules on the cell surface to surface cell the on molecules 5 receptors/cell) receptors/cell) >10 6

molecules/cell). 32 32 Department of Chemistry - “ -

(too toxic to be administered as a single agents) agents) single a as administered be to toxic (too Humanized More potent drugs Second generation of ADCs ” or fully humanfully - Voluntarily 2010in withdrawn Gemtuzumab - FDA 2000in approved mAbs

ozogamacin

33 Department of Chemistry myeloid leukemia (AML) Approved Anti CD33Anti Humanized Gemtuzumab

for the treatment of of treatment the for

ozogamacin picomolar breaks in resulting groove minor the Bind D3 oiie acute positive CD33 Calicheamicins n cl dah in death cell and concentration obe strand double

of DNA sub-

34 34 Department of Chemistry pae I satd n 04 fe a D accelerated- FDA and added a after 2004 ADC showed process in approval started III phase A drug/antibody with variable conjugates Mixture Mixture of ratios Gemtuzumab no benefit nobenefit over

conventional

in physiological solution in hreleased 24 6% at pH = 7.3 increased patient deathpatient increased ozogamacin

cancer therapiescancer

35 35 Department of Chemistry due topoor enrollment with patients in trial III phase A CD22+ NHL lymphoma)(Non-Hodgkin aggressive refractory with patients for study III phase a in survival improved demonstrated to failed Recently Anti CD22 Anti Humanized Inotuzumab

hs II tde cniu fr patients for leukemia) lymphoblastic ALL with (acute continue studies III Phase folicular

ozogamacin b-cell NHL has been terminated been has NHL b-cell

36 Department of Chemistry Third generation of ADCs (FDA approved(FDA in 2011) (FDA approved(FDA in 2013) ADCETRIS KADCYLA ® ® 37 37 Department of Chemistry • • afterphase (acceleratedprocess II) approval • ADCETRIS

Chimeric 53% patients. the of in94% Tumorreductions lymphomascell In ALCL, response rate of 86%, with with 86%, of rate response ALCL, In with 75%, of rate response HL, In with patients for Approved mebrane . Tumor reductions in 97% of the patients. patients. the of in97% .Tumor reductions monoclonal antibody (cAC10, which targets CD30, a cell CD30, cell a (cAC10,targets antibody which monoclonal protein of the tumor necrosis factor receptor family) receptor factor necrosis tumor the of protein (ALCL). ® (Seattle genetics, 2011) genetics, (Seattle Hodgkin’s complete remissions in 34% in remissions complete (HL) and (HL) opee eisos in remissions complete anaplasticlarge-

38 38 Department of Chemistry • •

Chimeric ADCETRIS for Hodgkin lymphoma ADCETRISlymphoma Hodgkin for (doxorubicin, ADCETRIS lymphoma – Non-HodgkinCHP and for Prednisone) (Cyclophosphamide, A A hs II lncl trial clinical III phase hs II lncl trial clinical III phase monoclonal antibody (cAC10, which targets CD30) (cAC10,targets antibody which monoclonal bleomycin ADCETRIS Hydroxydaunorubicin vinblastine, , s urnl cmaig CHOP comparing currently is s urnl cmaig ABVD comparing currently is dacarbazine ® ,

Oncovin ad V – AVD and ) [vincristine],

39 Department of Chemistry • •

mollusk pentapeptide Dolastatin by identified synthetic Dolastatin Auristatin Dolabella ADCETRIS compounds that were that compounds 10 10 agents sltd rm the from isolated SAR s lipophilic a is

auricularia

studies are fully of

® - auristatin HN N MMAE = Mono Methyl = Mono Methyl MMAE MMAF = Mono Methyl = Mono Methyl MMAF O O N H N H Dolastatin Auristatin Auristatin O O N N O O O O N N

10 E O F O O O N H N H Ph S COOH Ph N 40 Department of Chemistry • • •

oyeiain eutn i G2/M in resulting polymerization tubulin inhibit that tubulin Vinca cytotoxic than MMAE MMAE than cytotoxic MMAE than death cell and arrest MF s 0 tms es cytotoxic less times 100 is MMAF MMAF- ihy oet ( potent Highly -domain OMe ADCETRIS in vitro s 0 tms more times 100 is idr of binders nM-pM IC ® β 50 - - ) auristatins HN N MMAE = Mono Methyl = Mono Methyl MMAE MMAF = Mono Methyl = Mono Methyl MMAF O O N H N H Dolastatin Auristatin Auristatin O O N N O O O O N N 10

E O F O O O N H N H Ph S COOH Ph N 41 Department of Chemistry • • Auristatins

trials use MMAF trials use MMAF trials use MMAE / 3 Ds n clinical in ADCs 43 / 2 clinical in ADCs 43 / 16 ADCETRIS in other ADCs: ® -

auristatins HN N MMAE = Mono Methyl = Mono Methyl MMAE MMAF = Mono Methyl = Mono Methyl MMAF O O N H N H Dolastatin Auristatin Auristatin O O N N O O O O N N 10

E O F O O O N H N H Ph S COOH Ph N 42 Department of Chemistry • • • • but have not yet been tested in clinic studies). studies). inclinic tested been yet not have but auristatins than easier 12 as exist that residues cysteine the of conjugation Interchain mAbs Free itiuin f ojgts r otie, ih an with obtained, are conjugates of Distribution intrachain ADCETRIS do not generally contain free free contain generally not do thiol intrachain moieties disulfide per

disulfide bonds for a humanIgGa for bonds disulfide mAb maleimide disulfides ( disulfides

on the the on (the separated species can be purified by HILIC by purified be can species separated (the can be reduced and maintained as free free as maintained and reduced be can ® - conjugation site -conjugation -bearing -bearing disuflide 2 to 8 free 8 to 2 mAb thiols

r required are auristatin but they contain endogenouscontain they but MMAE = Mono Methyl = Mono Methyl MMAE pairs thiols 1 Auristatin ) derivatives derivatives ( 4 ) interchain avarage o nbe the enable to E of thiols

and 4

43 Department of Chemistry • • • • has a negative influence on the kinetics of the peptide hydrolysis). hydrolysis). peptide the of kinetics the on influence negative a has cleavage enzymatic of site the from drug form. active donating hydrolyzed stability The The cleavage, enzymatic After A pn internalization Upon valine-citrulline self- ADCETRIS immolative p (half-lives 6 days in mice and 10 days inmonkey) 10and days inmice days 6 (half-lives - aminobenzyl by the the by Val cathepsin Cit linker

spacer

group ( 1,6-elimination 1,6-elimination , the dipeptide linker is is linker dipeptide the , ® is designed to provide provide to designed is PAB - smart linker is needed to to needed is B proteases B PAB

) occurs, releasing MMAE in itsin MMAE releasing occurs, ) MMAE = Mono Methyl = Mono Methyl MMAE spatially separate theseparate spatially of the strongly electron- strongly the of Auristatin te uk payload bulky (the ih plasmahigh efficiently E

44 Department of Chemistry ADCETRIS H 2 N MMAE - O O O O HN N N O O O N H N H N H ® O O O - smart linker N N N - CO Cathepsine Cathepsine B 1,6 1,6 immolation O O O 2 O O O N N N O O O O O O N H N H N H OH OH OH Ph Ph Ph 45 Department of Chemistry • • currently in currently mAb of favor in lymphoma non-Hodgkin or vorsetuzumab the monomethyl to conjugated molecule, etl Gntc has Genetics Seattle Humanized Vorsetuzumab Stable linker , different drug, drug, different , phase I

auristatin oolnl nioy wih agt CD70) targets (which antibody monoclonal

mafodotin for the same tumors) tumors) same the for phenylalanine (MMAF) (MMAF) phenylalanine discontinued (SGN-75), for renal cell carcinoma cell renal for (SGN-75), i.e.

pyrrolobenzodiazepine

mafodotin auristatin MMAF h dvlpet of development the SGN-CD70A SGN-CD70A analogue

(same , 46 Department of Chemistry anti-CD70 • •

Cleavable linker currently in currently mAb of favor in lymphoma non-Hodgkin or vorsetuzumab the monomethyl to conjugated molecule, etl Gntc has Genetics Seattle CD70) targets (which antibody monoclonal Humanized

, different drug, drug, different , phase I

auristatin

mafodotin SGN-CD70A for the same tumors) tumors) same the for phenylalanine (MMAF) (MMAF) phenylalanine discontinued Pyrrolobenzodiazepine (SGN-75), for renal cell carcinoma cell renal for (SGN-75), i.e. (DNA-crosslinkingagents)

pyrrolobenzodiazepine auristatin h dvlpet of development the SGN-CD70A SGN-CD70A analogue (PDB)

(same , 47 Department of Chemistry than DC-81 invitro)DC-81 than cytotoxic more times 600 is DSB-120 potency in increase Dimers refuineus of broth the fermentation of constituent active the 1960s, the in when discovered first were Pyrrolobenzodiazepines anthramycin resulted in significant

a ioae as isolated was SGN-CD70A Streptomyces fr example (for Pyrrolobenzodiazepine (PBD) (DNA-crosslinkingagents) (PDB)

48 Department of Chemistry • multi-drug-resistant cancer cell lines cell cancer multi-drug-resistant vivo in result promising Gs otiig Ds showed ADCs containing IGNs • •

leukemia carcinoma G-D3 i in is SGN-CD33A in is SGN-CD70A gis bt nra and normal both against (AML) and with with and PDB-ADCs n vitro in hs I phase hs I phase non-Hodgkin lymphoma Pyrrolobenzodiazepine (DNA-crosslinkingagents) and for patients with with patients for

for patients with with patients for ct myeloid acute ea cell renal (PDB)

49 Department of Chemistry • • • •

20-25% of human breast cancer and is is and cancer breast human of 20-25% 2, receptor factor growth humanepidermal targets which following the binding binding the following Her2 is an excellent ADC target: it is is it target: ADC excellent an is Her2 Humanized complete a phase III phase a complete in1% remissions is AC prvd o a for approved ADC First In rat cancer breast KADCYLA monoclonal antibody ( antibody monoclonal , response rate of of rate response , ® oi tumor solid (Roche, 2013)(Roche, trastuzumab amplified 43% ucl internalized quickly and , clinically approved clinically , ih complete with frt D to ADC first , overexpress HER2 ) in 50 Department of Chemistry • • •

sltd rm h br o te Ethiopian the of shrub bark the from isolated a therapeutic benefit of ends the at located a has and As single agent, it it agent, single As the near tubulin binds It Maytansine Maytenus KADCYLA is a ih fiiy o tubulin for affinity high

ovatus failed benzoansamacrolide micrutubules

in1972 at tolerable dose dose at tolerable to Vinca demonstrate ® binding site binding

maytansine

Maytansine

51 Department of Chemistry •

Published semi-synthesis Published KADCYLA

® - maytansine

52 Department of Chemistry • • •

KADCYLA ru (MC, olwd y h rato o te oiid antibody modified the of the with reaction the by followed (SMCC), group bifunctional The ADCs are prepared by reacting the the reacting by prepared are ADCs The fully degradationfully of the ADC Non-cleavable linker: drug is released only after only released is drug linker: Non-cleavable of Average MCC = thiol N 3.5 DM1 -Maleimidomethylcyclohexane-1-carboxylate linker composed of an activated ester and a and ester activated an of composed linker containing MCC per antibody antibody per ® conjugation &linker conjugation

maytansinoid

(DM1) antibody DM1 DM1 lysines maleimido

ih a with

53 Department of Chemistry DM1 DM4 Me Me Me Me R1 H DM DM side H H Me Me Me Other R2 H H H H mAB Me Me Me Me R3 H H H side Me Me Me R4 H H H H maytansinoid 0 0 0 q 1 1 1 1 * by DTT at pH = 6.5, at 37°C 6.5, = 37°C DTTat pH at by * Disulfide reduction rate (k rate 0.0006 0.014 . M 0.8 0.8 14 14 2 1 -1. min -1 )* Relative Relative stability 22000 DM1 or DM4) paylod matynsinoids use trials clinical in ADCs of 43 / 13 980 170 170 14 14 16 7 1 ADCs In vivo PK t (mainly n.d. n.d. n.d. 218 218

87 15 47 47

as 1/2 (h) 54 Department of Chemistry Other cytotoxic Otherdrugs cytotoxic as payload Pyrrolobenzodiazepine Total ofTotal 43 ongoing clinical trials with ADCs: Duocarmycin Calicheamicin Maytansinoid Doxorubicin Auristatins Drug analog

dimer dimer

clinical trials ADCs inADCs 18 18 13 2 2 1 1 Microtubule disruptingagents Microtubule disruptingagents DNA minor groove binder binder minor DNA groove binder minor DNA groove Mechanism of action DNA intercalating agent intercalating DNA Alkylating agent agent Alkylating

55 Department of Chemistry •

• •

receptor 2 targets which approved Humanized 21, 2014 tumors solid metastatic A Advantage compare KADCYLAto hs I phase ) ,HER2) clinical trial for patients with with patients for trial clinical oolnl nioy ( antibody monoclonal Duocarmycin ua eieml rwh factor growth epidermal human a satd n mnh g ( ago month one started has in preclinical studies studies inpreclinical trastuzumab oal avne or advanced locally

clinically , October 56 Department of Chemistry • •

h mnr roe f DNA of groove minor the Streptomyces cell death architecture alkylation of DNA cause subsequently This Duocarmycins irps h ncec acid nucleic the disrupts atra n 1988, in bacteria , which leads to to leads which , frt sltd from isolated first , Duocarmycin irreversible id to bind tumor and

57 Department of Chemistry • • •

ellr yl (uui bnes attack binders (tubulin cycle cellular phase any at action of mode tumor cells in a mitotic state) state) mitotic ina cells tumor Duocarmycins as payloads ADC developing for essential duocarmycins Lessons learned in the the in learned Lessons activity therapeutic Limit as a single agent agent single a as are able to Duocarmycin SAR duocarmycins xr their exert ae been have in clinic of clinic in n the in

58 Department of Chemistry • • quickly of various types of preparation chloromethyl N ig closure Ring The N O cyclopropapyrroloindolo O in vitro O N HN Cl om n h peoi state phenolic the in form , either chemically or enzymatically, enzymatically, or chemically either , and Duocarmycin NH 2 in vivo HO moiety is is moiety prodrugs

O N HN Cl derivatized . . NH 2

wih loe the allowed which ,

in its its in O O ring openring N occurred HN NH 2 59 Department of Chemistry lines apoptosis ofvariouscell polymerization microtubule Tubulysins concentrations at resistance) drug multi with those (including Promising drug (preclinical) , natural subnanomolar

tetrapeptides Tubulysins

Amanitin n induceand perturb cell death transcription DNA of inhibition II polymerase RNA genus the of species several in found α - Amanitin mushrooms,

a , cyclic togy inhibits strongly laig to leading , octapeptide Amanita

NH and

60 Department of Chemistry estimated cost of a course of of course a of cost estimated example (for The costs for therapeutic applications are very high • immunogenic) are ingeneral proteins (recombinant • • • ofMost the ADCs:

regulations regulations thereby integrins) with the liverand the membranes need to be be to need elicit may induce can a have etitd blt t cos nohla cell endothelial cross to ability restricted severe immune responses rigorously purified rigorously Drawbacks of ADCs Kadcyla cell surface receptor clustering receptor surface cell by passive diffusion and non-specific uptake by uptake non-specific and diffusion passive by reticuloendothelial n h U i $,0 pr ramn. The treatment. per $9,800 is US the in enhance tumor angiogenesis Kadcyla is approximately $94,000) approximately is in order to comply with the with comply to order in system. in a patient patient ina

(for example(for 61 Department of Chemistry needed to characterize their complex structures. structures. complex their characterize to needed performance homogeneity Greater •

conjugates: eeoig e tcnlg t create to technology new Developing • • • •

hc i imblzs h atbd o a oi support, solid a on antibody the form. soluble ina ADC the releases it,then and conjugates immobilizes it which points linkage a as antibody inthe acids maintain the to attached) the reconnects ADC Bio has developed a “lock and release” technology through technology release” and “lock a developed has Bio ADC Reedwood’s Similarly, PolyTheric’s hl gety ipiyn te eadn analytics demanding the simplifying greatly while Igenica’s

SMARTag ThioBridge What’s next? interchain cysteines is expected to to expected is SNAP technology uses uses technology SNAP ehooy o nrdc nw amino new introduce to technology t wih txc ala i already is payload toxic a which to , is 3 carbon bonding bonding improve eldfnd drug well-defined

bisthiol ADC bifunctional stability linker that linkers and 62 62 Department of Chemistry which in turn would would inturn which in changes Thus, linker The •

conjugates: eeoig e tcnlg t create to technology new Developing • • • •

hc i imblzs h atbd o a oi support, solid a on antibody the form. soluble ina ADC the releases it,then and conjugates immobilizes it which points linkage a as antibody inthe acids maintain the to attached) the reconnects ADC Bio has developed a “lock and release” technology through technology release” and “lock a developed has Bio ADC Reedwood’s Similarly, PolyTheric’s determines the nature of the metabolite produced metabolite the of nature the determines linker design linker Igenica’s

SMARTag influence clinical toxicity ThioBridge What’s next? interchain cysteines SNAP technology uses uses technology SNAP ehooy o nrdc nw amino new introduce to technology t wih txc ala i already is payload toxic a which to , is 3 carbon bonding bonding would alter the the alter would eldfnd drug well-defined

metabolite profile metabolite bisthiol bifunctional linker that linkers , . 63 Department of Chemistry • • •

tubulins tubulins maytansines the to action alternatives of mechanisms different t h sm time, same the At antigen expressions, heterogeneity and internalizationandrate heterogeneity expressions, antigen diseases and autoimmune Develop ADCs outside oncology in areas such as such areas in oncology outside ADCs Develop Identification Identification • •

conjugation highly hydrophobic drugs without aggregations aggregations without drugs hydrophobic highly conjugation pyrrolobenzodiazepines

amanitin from are the most promising promising most the are of the the of What’s next? ImmunoGen oe oet lse o toxins of classes potent more auristatins e cl srae targets surface cell new

rm etl Gntc ad the and Genetics Seattle from al f hc at y inhibiting by act which of all , , duocarmycins ae mrig as emerging are ,

inflammatory bsd on based , having ,

, α - 64 64 Department of Chemistry • •

penetration whole of rpniy o cuuae n iny rqie half-life requires kidneys extension strategies ( in accumulate to propensity Use of smaller scaffold proteins proteins scaffold smaller of Use h fse cerne f hs famns n the and fragments these of clearance faster The •

level of penetration penetration of level intact the while sFv o eape te mlet rget snl vral domain, variable (single fragment smallest the example, For , eosrtd aia tmr eerto a 05 hours, 0.5 at penetration tumor maximal demonstrated ), IgGs , may offer the advantage of of advantage the offer may , What’s next? IgG took 48 to 96 hours to achieve the same the achieve to hours 96 to 48 took e.g. conjugation with polymers) or antibody fragments antibody or

rae tumor greater , instead, 65 Department of Chemistry • •

clinic: failure in the clinic clinic inthe failure More preclinical evaluation Setting such such Setting • •

doses achievable in the clinic (2–7 mg (2–7 clinic inthe achievable doses the to be It is important to demonstrate antitumor activity invivo activity at antitumor demonstrate to important Itis have studies efficacy for models preclinical therapy, ADC For xenograft carefully selected carefully selected higher preclinical bars

reflects the clinicalreflects population What’s next? such that the antigen expression on expression antigen the that such prior to advancement to the the to advancement priorto may mitigate the chance of chance the mitigate may . kg -1

for most ADCs) ADCs) most for to betreated 66 Department of Chemistry • • • •

Small molecule drug conjugates C4) tasern eetr (D1, rwh factor growth (CD71), (EGFR,receptors VEGFR, PDGF) receptors transferrin (CD44), cell’s diseased the to receptors bind selectively that molecules Folate The most used receptors are: The Linker Targeting ligand agtn ligands targeting receptors and cytotoxic drugcytotoxic itgis hauoi ai receptors acid hyaluronic integrins, , smart linker oss o hg-fiiy small high-affinity of consist similar to ADCs ADCs similarto

cytotoxic cytotoxic drug

67 Department of Chemistry • • •

(NSCLC) with patients vintafolide to linked drug anti-mitotic the of Phase Vintafolide hs II td ws stopped was study III Phase IIb folic acid is still ongoing is still int eosrt efcec we treating when efficiency demonstrate didn’t study in non-small-cell lung carcinomalung non-small-cell in study , platinum-resistant ovarian cancer

the most advanced SMDC, is a derivativea is SMDC, advanced most the Vintafolide vinblastine

which is chemically is which n a because May in 68 Department of Chemistry •

H EC1456 (structure not (structure released): EC1456 (HCC) Carcinoma Hepatocellular Cancer; Ovarian (NSCLC); (TNBC); Breast Cancer Cancer Lung Cell Non-Small Advanced EC1456: 2 N phase I clinical trials, sponsored by Two HN O N N N folate patients with with patients Solid Advanced Tumors; Triple-Negative H N Other O H N ojgts r crety n active a in currently are conjugates CO 2 H O N H folate O CO H N 2 H HN O N H NH NH O CO 2 H N 2 H conjugates patients with prostate cancer cancer prostate with patients CO S S 2 H O HN O H N O Ph N H Endocyte O N S OAc

O O N O NH

O N 69 Department of Chemistry • • • •

releases the vinblastine the releases selectively linker stable endocytosis via internalized subsequently folate Once inside the cell, the serum- the cell, the inside Once Folate iess icuig te agesvl goig acr and cancers growing aggressively disorders. inflammatory other including diseases, folate express often cells Elevated expression of the the of expression Elevated Folate Mechanism of to support rapid cell growth. growth. cell rapid support to ojgts id o the to bind conjugates s eurd o cl dvso, n rpdy iiig cancer dividing rapidly and division, cell for required is receptor and are folate Targeted 2011, Inflammation. and for Cancer Strategies Drug eetr i odr o atr enough capture to order in receptors folate folate eetr cus n many in occurs receptor conjugates pp 135-150 135-150

70 Department of Chemistry extracellular matrix and mediate several intracellular signals intracellular several matrixmediate and extracellular Integrins are cell surface receptors that interact with the the with interact that receptors surface cell are and to the the to and • ( cellular migration The extracellular • The Integrins programmed Attachment of the of Attachment Signal main signals

, functions differentiation extracellular transduction cell involve:

matrix

cell

division

death of to the the to integrins cell cell ). ,

matrix

cell

cell , to growth

apoptosis rm the from . . other

are: survival

cells cell ,

70 71 Department of Chemistry ait o ptooia poess hc makes which processes targets attractive pathological of variety function integrin of Disturbance Integrin for pharmacological research research pharmacological for

disturbance s once t a large a to connected is

integrins

71 72 Department of Chemistry α IIb Adapted from: S. L. Adapted Goodman, M.Picard β 3 Integrins

, Trends in Pharmacological Sciences,in Trends as

therapeutic , 2012, targets Symbols discontinued centers 33 ,405-412 with with represent Discontinued Phase Phase Phase Market trials black I II III 72

73 Department of Chemistry 2014.10.021 Letters Chemistry Medicinal and Bioorganic AAPS J. News & Engineering Chemical TodayPharmaceutical Today Discovery Drug Biomedicines Angewandte Hematology Antibody http:// https:// References future readings& adcreview.com clinicaltrials.gov

2014 2013

Chem. Int.Chem. Ed.

2013

2014 ,

16 2 , 113-129 , 306-310 , 306-310 ,

899-913 899-913 , 1-13 and references herein herein references and /knowledge-center/ /knowledge-center/

/ 2014 2014

, 869-881 , 869-881 , 42-47 , 42-47 2014

2014 , 53 , , 3796-3827 , 3796-3827 92 , 13-21 , 13-21 10.1016/j.bmcl.

74 Department of Chemistry Eli Lilly – LIFA (Lilly innovation fellowship award) Prof. Prof. Wipf Gennari group past present & Thanks! Prof. Prof. &

Wipf Gennari

group 75 Department of Chemistry Department of Chemistry Mechanism of Department of Chemistry Department of Chemistry Department of Chemistry Hydrophobic Interaction Chromatography (HILIC) Measures Heterogeneity Department of Chemistry h sa o te ih dmntae cmlt eiiain of elimination complete demonstrates normal are bladder). (brain, right the kidneys,and the right the tumours on scan The in cancer) of (spread disease metastatic (70 patient the of the on extent image the The shows scans. left treatment (right) after and (left) Before two weeks after treatment (the black blobs in the scan onscan the in blobs black (the treatment after weeks two tumours ADCETRIS ADCETRIS from Non-Hodgkin’sfrom Lymphoma). Department of Chemistry Trastuzumab – tubulysin ADC Department of Chemistry (predicted from preclinical tests) are expected expected are tests) preclinical from (predicted toxicities off-target less consequence, a as route; excretion biliary the through transit will drug less clearance, hepatic reducing liver. By the through properties clearance non-specific limited by sponsored trials clinical latest the is EC0489 Improved folate analogue analogue folate -targeted chemotherapeutic to enter enter to chemotherapeutic -targeted Endocyte – vinblastine –vinblastine , that was designed to have to designed was , that Department of Chemistry Department of Chemistry 19 Oct 2009 Oct 19 Department of Chemistry Of the 24 different heterodimers known, the heterodimers 24 different the Of Integrins α V β 3 , α V β : 5 , angiogenesis α 5 β 1 are key-factors of angiogenesis of key-factors are RGD-binding integrins

key-factors

85 Department of Chemistry • •

and significantly and integrins an containing ligands (RGD) recognise to ability the Synthetic RGD-ligands can bind selectively selectively bind can RGD-ligands Synthetic five All metastasis R = R = Arg tripeptide α V

RGD- integrins . active site active binding , two , inhibit β

integrins

angiogenesis

G =Gly integrins ( α 5 , α 8 ) α and D = Asp , V β tumor growthtumor

3 Arg , α α IIb - V Gly β β 3 5 share , -Asp α 5 β 1

86 !"#$%&'"(&)*+),-"'./&%01 Department of Chemistry Department of RGD Chemistry RGD

Integrin Integrin Asp150 Asp150 electrostatic clamp electrostatic electrostaticclamp The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the ﬁle again. If the red x still still x red theIf again. ﬁlethe thenopen and computer, your Restart corrupted. havebeen imagethemay or image, the open have enoughnotto may memory computer Your displayed. be imagecannot The appears, you may have to delete the image and then insert it again.then it insert image theand delete haveto may you appears, _ _ H H 2 2 N N NH NH Asp218 Asp218 N H N H C( C( " β )-C( )-C( NH NH O O " β Distance: ) 9 9 ≈ Distance: ) N H N H O O Ca Ca 2+ 2+ 2+ O O H N H N MIDAS MIDAS MIDAS ! 9 O O Å Å #

OH OH 87 Department of Chemistry The The cyclo α receptor integrin to binding Inhibitionof (H. Kessler et al., et (H.Kessler Cilengitide with glioblastoma multiforme V III H β potent O [ 3 Arg O clinical IC C O P 50 - h

Gly α 0.58 N Cilengitide V H biotinylated O N O

J -Asp- β E is . H trials for M Med M N 3

D nM currently H e ligand RGD- 1 N H 2 . D

Chem 1 N

- 9 Phe 7 O O 4 , .

cyclic 1999

vitronectin N patients H in N (Me)-Val] (Me)-Val]

, binding 42 peptide peptide N phase N , 3033) , H H 2

integrins (C. Scolastico et al., et (C.Scolastico α α receptors integrin to binding Inhibitionof (C. Scolastico et al., et (C.Scolastico Cyclic V V β β

cyclo 5 3 IC IC RGD 50 50 [ 1.4 ±0.8 1.0 ±0.5 Arg biotinylated pentapeptide ST1646 ST1646 H ChemMedChem - S N Gly Org T N

1 6 O A -Asp- . 4 s Lett 6 nM nM p G A O l r . y

g 2001

lactam

vitronectin

2009

mimics ,

3 ,1001) ] , 4 , 615) ,

Department of Chemistry RGD RGD • •

α α delivery V V

integrins integrins Conjugate tumor ligands of potent cytotoxic drugs ( drugs cytotoxic potent of -homing are overexpressed on the surface of cancer cells cells cancer of surface the on overexpressed are can be internalized by cells cells by internalized canbe

conjugate RGD ligands can be employed as employed canbe RGDligands peptides for peptides to to anticancer site-directed e.g. Paclitaxel) Paclitaxel)

drugs 89

Department of Chemistry Department E. R. Colombo RGD RGD CYTOTOXIC [DKP-RGD] [DKP-RGD] Scanziani SPACER SPACER LINKER LINKER of DRUG DRUG Chemistry , M. C. ligands Gennari Mingozzi

. , L. J. Med. Chem. Belvisi , D.

H conjugate HN Arosio 2

2012 N , , U. NH 55 , 10460-10474. , 10460-10474. Piarulli O , N. O NH Carenini O HN H N to to , P. N O Perego HN anticancer O , N. HN NH O Zaffaroni O Ph Ph O O , M. De O OH O NH Cesare O O O HO

Ph , V. drugs Castiglioni O AcO H OAc O , O OH

90 Department of Chemistry Boc HO Mtr = O HN O H N R R 1 1 = H, R = N DKP- Boc-HN O O Synthesis HO 2 R = O 1 f 2 or O N NH-Mtr N H N O R O O S 2 Mtr , R NH-Mtr 2 = H methyl, benzyl, methyl, 5 5 steps Boc HO and O HN O H N of Cbz Mtr N orthogonal to: orthogonal DKP- N H protecting groups protecting O O NH cyclo f O N H 3 HN O NH allyl O R O H N H N 1 HN , O N t Mtr Bu O [DKP-RGD] [DKP-RGD] , N Boc Mtr O R 2

, N H Phenol 1% Phenol EDT 5% 10% Thioanisol TMSBr 14% TFA 70% 70-80% rt, 2h DKP- Boc HO f 5 O cyclo cyclo cyclo cyclo O H N N [DKP- [DKP- [DKP- [DKP- H linker = linker NH 2 N O O NH f f f f 6-RGD]: 3 6-RGD]: 3 4-RGD]: 3 3-RGD]: 3 2-RGD]: N H O HO HN NH ligand Mtr O R O H N S R S R 1 N H HN , 6 , 6 , 6 , 6 O N 2 R, R, S, S, O 3 6 R R R R 5 DKP- N 1 1 1 1 = linker, R = linker, R = H, R = H, R NH O R Boc 2 HO f 2 6 O

2 2 = linker = linker O H N N 2 2 = H = H NH O 91 Department of Chemistry 10 h0° CH Ph O 3 CN / phosphate buffer /CN phosphate = (pH 7.3 -7.6) O O HO HN AcO NH N H C C to rt O O Ph HN R O H N O HO 1 HN O C N NH OH O O NH 60-75% N 2 O R OH H 2 CF O OAc O R O R 3 Synthesis 1 1 COOH = H, R = Ph 2 O = or overnight DCM, DCM, Py 92% NH O Paclitaxel 2 , R 2 NH O = H 2 Ph O O AcO of N H O O Ph HO HN O HO NH O O O cyclo HN R O H N O 1 HN O C N NH OH 2 H

O 3 6 O NH conjugates O O 5 OAc N O 2 Ph O R OH 2 Linker-PTX = Linker-PTX cyclo cyclo cyclo cyclo [DKP-RGD]- overnight, RTovernight, NaO DIC, DMF [DKP- [DKP- [DKP- [DKP- quant O S O f f f f 6-RGD]-PTX: 6-RGD]-PTX: 3 4-RGD]-PTX: 3 3-RGD]-PTX: 3 2-RGD]-PTX: 3 Bz O AcO N H N O O Ph Ph OH O HO

O O O AcO S R S R N H O , 6 , 6 , 6 , 6 O Ph OH R, R, S, S, H O HO O O R R R R O OAc O O 1 1 1 1 O = linker-PTX, R = linker-PTX, R = H, R = H, R Ph O N H OH H 2 2 O = linker-PTX = linker-PTX O O OAc O O Ph O N 2 2 = H = H O O S O Na O 92 + - Department of Chemistry cyclo cyclo cyclo cyclo Inhibition of Inhibition cyclo cyclo cyclo cyclo [DKP- [DKP- [DKP- [DKP- cyclo binding to to binding Compound [DKP-6-RGD] [DKP-4-RGD] [DKP-2-RGD] [DKP-3-RGD] ( f f f f RGDfV 6-RGD]-PTX 4-RGD]-PTX 2-RGD]-PTX 3-RGD]-PTX )

biotinylated α v β 3 and IC 0.9 ±0.6 8.5 ±0.8 5.2 ±2.3 7.6 ±4.3 3.2 ± 2.7 3.2 ±2.7 2.1 ± 0.6 ± 2.1 3.2 ±1.3 4.5 ±1.1 1.1 ± 0.1 ± 1.1 α 50 [ v β nM α 3

]

5 vitronectin receptors IC 219 ± 124 ± 219 7.5 ±4.8 149 ± 25 ± 149 518 ±10 114 ± 99 ± 114 216 ± 5 ± 216 76 ±32 79 ±3 22 ± 3 ± 22 α 50 [ v β nM 5

]

93 Department of Chemistry Integrin α α v v β β Ph O 3 5 AcO

N H

cyclo O Ph O HO O O [DKP- O OH H O f 3.4 ±0.9 O 4.8 ±1.9 3-RGD]-PTX IGROV-1 O OAc O Ph Integrin N H

O O N 23.3 ± 5.0 ± 23.3 O IGROV-1/Pt1 IGROV-1/Pt1 NH 3.3 ±0.5 OH O NH H N HN O H HN HN 2 N O

expression

O NH Mean 27.4 ± 0.1 ± 27.4 1.8 ± 0.6 ± 1.8 U2-OS

fluorescence accessibility on a multi-gram scale multi-gram aon accessibility mainly synthetic straightforward its conjugate of because lead as chosen cyclo integrin becouse IGROV-1/Pt1 cellular

6.4 ±0.05 of line [DKP- 4.4 ±0.5 SKOV3

intensity its

was high expression of of expression high f

3-RGD]-PTX

chosen

25.7 ± 6.5 ± 25.7 7.9 ±2.8 PANC-1

in vivo in

Department MIA-PaCa2 5.6 ±0.9 model 1.2 ± 0.1 ± 1.2 Chemistry

was α v β of 3

Department of Chemistry Department of Chemistry of Department as single agent decreased over time over decreased agent as single Paclitaxel with treatment after observed cells mitotic aberrant of amount The after the 2 with observed were mitoses aberrant of levels High Evaluation of RGD]-PTX cyclo RGD]-PTX cyclo Paclitaxel Treatment [DKP- [DKP-

f f

treatment and persisted after the 4 persisted treatment and

3- 3-

c [DKP- c [DKP- f 2

3-RGD] (mg/kg) f

nd 3-RGD]-PTX Dose Dose 30 30

c [DKP-

c ( [DKP- mmol f3-RGD] in vivo Dose Dose 4 f 35.1

3-RGD]-PTX 19.1 9.6 th

/kg)

TVI% 64 85 76

antitumor

c [DKP- c [DKP-

2 f3-RGD] th

nd f 3-RGD]-PTX * 0/8 2/8 3/8 15 mg/kg CR treatment. **

cyclo

c c

[DKP- [DKP- c [DKP-

f3-RGD] NED 0/8 2/8 *** 4 f3-RGD]-PTX f - th 3-

[DKP- *

activity BWL% f 3-RGD]-PTX 0 4 3

D/T 0/4 0/4 0/4

Department of Chemistry C. R. Colombo Department of Chemistry of Department h aon o aern mttc el osre atr ie ramn with treatment mice after observed cells mitotic time over decreased Paclitaxel aberrant of amount the 2 the after with observed were mitoses aberrant of levels High Gennari . , M. J. Med. Chem. Mingozzi nd 2

treatment and persisted after the 4 the after persisted and treatment , L. nd

2012 Belvisi Immunohistochemistry , 55 , D. , 10460-10474. , 10460-10474. Arosio , U. Piarulli 4 th , N.

Carenini , P. Perego , N. Zaffaroni th treatment. On the contrary, the On treatment. , M. De 2 nd * cyclo ** Cesare [DKP- , V. Castiglioni

f 3-RGD]-PTX *** 4 , E. th * Scanziani

96 Department of Chemistry Ph Cyclo O AcO N H cyclo O Ph O HO O O [DKP- [DKP- O OH H O f O 3-RGD]-PTX O OAc O Ph N H f 3-RGD]-PTX 3-RGD]-PTX O O N O NH OH O NH H N HN O H HN HN 2 N O O NH HOOC vs O O NH PTX-E( PTX-E(cyclo[RGDfK] PTX-E(cyclo[RGDyK] N H H N O HN HN HN O H 2 N R O NH O N H 2 2 Ph ) ) O

Ph R=H(Ryppaandco-workers)

cyclo HN R =OH NH O R O O

(Chen andco-workers) O O O N H AcO Dipartimento di Chimica di Dipartimento O HO [ O RGDfK NH HOOC Ph O O NH O H O OH OAc H N HN HN O O HN O NH

] 2 NH ) 2